QUALITY ASSURANCE PLAN FOR THE CHESAPEAKE BAY PROGRAM
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QUALITY ASSURANCE PLAN FOR THE CHESAPEAKE BAY PROGRAM
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INTRODUCTION
The overall goal of the Chesapeake Bay Program (CBP) is to protect and
enhance the quality of the Chesapeake Bay. To this end, the present condition
of the Bay-must: be .recordedso- that trends can_be established. ~_This. requires "-
the determination of a variety of parameters such as toxicant and nutrient
concentrations in water, sediment and biota. The data resulting from these
studies will be used to develop a model to predict the future of the Bay. A
control program can then be formulated to optimize the Bay's uses and resources
while preserving or improving its quality.
The following three study plans have been proposed for the Chesapeake Bay
Program:
"Plan of Action - Toxics Accumulation in Food Chain", prepared by the
Toxics Work Group of the CBP;
"A Plan for Ecological Studies of Submerged Aquatic Vegetation and
Associated Living Resources of Chesapeake Bay", prepared by the
Working Group on Submerged Aquatic Vegetation of the Chesapeake Bay
Program; and
"Eutrophication Work Program for Chesapeake Bay", prepared by the
CBP Eutrophication Work Group.
The activities proposed in the program plans listed above consist of. lit-
erature reviews, accumulation and evaluation of available data, extensive sam-
pling in the Bay combined with analyses of the samples, some experimental, lab-
oratory work, and management tasks. The sampling and analytical activities
described in the various plans are partially overlapping and often not clearly
defined. It is desirable to combine sampling events proposed under the various
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programs to the highest extent possible. This will not only be more economical
and reduce duplication of efforts but concurrent data will be obtained which
will greatly facilitate interpretation and modeling (the media to be sampled,
as well as the parameters to be determined, are summarized in Appendix A).
Most of the work proposed in the study programs will be performed by as
yet unspecified contractors and/or grantees. The data generated will form the
input for the model mentioned above,.which in turn will be the basis for
management-.decisions on action to be taken. These'management decisions can -."--
be only as good as the quality of the data they are based upon, and it is
imperative that the precision and accuracy of the data be assured. To achieve
this, a comprehensive qualityi assurance (QA) program must be developed covering
all work undertaken, and uniform quality control must be imposed on the data
collection activities of all contractors and grantees involved.
The purpose of this plan is to outline coordinated and comprehensive QA
guidelines for the Chesapeake Bay Program. It should encourage project officers
and individual investigators to give adequate thought and sufficient planning
to the quality control measures, techniques, and procedures to be used before
initiating a project, task, experiment, or contract. It is suggested that this
plan, together with copies of the relevant parts of the referenced literature,
be issued to all contractors and grantees, to be used as a guideline for the
preparation of the QA plans that are required for all experiment, task, project,
or contract protocols.
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Quality Assurance in Surveillance Programs
The first step of the CBP, an inventory and baseline determination, con-
sists basically of an extensive surveillance program, which will then "be '..-.
followed by monitoring programs as needed.
The role of such an environmental surveillance program is to provide
qualitative and quantitative data on selected environmental parameters. The
subjects of such programs are dynamic systems which undergo physical, biolog-
ical, climatic and man-made changes. The programs usually include the fol-
lowing operational steps:
Planning
Sample Collection .
Sample Storage
Sample Preparation
Sample Analysis
Data Manipulation
Data Interpretation .
Reporting
However, in order to obtain valid daa, an overall QA program must apply
quality control to all pertinent operational steps. In addition to the usual
analytical and equipment QA procedures, a comprehensive QA program should
include details on the reliability of the sampling program. Sampling schemes,
data analysis strategies, and the objectives of the surveillance program must
be well defined in order for a statistician to assist in the development of an
efficient collection program. Finally, proper attention must be given to
climatic, seasonal, and long-range changes in environmental conditions.
The following items and procedures are obviously important to the succes:;
of a comprehensive QA program. They are not intended to be an exhaustive list,
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merely representative:
Satisfactory facilities and equipment
Adequately trained and experienced personnel
Use of standard methods
Routine analysis of| control and replicate samples, reagent.
blanks and standards
Frequent calibration and servicing of instruments and equipment
Participation in "round-robin" programs. -____-
Methodology and Quality Assurance for the Chesapeake Bay Program
An outline which can form the basis for the preparation of QA sections of
specific protocols for the CBP plans is presented as Appendix B*. This outline
is valid for all media. Special items that go beyond the scope of Appendix B
or that supplement the outline are addressed below.
Evaluation of Literature and Unpublished Data
A substantial part of the data to be used in the CBP have been recorded in
the past by different researchers in a variety of studies. While EPA cannot
impose any rigid a priori criteria for acceptance or rejection of these.data,
. it should be stressed that they must be closely scrutinized. In many instances,
data may be of limited value or even useless because precision and accuracy
were not reported, or because of inadequate reporting of other parameters
(sampling site location, date and time, tide, water temperature, etc.).
Site Selection
For the northern part of the Chesapeake Bay, 640 sampling sites that have
been randomly selected are at present being used to monitor submerged aquatic
vegetation (SAV). It has been suggested to extend this random site selection
into the southern part of the Bay for SAV research. It should be seriously
considered if the same sites (or a certain defined and described selection of
*It is recognized that certain requirements listed in Appendix B might sound
superfluous or redundant. However, it should be kept in mind that the manner
in which baseline data are determined now might be challenged in court at any
time in the future should these baseline data ever be used to demonstrate
pollution above the baseline level.
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these sites) could be used for all sampling tasks to be carried out.. This
approach would have obvious logistic advantages because a variety of samples
could be taken simultaneously at the same sites. Also,, the determination, of .a
wide variety of parameters of the same site would provide a comprehensive site
description and thus greatly facilitate the correlation and interpretation of .
the data. However, the feasibility of using a pattern of randomly selected
sites throughout the Bay area must first be evaluated on the basis of antici-
pated,, sample.,numbers and. frequency of sampling. . . '[: _ _......'._. .-'-..
Supplemental Data Acquisition .
Whenever samples are collected at field sites, a variety of parameters
important for interpretation and correlation of all data must be recorded.
These parameters include: sampling site location (coordinates, site number),
sampling depth, flow data, date and time of day, tide, meteorological con-
ditions (air temperature, wind speed and direction, percent cloud cover, pre-
cipitation, fog), water temperature, water quality parameters (to be determined
in the lab or field from collected samples: pH, conductivity, turbidity,
color, dissolved oxygen, .acidity, alkalinity, BOD, etc.). These data when
adequately correlated and evaluated might facilitate trend recognition and
might explain patterns that become apparent during the studies.
Interface Measurements
The CBP is basically a multimedia surveillance and monitoring program.
One of the proposed study programs includes an investigation of the
water/sediment interface (interstitial water, etc.). The water/air interface
has not been addressed by any of the proposals. While Bay water contamination
resulting from air'pollution might indeed pose no problem at present, possible
future effects.of air pollution on the Bay quality should be evaluated.
Remote Sensing
The use.of remote sensing has been considered in one of the program plans
to map the general distribution of SAV. More extensive use of remote sensing
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by aircraft should be considered. The present state-of-the-art allows remote
recording of water temperature, water surface roughness, plumes of a variety
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of materials, algae blooms, and oil films as thin as 10" .to 10~ y. EMSLrLV
has used remote sensing for water body evaluation for years and has first-hand
knowledge of QA requirements in this field.
Sampling '.
Sampling should be highly coordinated among the grantees and contractors,
to collect a maximum of samples per sampling trip. It is impossible to give
directions covering all conditions, and the choice of sampling technique must
often be left to the analyst's judgment. However, samples should be truly
representative of existing conditions. This can, depending on circumstances,
sometimes be achieved by making composites of samples that have been collected
over a period of time, or at different sampling points.
In addition to details pertaining to the site and atmospheric conditions,
the water current vector should be recorded. Discussions of water movement
determinations are presented in limnology textbooks, e.g.., "Limnological
Methods" by Paul S..Welch, McGraw-Hill Book Co., 1948, p. 141-159; however, no
standard method seems to be available. The same book contains a discussion on
water sampling methods (p. 199-206) which allow samples to be taken at pre-
determined depths (sample containers and sample preservation will be addressed
later).
All sample containers should be sealed and tagged before they are shipped
to the laboratory. Pertinent information should be recorded on a sample tag,
e.g., sample number, date and time taken, source of sample, preservative,
analyses to be performed, and name of sample collector.
Water Samples
The "Manual of Methods for Chemical Analysis of Water and Wastes"> EPA-
625/16-74-003, prepared and published by the Methods Development and Quality
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Assurance Research Laboratory, EMSL-Cincinnati, provides standard methods for
almost all aspects of water analysis. The following points of importance, to
the CBP are discussed in detail:
Sample volume, sample preservation, holding containers,
holding time, sample analysis (description of analytical
procedures including precision and accuracy discussion).
The methods-and procedures described also include physical parameters such as
. temperature) turbidity, pH, conductivity-, color, dissolved oxygen, acidity
alkalinity, BOD, chemical speciation and oil.
The "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", prepared and published (June 1972) by the Analytical Quality
Control Laboratory, EMSL-Cincinnati, addresses in detail the following areasr
laboratory facilities, instruments, glassware requirements, and reagents.. It
further deals with control of analytical performance and data handling and
reporting. A separate chapter is devoted to the special requirements for trace
organic analysis. Copies of the two handbooks are included as Appendices F and
G.
The handbook "Standard Methods for the Examination of Water and Waste-
water", 14th edition (1975), published by the American Public Health Associ-
ation, 1015 Eighteenth Street NW, Washington, DC 20036, describes standard
methods for practically all parameters to be determined in Bay water. It
provides methods for the determination of color, conductivity, salinity,
temperature, turbidity, pH, dissolved oxygen, acidity, alkalinity, and BOD. It
covers essentially all elements listed in Appendix A, except Mo and Sn. One
chapter is devoted to the determination of organic constituents including oil,
pesticides, phenols, surfactants, and tannin and lignin.
Sediment Samples
No standard or tentative EPA-approved or recommended methods are available
for sediment sampling or analysis, and the individual investigators must choose
their own methods, according to the best of their experience and knowledge.
However, it is suggested that individual research groups involved in sediment
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sampling and analysis agree in advance on a particular set of methods. Indi-
vidual skills and the merits of methods could be evaluated in round-robin
programs using actual Chesapeake Bay sediment samples and reference material.
The National Bureau of Standards is at present certifying a river sediment
standard reference material that will become available in late 1978 as SRM
1645.
Every, effort must be.made .to ensure that: sediment samples collected are __L'.-.._..
representative. A number of sediment sampling devices and techni<3ues have been
described in the literature. The Petersen and the Ponar dredge ajre preferred
for compacted or gravelly sediment, whereas the Ekroan dredge is commonly used
for soft, mud, silt, or finely divided sand bottoms (O. T. Lind, Handbook of
Common Methods in Limnology, p. 120. The C. V. Mosby Co., St. Locals, 1974).
Bottom samples involving consideration of bottom stratification can be collected
with vertical core samplers. An example has been described by P. S. Welch
(Limnological Methods, p. 182. McGraw-Hill Book Co., Inc., 1948)..
A standard method for extraction of oil and grease from sludge sample and
subsequent isolation of the hydrocarbon fraction has been described in "Stand-
ard Methods for the Examination of Water and Wastewater", p. 517-2:3,. A manual
cold vapor technique for the determination of mercury in sediment lias been
described in the "Manual of Methods for the Chemical Analysis of Water and
Wastes", p. 134-138, and recently the Central Regional Laboratory, Region V,
has developed an automated method for the determination of mercury in sediments
(Appendix H) . EMSL-Cin. has prepared some interim general guidelines as to
sediment analysis (Appendix I); however, no tentative method or methods have so
far been developed. It should be noted that neither Appendix H nor Appendix I
represent EPA-approved methods but are included as examples only.
Aquatic Vegetation, Mollusks, Fish and Birds
The manual "Biological Field and Laboratory Methods for Measuring the
Quality of Surface Waters and Effluents," C. I. Weber, ed., EPA-67O/4-73-001,
NERC-Cin., i;s at present available, with issuance of a new edition being planned
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for the summer of 1978. Wh'ile the methods described in this manual are con-
sidered to be the best available at this time, the manual does not represent
the final position of the EPA in this matter. A comprehensive manual, entitled
"Quality Assurance Guidelines for Biological Research and Environmental
Monitoring" is presently being prepared for publication at EMSL-LV and will
probably become available in late 1978. These two manuals complement each
other in describing in detail methods and QA procedures covering the following
items ..of. importance to; the .CBP: ....'>/...; ;':J:7.'-r., "'..-. -' ".- .... .... .. -;.[_- .. . '-'" ': - .-.-'.-:.
Sampling methods for plankton, periphyton, macrophyton, macroinverte-
brates, fish, birds, mammals, plants.
Sampling frequency.
Sample preservation and preparation, handling, holding time, container
choice and analysis for benthic macroinvertebrates, fish, macro-
phytes and macroalgae, periphyton, phytoplankton and zooplankton.
Calibration and maintenance of sampling equipment and field instruments.
Culturing, identification, estimation of population size, biomass and
productivity.
Field bioassay tests.
A variety of sample forms covering aspects of sampling and analysis are displayed
in the appendices of the two manuals.
No compilation of standard methods for the analysis of aquatic vegetation
for the pollutants of interest is available. However, scattered throughout the.
scientific literature, analytical methods have been published for some pollu-
tants of interest; an example is "Environmental Pollutants - Selected Ana-
lytical Methods," compiled by W. Gallay, et al., Ann Arbor Science Publishers,
Inc., 1975. This book details methods for the determination of Hg in biological
media, methylmercury compounds in fish, total Pb and Cd in biological media, Se
in biological media, and an estimation of DDT and related compounds together
with PCBs in biological media.
Individual investigators will have to select their own methods. Again, it
should be attempted to correlate the analytical methodology among the research
groups as. much as possible.
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The Role of EMSL-LV in the QA Section of the CBP
The preceding discussion has demonstrated the importance of a compre-
hensive and coordinated QA plan for the Chesapeake Bay Program. It is
estimated that approximately 10 to 15 percent of the available funds should be
spent for QA aspects. The EMSL-LV would act as coordinator and serve as a
focal point for all QA activities. In this capacity, the EMSL-LV could assist
the CBP management in the following" areas: ",...._..---.,-,/.. .""-. .'."_=; ._.j/--.-. r.."---;'---'.- -"":
1. Assist contractors and grantees in identifying and coordi-
nating methods and QA procedures.
2. Evaluate and coordinate the QA parts of all submitted
detailed protocols. Define data acceptability for the
various tasks and ascertain uniform data reporting (in-
cluding uncertainties).
3. Provide or arrange for the provision of reference materials
as available.
4. Conduct round-robin evaluations as needed to evaluate
analyst skills and methods adequacy.
These control and guidance measures would ascertain that all contractors
adequately address the use of standardized or comparable procedures in their
activities, such as sampling, sample preservation, handling and storage, sample
analysis, reference methods, instrument calibration routine, use of reference
materials, duplicates, blanks and split samples, chain-of-custody procedures
and record-keeping, etc. The EMSL-LV has available the following biological
reference materials from various sources that could be distributed to the
analytical laboratories involved in the program: fish solubles, oysters, copepod
homogenate, animal blood, animal muscle, bovine liver, animal bone, orchard
leaves, pine needles, tomato leaves, and spinach. These materials are certified
for a variety of stable elements at environmental levels. Reference materials
consisting of water hyacinth leaves and a variety of goat tissues, certified
for Hg, Pb, As and Cd at environmental and several higher levels are at
present being prepared under grants monitored by EMSL and will become
available in FY 1979. A round-robin program on water analysis is being con-
ducted by EMSL-Cinc., and all contractors doing this kind of analysis should
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be required, at no additional cost to the contractor or the CBP, to partici-
pate in this program.
EMSL-LV has years of extensive experience in conducting laboratory
intercomparison study programs. Within the framework of .the CBP, such a
program could be conducted using actual samples collected.from thte Chesapeake
Bay, to demonstrate the comparability and compatibility of analytical data
between participating laboratories. ' ... -,'-<-'.'-->;- ..IT". . . ''.".''-'.-' '""'"': : . - : -
To conduct a QA Coordination program as proposed, EMSL-LV would require
the following:
FY 1978 FY 1979 FY 19SO FY 1981
Personnel
Permanent
Temporary
Funds
Travel Funds
Material
This estimate is based on the following assumptions:
1. The QA plans of a total of 10 contractors will be
reviewed and evaluated.
2. Reference materials delivered consist of materials available
at EMSL-LV or other government agencies.
3. A total of round-robins involving the 10 contractor
laboratories will be conducted during a -year period.
4. The performance and cooperation of the contractor labora-
tories is generally good.
Not included in the cost estimate are outside expertise used as well as special
studies and requests.
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APPENDIX A
Major media to be sampled (according to the combined CBP plans)
""-..; Bay. ""waiter (at various depths) and interstitial water, .suspended -'':";:-''
sediment and fluid mud, sediment, benthic vegetation, submerged aquatic
vegetation, phytoplankton, mollusks, shellfish, finfish, birds.
Parameters to be determined in the various media (according to the combined
CBP plans)
Water:
Temperature, turbidity, water current vectors, pH, conductivity
As, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Se, Sn<» Zn
Dissolved species of S, C, P, N, Si, Cl
Speciaticn of As, Kg, Sn
Nutrients (including dissolved organic and inorganic forms of B3 and P)
Herbicides, insecticides, miticides, bactericides, fungicides, nemato-
cides, wood and metal preservatives, selected persistent organics
Selected compounds from industrial effluents in VA and MD
Low-molecular-weight toxic halogenated hydrocarbons
PCBs
Other toxic substances known to be present in the Bay
Priority toxic organic pollutants
Oil and oil fractions
Previously unidentified organic compounds that show significant changes in
concentration or areal distribution during the monitoring period.
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Suspended sediment and fluid mud:
Toxicants.
Sediment:
Grain size, bulk water content, fecal pellet analysis, seeds
Heavy metals, bioactive trace metals, heavy minerals, nutrients
Pollen, Pb-210 and C-14 for dating
Toxic substances known to be present in the Bay
Selected persistent organics
Pesticides
PCBs
Oil and oil fractions
Previously unidentified organic compounds that show significant changes in
concentration or areal distribution during the monitoring period
Plant pigments
Selected microorganisms.
Submerged aquatic vegetation:
Distribution and abundance, biomass, biogenic structure, growtJi rate, COz
fixation, growth rate, respiration
Chlorophyll concentration
Heavy metals
As, Cd, Cu, Hg (inorganic and organic), Ni, Pb, Se, Sn, Zn
Pesticides, herbicides
PCBs
Nutrients
Oil fractions.
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Phy t(_>j->l
Dominant species
Chlorophyll concentrations.
Miscellaneous media:
Birds- ;~ determine specimen/ their physiological conditions;
analyze for contaminants
Finfish: determine number, biomass, length
Mollusks: monitor for toxic substances.
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APPENDIX B
General Outline of the Quality. Assurance Section of a Protocol*
; - The .Quality Assurance Section of every protocol should, contain,, when " "'""
applicable.* a discussion of each of the items addressed below. Wtien published
procedures or methods are being utilized without modification, a reference
(publication, volume, number, pages, date) to the method may be included.
A. FIELD SAMPLING
1. Describe the criteria used to select the sampling site(s). Explain
how the sampling sites are described, identified and recorded.
2. Describe the sampling method (s) used and show why grab; or composite
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sampling is the appropriate method for the particular task. Show that the
sampling is representative.
3. Estimate the number of samples to be taken per site and per study;
explain the sampling frequency and the rationale upon which it is fcased.
4. Detail the procedures to be used for the identification- and storage
of samples (all samples must be identified as to the exact time and date of
collection, the exact location, sampling depth, nature and purpose of sample,
sample preservation method, and name of sampler).
5. Describe the sample preservation'method(s) and the shipment pro-
visions (EPA recommendations for sample containers, preservation techniques and !
maximum sample holding time must be followed).
6. Define the types of field log books and forms, and the procedures
used in recording and maintaining field data (a field sheet or bmind notebook
must be used for recording all aspects of sample collection, flow measurement,
in-situ field testing and other field data collection activities) .
*This outline is largely based on the quality Assurance plan requirements
currently in use at EMSL-LV.
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7. If there is any probability that a sample or set of samples may be
required for any judicial or quasi-judicial proceeding, then formal chain of
custody procedures must be followed (an excerpt from the NEIC Compliance
Monitoring Procedures Handbook is included as Appendix C).
8. List the procedures to be used for the maintenance and calibration of
field instruments, including frequency and record-keeping (sampling, equipment,
flo.w measuring devices, and direct-reading field instruments must be calibrated
according:to.proper specifications . immediately, before-andUafter use^in-the'-;-,,£-f_:__
field. If samples are collected and analyzed over a long period of time, more
frequent calibration is necessary. In-situ continuous monitoring devices must
be calibrated according to manufacturer's specifications and these calibrations
verified by approved manual methodology. Sensor calibration must be verified
at least on a daily basis).
9. Define what written instructions and other information will be
provided to the sampler(s). |
B. STANDARDS, INSTRUMENT CALIBRATION, AND INSTRUMENT MAINTENANCE
1. Identify the standard(s) required for each experiment, task, project,
or contract, and the source (or sources) of the required standards; describe
the way in which the standards are to be utilized, and their frequency of use.
2. Discuss the known or expected uncertainty of each standard and the
effect of this uncertainty on the objectives and the acceptable overall error
of the project, experiment or study.
3. Describe or refer to the instrument calibration procedures to be
employed and the proposed frequency of calibration (guidance on calibration
procedures and suggested frequency for a variety of laboratory instruments
is included in Appendix D). Include plans for periodic instrument checks
and maintenance.
4. Define the logbooks, forms, records, and/or control charts to be
employed in conjunction with the maintenance and calibration of instruments.
C. ANALYTICAL PROCEDURES
1. Outline the responsibility for the custody of samples if applicable
(see this Appendix, A-7).
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2. Describe formalized handling procedures in the laboratory (the move-
ment of. samples through the laboratory should be well organized and controlled,
and forms should be prepared for both requests |and the reporting of results.
Samples which have been held for a period in excess of the maximum recommended
holding time should be rejected).
3. Define or reference all analytical procedures (laboratory analyses
must be conducted using reference or equivalent methods as described in the
-.Federal. Register, or by using EPA-approved alternative .test procedures. ..All ":'."
reference or equivalent methods must state the precision and accuracy possible
with the method. Modifications of referenced methods should be described) .
4. Describe the number (or percentage) and the types of samples to be
recycled for control purposes. Establish specific control programs for
each sampling procedure and analytical test. Provide for record-keeping on all
analytical tests such as replicate analyses, parallel testing, and the con-
firmation or verification of tests (based on sound statistical techniques) .
5. Assign responsibility for the preparation of reagents, jmaterials, and
pertinent|records.
"6. Estimate all analytical and instrumental uncertainties, and indicate
any expected or real bias. On all new or proposed projects, where preliminary
. experimental data are unavailable, all factors which may contribute to uncer-
tainties, and the procedures and calculations to be used in estimating error
shall be described. The acceptable error, based on the objectives of the
experiment or project, should be so indicated.
7. Specify the criteria to be utilized for the acceptance or rejection
of anomalous data. Normally, data sets must be within plus or minus 2 standard
deviations of the established precision and accuracy (bias).
8. Describe procedures for the analysis and formal reporting of the data.
9. Discuss plans for the use of control charts or other techniques.for
monitoring daily performance.
10. Describe plans for participating in intercomparisons, cross-checks,
performance studies, and interlaboratory calibrations.
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11. Identify provisions for recording and storage of actual laboratory
and supporting data in bound ledgers and for the maintenance of records of
analyses (to be kept for not less than 10 years, as specified by legal mandate)
A check-list, designed to assist investigators and project officers to
assess the adequacy of their internal and external controls, is included as
Appendix E. . . .
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APPENDIX C
ENVIRONMENTAL PROTECTION AGENCY
Office Of Enforcement
NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Building 53. Box 25227, Dqr«*er FeoerolCenlor -
Denver, Colorodo 80225
June 1, 1975
.CHAIN OF CUSTODY PROCEDURES . ~^^£&-:^: o-rl^.:?V ;/. V
General: .-'..
The evidence gathering portion of a survey should be characterized by the
minimum number of samples required to give a fair representation of the
effluent or v/ater body from which taken. To the extent possible, the quan-
tity of samples arid sample locations will be determined prior to the survey.
Chain of Custody procedures must be followed to maintain the documentation
necessary to trace sample possession from the time taken until .the evidence
is introduced into court. A sample is in your "custody" if:
% 1. It is in your actual physical possession, or
2. It is in your view, after being in your physical possession, or
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3. It was in your physical possession and then you locked it up in
a manner so that no one could tamper with it.
All survey participants will receive a copy of the survey study plan and will
be knowledgeable of its contents prior to the survey. A pre-survey briefing
will be held to re-appraise all participants of the survey objectives, sample
locations and Chain of Custody procedures. After all Chain of Custody samples
are collected, a de-briefing will be held in the field to determine adherence
to Chain of Custody procedures and whether additional evidence type samples
are required. . "..-..
Sample Collection: .
1. To the maximum extent achievable, as few people as possible should
handle the sample. .
2. Stream and effluent samples shall be obtained, using standard field
sampling techniques.
3. Sample tags (Exhibit I) shall be securely attached to the sample
container at the time the complete sample is collected and shall
contain, at a minimum, the following information: station number,
station location, date taken, time taken, type of sample, sequence
number (first sample of the day - sequence No. 1, second sample -
sequence No. 2, etc.), analyses required and samplers. The tags
must be legibly filled out in ballpoint (waterproof ink).
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Chain of Custody Procedures (Continued)
Sample Collection (Continued)
4. Blank samples shall also be taken with preservatives which will
be analyzed by the laboratory to exclude the possibility of
container or preservative contamination.
5. A pre-printed, bound Field Data Record logbook shall be main-
tained to record field measurements and other pertinent infor- ,;
;-..-"; ^ii^ination- necessa^^tavrefresh-'the-samplerls memory JT^ the ev&itS^rW
he later takes the stand to testify regarding his action's r
during the evidence gathering activity. A separate set of field
notebooks shall be maintained for each survey and stored in a
safe place where they could be protected and accounted for at
all times. Standard formats (Exhibits II and III) have been
established to minimize field entries and include the date, time,
survey, type of samples taken, volume of each sample, type of
analysis, sample numbers, preservatives, sample location and
field measurements such as temperature, conductivity, DO, pH,
flow and any other pertinent information or observations. The
entries shall be signed by the field sampler. The preparation
and conservation of the field logbooks during the survey will
' be the responsibility of the survey coordinator. Once the
survey is complete, field logs will be retained by the survey
coordinator, or his designated representative, as a part of the
permanent record. '
6v. The field sampler is responsible for the care and custody of the
samples collected until properly dispatched to the receiving lab-
oratory or turned over to an assigned custodian. He must assure
that each container is in his physical possession or in his view
at all times, or locked in such a place and manner that no one can
tamper with it.
7. Colored slides or .photographs should be taken which would visually
show the "outfall sample location and any water pollution to sub-
stantiate any conclusions of the investigation. Written documenta-
tion on the back of the photo should include the signature of the
photographer, time, date and site location. Photographs of this
nature, which may be used as evidence, shall also be handled
recognizing Chain of Custody procedures to prevent alteration.
Transfer of Custody and Shipment: '.
1. Samples will be accompanied by a Chain of Custody Record which
.Includes the name of the survey, samplers signatures, station
number, station location, date, time, type of sample, sequence
number, number of containers and analyses required (Fig. IV).
When turning over the possession.of samples, the transferor and
transferee will sign, date and time the sheet. This record shaet
-------
Chain of Custody Procedures (Continued)
allows transfer of custody of a group of samples in the field,
to the mobile laboratory or when samples are dispatched to the
NFIC - Denver laboratory. When transferring a portion of the
samples identified on the sheet to the field mobile laboratory,
the individual samples must be noted in the column with the
signature of the person relinquishing the samples. The field
laboratory person receiving the samples will acknowledge receipt.
;,.;by signing in the appropriate column. .
2. The field custodian or field sampler, if a custodian has not
been assigned, will have the responsibility of properly pack-
aging and dispatching samples to the proper laboratory for
analysis. The "Dispatch" portion of the Chain of Custody-Record
shall be properly filled out, dated, and signed.
3. .Samples will be properly packed in shipment containers such as
ice chests, to avoid breakage. The shipping containers will be
padlocked for shipment to the receiving laboratory.
4. All packages will be accompanied by the Chain of Custody Record
'"* snowing identification of the contents. The original will accom-
.".'' pany the shipment, and a copy will be retained by the survey
coordinator.
5. If sent by mail, register the package with return receipt request-
ed. If sent by common carrier, a Government Bill of Lading should
be obtained. Receipts from post offices and bills of lading will
be retained as part of the permanent Chain of Custody documentation.
6. If samples are delivered to the laboratory when appropriate person-
nel are not there to receive them, the samples must be locked in
a designated area within the laboratory in a manner so that no
one can tamper with them. The same person must then return to the
laboratory and unlock the samples and deliver custody to the
appropriate custodian. - '
Laboratory Custody Procedures:
1. The laboratory shall designate a "sample custodian." An alternate
will be designated in his absence. In addition, the laboratory
shall set aside a "sample storage security area." This should be
a clean, dry, isolated room which can be securely locked from the
outsjde.
2. All samples should be handled by the minimum possible number of
persons. -
3. All incoming samples shall be received only by the custodian, who
will indicate receipt by signing the Chain of Custody Record Sheet
-------
_Chain of Custody Procedures (Continued)
accompanying the samples and retaining the sheet as permanent
records. Couriers picking up samples at the airport, post
office, etc. shall sign jointly with the laboratory custodian.
4. Immediately upon receipt, the custodian will place the sample
in the sample room, which will be locked at all times except
: : :-^ v/hen samples are, removed1 or replaced by the custodian^. To the
"'" ""'"^''hiaximurh'extent'possible',''only.'the custodian should be permitted
in the" sample room.
5. The custodian shall ensure that heat-sensitive or light-sensitive
samples, or other sample materials having unusual physical
characteristics, or requiring special handling, are properly
stored and maintained. ..-
6. Only the custodian will distribute samples to personnel who are
to perform tests.
7. The analyst will record in his laboratory notebook or analytical
worksheet, identifying information describing the sample, the
procedures performed and the results of the testing. The notes
shall be dated and indicate who performed the tests. The notes
shall be retained as a permanent record in the laboratory and -, .
should note any abnormalities which occurred during the testing
procedure. In the event that the person who performed the tests
1s not available as a witness at time of trial, the government
may be able to introduce the notes in evidence under the Federal
Business Records Act.
8. Standard methods of laboratory analyses shall be used as described
in the "Guidelines Establishing Test Procedures for Analysis of
Pollutants," 38 F.R. 28758, October 16, 1973. If laboratory
. personnel deviate from standard procedures, they should be prepared
to justify their decision during cross-examination.
9. Laboratory personnel are responsible for the care and custody of
the sample once it is handed over to them and should be prepared
to testify that the sample was in their possession and view or .
secured in the laboratory at all times from the moment it was
received from the custodian until the tests were run.
10. Once the sample testing is completed, the unused, portion of the
.sample together with all identifying tags and laboratory records,
should be returned to the custodian. The returned tagged sample
will be retained in the sample room cintil it is required for trial.
Strip charts and other documentation of work will also be turned
over to the custodian.
-------
Chain of Custody Procedures (Continued)
11. Samples, tags and laboratory records of tests may be destroyed
only upon the order of the laboratory director,, v/ho will first
confer vnth the Chief, Enforcement Specialist Office, to make
certain that the information is no longer required or the samples
have deteriorated.
-------
EXHIBIT I
CHAIN OF CUSTODY RECORD
ENVIRONMENTAL PROTECTION AGENCY
National Field Investigations Canter-Denver
Denver Federal Center . " ' - -_r-V-v--:'j '.
Denver, Colorado 80225
SAMPLE NO.
TIME TAKEN (hours)
DATE TAKEN
SOURCE Of SAMPLE
PRESERVATIVE
SAMPLE COLLECTOR
WITNESSES)
REMARKS: (Analyses Required. Sample Type, etc.)
1 hereby certify trat 1 received thij sample end deposed of it as noted be!o-«r:
ss
ft*
§3
RECEIVED FROM
DISPOSITION OF SAMPLE
DATE RECEIVED TIME RECEIVED
SIGNATURE
1 hereby certify that 1 received this sample and disposad of it as noted belcw:
5u
at
u<
a5
cc
RECEIVED FROM
DISPOSITION OF SAMPLE
DATE RECEIVED TIME RECEIVED
SIGNATURE
1 hereby certify that 1 obtained this umple and d>sp»tclv»d it as shown below:
[ DISPATCH OF
1 SAMPLE
DATE OBTAINED TIME OBTAINED
SOURCE
DATE DISPATCHED TIME DISPATCHED METHOD OF SHIPMENT
SENT TO
SIGNATURE
-------
EXHIBIT IV
ENVIRONMENTAL PROTECTION AGENCY
Office Of Enforcement
NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Building 53. Box 25227, Denver fcevol Cenier
Denver, Colorado 80225
CHAIN OF CUSTODY RECORD
SURVEY
;l±-;~.v" :.
STATION
NUMBER
--
1
STATION LOCATION
.
DATE
Relinquished by: (signature)
Relinquished by: (Signature)
' Relinquished by: (Signature)
Relinquished by: (Signature)
Dispofched by: (Signcturej
^f
Method of Shipment:
Date,
TIME
SAMPLERS: [Signature) . . ,
- . .-- , ':'.' '' - .. :_.'. ' '-. --- - i
SAMPLE TYPE
Water
Comp.
Grab.
*
Air
SEO.
MO.
NO. Of
CONTAINERS
s.
ANALYSIS
i
*
'
i
Received by: (signature)
Received by: (s;3naiure)
Received by: (S^noiurc)
Received by Mobi e Lcbora'ory for field
analysis: (S.gnoiurtj
/Time
Received for Laborcfory by:
Dote/Time
Dote/Time
Date/Time
Dcte/Ti;n3
Dcte/Time:
Ji:frihu!ic.n
1 Copy Survey CoQnJii:o!ct f1;;.:':! r;;/:-;
-------
APPENDIX D
INSTRUMENT CAL1ERATIONS
Instrument
Procedure
Frc-cuencv'
1) Analytical Balances
2) pH Keters -
3) Conductivity Xeters
A) Nepheloseter/
Turbidineters
5} Colorineters/Filter
Photometers
6) UV/Visible.
Spectroohotoir.eters
(a) Zero
(b) Standards-eights
Xc}--Full calibration ""and;
adjustment:.
At pH 4,7, and 10
(a) Obtain cell constant
. with potassium chloride
reference solutions
(b) Construct temperature
curve if i^easuretr.ents
are to be r-ade other than
at 25.± 0.5°C
(a) Check instrument scales
or develop calibration '
curve vith forcazine stds.
(<_ 40KTU)
(b) If nsr:uf£ct;urerTs stds.
are not forr:azine, check
against forniazine stds.
(5 40NTU)
Curves detemined with 5-6
laboratory-prepared std.
solutions for.each parameter
in cone, range of samples
(a) Wavelength calibretion
v?ith holinura oxide glass.
or solution, low-pressure
mercury arc, benzene vapor
(UV), or hydrogen arc
(visible)
(b) Absorbance vs. concentration
curves with 5-6 stc. Solu-
tions for each parameter.
at analytical wavelength
iTi COnC. *~.r»nc*r- r. r er^:;?"*.^o
(c) Full servicing and adjust-
ment
(a) Before each vcighii
(b) llonthly
(c)
Daily
Daily
Monthly.
2-'ontiily
Annually
Daily
Quarterly "
Daily
Annually
-------
INSTRUMENT CALIBRATIONS
Instrument
Procedure
Frequency
7) infrared Spectro-
photonjetrers
8) Atomic Absorption
Spectrophotoneters
9) Carbon Analyzers
10) DO Meters
11) Other Selective
Ion Electrodes and
Electrometers
12) Themonaters
13) Technicon Auto
Analyzers
(a) Wax'elength calibration with
polystyrene or indene
(b) Absorbance vs. concent; rat ion
curves with 5-6 std. solu-
tions ..for." each .parameter
- at analytical wavelength in
- cone, range of samples
(c) Full servicing and adjust
Eent
(a) Response vs. concentration
curves with 6-8 std. solu-
tions for each metal (std.
mixtures are acceptable,
but with same acid as
sairples to be run) in cone.
range of sacples
(b) Full servicing and adjust-
ment
Curves determined with 5-6 std.
solutions in cone, range of
samples
Calibrated against modified
Vinkler method on aerated
distilled or tap water
Curves determined with 5-6 std.
solutions in cone, range of
samples
Calibrate in constant temper-
ature baths at two temper-
atures againsi. precision
. themoneters certified by
NBS.
(a) Curves determined with
L*td. solutio:xs for each
parameter.
(b) Full service ar.d adjusti-
(asp. colorimeter)
Daily
Daily
Semi-annually
Daily
Annually
Daily
Daily
Daily
Quarterly
Each set of sasoles
Annually
-------
Instrument
Procedure
cir- Chrcmatographs
(a) Retention tines end detector
response checked vith std.
solutions
i(b) Response-:cuirv*es;ifor each :
'--" ' parameter determined with
std. solutions
Freaur-ncv
Quaxierly
15) Sulfur Dioxide in (a) Calibrate flovraeters and
Air Sampler/Analyzers . hypodermic needle against
(Pararosaailine £ wet test rceter
Method) (b) Spectrophotcnetric calibre.
tion curve with'5-6 std..
sulfite-TCM solutions at
controlled tenperature
(c) Sampling calibration curve
vith 5-6 std. atOTT.ospheres
froE perir.cRtion tul/es cr-
cylinders
(d) Calibrate associated thernio Quarlterly
Eeters, barp-Tteters, ar»d
spectrophototneter ('.cave
length) ' . :.
16) Suspended Particulates (a) Calibrate sampler (curve.of KontSly
(High-voluse Sampler
Method)
. true airfloT-7 rate vs. rota
r.eter or recorder resding)
with orifice calibration .
unit and differential mano
. ceter at 6 air flov; rates.
(b) Calibrate orifice calibra-
tion, unit vrith positive
displacement primary
standard and cifferenuial
nanor.eters
Cc) Calibrate relative hu-icity
iriJicctcr ir. the ccr.ditlon
ing environr.ent a~^inst T..-e
b-jlb/cry-bulb psychrc;~e;:er
(d) Check elapsed tints ir.dicaty
(e) Calibrate associated £r»-Iy
tical balances, therro-
netcrs, bai'or.eters
Anr.ually
Sealanuaall y
Semiannually
As needed
-------
INSTRUMENT CALIBRATIONS
17) Carbon-ir.onoxide
(Non-dispersive IR)
18) Photochemical
. Cxidants (Ozone)
;19) Hydrocarbons
(corrected for
Methane)
20) :.Tirrog=n Dioxide
(Arsenite 24 hr.
Sampling Method)
Monthly
(a) Determine linearity of
detector response (cali-
bration curve) with cali-
bration, gases (0, 10, 20,
".v; 40, and 80% of full scale, -; >:
certified to ±27, and checked
against auditing gases certi-
,.. fied to +1%)
(b) Perform zero and span cali-
brations
(c) Calibrate rotair.eter and
sample cell pressure gauge
(a) Calibrate standard KI/I
solutions in terms of
calculated 0_ equivalents
at 352 nn
(b) Calibrate instrument response Monthly
with 6-8 test atmospheres
froni ozone generator, span-
ing e>:pectec? ranged of sample
concentrations (usually 0.05-
0.5 ppm 03)
(c) Calibrate rlovoeters, baro-
Before and after each
sampling period
Semi-^annually
Weekly
j thernor.eter
(d) Calibrate and service spec-
trophotoneter
(a) Determine linearity of detec-
tor response with calibra-
tion gases (0, 10, 20, 40,
and 80% of full scale, certi-
fied to +2%)
(b) Perform zero and span cali-
brations
(c) Calibrate floi.ineters and
other associated apparatus
(a) Calibrate flovir.eter with wet
test meter
(b) Calibrate hypcderaic nesdle
(flow restrictcr) vith
flowneter
»'c) Obtain colorir-.etric calibra-
tion curves v:ith 5-6 std.
nitrite solutions
S eiai-annually
As specified
Monthly . .
Before and after eac:
sampling period.
Sei?:i-anr.ua2 ly
Monthly
Each ne-.w needle
Weekly
-------
INSTRUMENT CALIEIUTIO::3
Instrument
Procedure
Freemen.~v
21) Nitrogen Dioxide
(Griess-Saltzuum
ColcriiT.etric,
...-;-. Continuous)^-':;
22) Nitrogen Dioxide
(Chenilviniinescen ce,
Continuous)
Monthly
(a) Dynamic calibration with
std. atmospheres (e.g.,
froa permeation tubes)
[ry-^i:. spanning the range of
observed concentrations
(b) Static colorimotric calibra-
tion with 5-6 std. nitrite
solutions
(a) Calibrate std. NO cylinder. '
with ozone generator (pre-
calibrated by iodoinetric
procedure)
(b) Calibrate NO monitor v;ith
std. NO cylinder at several
concentrations
(c) Calibrate NO monitor .with
std. NO cylinder (diluted
NO concentrations determined "
with NO monitor) and calibra-
ted ozone generator
(d) Calibrate associated flow- Seci-annually
meters
Weekly
Each r.ew cylinder
Monthly
Monthly
23) Autoclaves and
Sterilizers
(a) Sterilization effectiveness
checked (e.g., E. stearo-
theTTnophilus, color-indi-
. cator tape for ethylene
oxide)
(b) Temperature-recording device
calibrated
Daily
S emi-annual ly
-------
INSTRUMENT CALIBRATION
Instrument
Procedure
Frequency
24) Gamma Spectroscopy
25) Gas Flow Proportional
26) Radon System
27) Liquid Scintillation
28) Alpha Spectrometer
29) Alpha Scintillation
30) Beta Scintillation
(a) Background
(b) Efficiency
(c) Time base
(d) Check source - for
energy and efficiency
(Bi-207, or Cs-137.,
Co-60)
(a) Plateau
(b) Background
(c) Check source
(d) Efficiency vs mass
(Am-241, Sr-90)
(a) Calibration of cells,
photomultiplier,
concentrator, and
collector
(b) Background
(c) Performance standard
(a) Performance standard
(b) Background
(c) Channels ratio for
each radionuclide
(H-3, Sr-90, Sr-89,
Cs-134, 1-131, C-14)
(a) Performance check
(b) Background
(c) Efficiency
(Am-241, Pu-239)
(a) Performance check
(b) Background
(c) Efficiency (Am-241)
(a) Performance check
(b) Background
(c) Efficiency (Sr-90)
(a) Weekly
(b) Annually, and.for every
change in library radio-
nuclides or matrix
(c) Annually
(d) Daily
(a) Every change of gas
(b) Daily, when in use
(c) Daily, when in use
(d) Semi-annually
(a) Annually
(b) Daily, when in use
(c) Daily, when in use
(a) Daily, or every 20 samples
(b) Daily, or every 20 samples
(c) Annually
(a) Daily
(b) Weekly
(c) Semi-annually
(a) Daily
(b) Daily
(c) Semi-annually
(a) Daily
(b) Daily
(c) S emi-annua1ly
-------
APPENDIX E
INTERNAL AND EXTERNAL CONTROLS
3.
Control of Analytical Methods and Instruments
(1) Written Instrument Maintenance and Calibration
:--. '.:. Procedures and Log Books ; - . - ; .
(2) Written Operating Procedures
Control of Sampling
(1) Written Sampling Procedures Covering:
Sampling Plans and Sampling Equipment
Sample Collection and Preservation
Identification and Storage of Samples
Laboratory Handling of Samples (Request for
analysis, sample preparation, timely
performance, etc.)
(2) Written Description of the Chain of Custody of
Samples
(3) Written Procedures for Field Measurement (Flow,
critical tests: D.O., Residual Cl, etc.)
(4) Written Procedures for Monitoring (Water supply,
effluents, ambient air, stacks, mobile vehicles,
pesticides, radiation, etc.)
Quality Control
(1) Written Quality Control Program Covering:
Quality Policy
Assignment of Responsibility
Training in Quality Control Methods
Control of Purchased Chemicals/Reagents
Internal Field and Laboratory Checks:
Precision/Accuracy
Routine Duplicates, Spiked, and Standard
Samples
Statistical Methods, Including Control
Charts and/or Computer Methods
(2) Written Description of Lab Record System (Data
handling/calculations, data review, validation
(3)
and audit)
Written Description of Lab Report Systems
Available
Yes
No
-------
APPENDIX F
ElJA-625-/6-74-003
METHODS FOR CHEMICAL ANALYSIS
OF WATER AND WASTES
METHODS DEVELOPMENT AND QUALITY ASSURANCE
RESEARCH LABORATORY
National Environmental Research Center
Cincinnati, Ohio 45268
U. S. ENVIRONMENTAL PROTECTION AGENCY
Office of Technology Transfer
Woshington D.C. 20460
1974
-------
FOREWORD
The accomplishment of our objectives in protecting the environment requires an
awareness of the interdependence of the components we seek to protect - - -air,
water, and land. Through individual and joint efforts the National Environmental
Research Centers provide this multidisciplinary focus through programs engaged in
studies on the effects of environmental contaminants on man and the
biosphere,
the development of efficient means of monitoring these contaminants, and
a search for more effective ways to prevent undesirable modification of the
environment and the recycling of valuable resources.
This chemical methods manual was developed by the staff of the Methods Devel-
opment and Quality Assurance Research Laboratory of the National Environmental
Research Center, Cincinnati, to provide methodology for monitoring the quality of
our Nation's waters and to determine the impact of waste discharges. The test
procedures have been carefully selected to meet the needs of laboratories engaged
in protecting the aquatic environment. The contributions and counsel of scientists
in other EPA laboratories are gratefully acknowledged.
The manual is published and distributed by the Office of Technology Transfer, as
one of a series designed to insure that the latest technologies developed by EPA
and private industry are disseminated to states, municipalities and industries who. are
responsible for environmental pollution control.
The other manuals in this series are:
Handbook for Monitoring Industrial Waste-water
Handbook for Analytical Quality Control in Water and Waste-water Laboratories.
These are also available through the Office of Technology Transfer, Washington, D.C. 20460.
Robert E. Crowe, Director Andrew W. Breidenbach, Ph.D.
Office of Technology Transfer Director, National Envrionmental
Washington, D.C. Research Center, Cincinnati, Ohio
m
-------
INTRODUCTION
This second edition of "Methods for Chemical Analysis of Water and Wastes" contains the
chemical analytical procedures used in U.S: Environmental Protection Agency (EPA)
laboratories for the examination of ground and surface waters, domestic and industrial
waste effluents, and treatment process samples. Except where noted under "Scope and
Application," the methods are applicable to both water and wastewaters, and both fresh and
saline water samples. The manual provides test procedures for the measurement of physical,
inorganic, and selected organic constituents and parameters. Methods for pesticides,
industrial organic waste materials, and sludges are given in other publications of the Agency.
The methods were chosen through the combined efforts of the EPA Regional Analytical
Quality Control Coordinators, the staff of the Physical and Chemical Methods Branch,
Methods Development and Quality Assurance Research Laboratory, and other senior
chemists in both federal and state laboratories. Method selection was based on the following
criteria:
(1) The method should measure the desired property or constituent with precision,
accuracy, and specificity sufficient to meet the data needs of EPA, in the presence
of the interfering materials encountered in water and waste samples.
(2) The procedure should utilize the equipment and skills available in modern water
pollution control laboratories.
(3) The selected method is in use in many laboratories or has been sufficiently tested
to establish its validity.
(4) The method should be rapid enough to permit routine use for the examination of
a large number of samples.
Instrumental methods have been selected in preference to manual procedures because of the
improved speed, accuracy, and precision. In keeping with this policy, procedures for the
Technicon AutoAnalyzer have been included for laboratories having this equipment
available.
Precision and accuracy statements are provided where such data are available. These
statements are derived from interlaboratory studies conducted by the Quality Assurance
and Laboratory Evaluation Branch, Methods Development and Quality Assurance Research
Laboratory; the American Society for Testing Materials; or the Analytical Reference Service
of the US Public Health Service, DHEW.
IV
-------
These methods may be used for measuring both total and dissolved constituents of the
sample. When the dissolved concentration is to be determined, the sample is filtered through
a 0.45-micron membrane filter and the filtrate analyzed by the procedure specified. The
sample should be filtered as soon as possible after it is collected, preferably in the field.
Where field filtration is not practical, the sample should be filtered as soon as it is received
in the laboratory.
Many water and waste samples are unstable. In situations where the interval between sample
collection and analysis is long enough to produce changes in either the concentration or the
physical state of the constituent to be measured, the preservation practices in Table II are
recommended.
This manual is a basic reference for monitoring water and wastes in compliance with the
requirements of the Federal Water Pollution Control Act Amendments of 1972. Although
other test procedures may be used, as provided in the Federal Register issue of October 16,
1973 (38FR 28758), the methods described in this manual will be used by the
Environmental Protection Agency in determining compliance with applicable water and
effluent standards established by the Agency.
Although a sincere effort has been made to select methods that are applicable to the widest
range of sample types, significant interferences may be encountered in certain isolated
samples. In these situations, the analyst will be providing a valuable service to EPA by
defining the nature of the interference with the method and bringing this information to the
attention of the Director, Methods Development and Quality Assurance Research
' Laboratory, through the appropriate Regional AQC Coordinator..
-------
SAMPLE PRESERVATION
Complete and unequivocal preservation of samples, either domestic sewage, industrial
wastes, or natural waters, is a practical impossibility. Regardless of the nature of the sample,
complete stability for every constituent can never be achieved. At best, preservation
techniques can only retard the chemical and biological changes that inevitably continue
after the sample is removed from the parent source. The changes that take place in a sample
are either chemical or biological. In the former case, certain changes occur in the chemical
structure of the constituents that are a function of physical conditions. Metal cations may
precipitate as hydroxides or form complexes with other constituents; cations or anions may
change valence states under certain reducing or oxidizing conditions; other constituents may
dissolve or volatilize with the passage of time. Metal cations may also adsorb onto surfaces
(glass, plastic, quartz, etc.), such as, iron and lead. Biological changes taking place in a
sample may change the valence of an element or a radical to a different valence. Soluble
constituents may be converted to organically bound materials in cell structures, or cell lysis
may result in release of cellular material into solution. The well known nitrogen and
phosphorus cycles are examples of biological influence on sample composition.
Methods of preservation are relatively limited and are intended generally to (1) retard
biological action, (2) retard hydrolysis of chemical compounds and complexes and (3)
reduce volatility of constituents.
Preservation methods are generally limited to pH control, chemical addition, refrigeration,
and freezing. Table 1 shows the various preservatives that may be used to retard changes in
samples.
VI
-------
Preservative
TABLE 1
Action
Applicable to:
HgCl;
Bacterial Inhibitor
Nitrogen forms,
Phosphorus forms
Acid(HNO3)
Acid(H2SO4)
Alkali (NaOH)
Refrigeration
Metals solvent, pre-
vents precipitation
Bacterial Inhibitor
Salt formation with
organic bases
Salt formation with
volatile compounds
Bacterial Inhibitor
Metals
Organic samples
(COD, oil & grease
organic carbon)
Ammonia, amines
Cyanides, organic
acids
Acidity-alkalinity,
organic materials,
BOD, color, odor,
organic P, organic
N, carbon, etc.,
biological organism
(coliform, etc.)
In summary, refrigeration at temperatures near freezing or below is the best preservation
technique available, but it is not applicable to all types of samples.
The recommended choice of preservatives for various constituents is given in Table 2. These
choices are based on the accompanying references and on information supplied by various
Regional Analytical Quality Control Coordinators.
vii
-------
TABLE 2
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT (1)
Measurement
Acidity
Alkalinity
Arsenic
BOD
Bromide
COD
Chloride
Chlorine Req.
Color
Cyanides
j
Dissolved Oxygen
Probe
Winkler
Vol.
Req.
(ml)
100
100
100
1000
100
50
50
50
50
500
300
300
Container
P,G<2)
P,G
P,G
P,G
P,G
P,G
P,G
P,G
P,G
P,G
G only
G only
Preservative
Cool, 4°C
Cool, 4°C
HNO3 to pH <2
Cool, 4°C
Cool, 4°C
H2SO4 topH<2
None Req.
Cool, 4°C
Cool, 4°C
Cool, 4°C
NaOHtopH 12
Det. on site
Fix on site
Holding
Time(6)
24 Hrs.
24 Hrs.
6 Mos.
6Hrs.(3)
24 Hrs.
7 Days
7 Days
24 Hrs.
24 Hrs.
24 Hrs.
No Holding
No Holding
Vlll
-------
TABLE 2 (Continued)
Measurement
Fluoride
Hardness
Iodide
MBAS
Metals
Dissolved
Suspended
Total
Mercury
Dissolved
Total
Vol.
Req. Holding
(ml) Container Preservative Time(6)
300 P, G Cool, 4°C 7 Days
100 P, G Cool,4°C 7 Days
100 P, G Cool, 4° C 24Hrs.
250 P, G Cool, 4°C 24Hrs.
200 P, G Filter on site 6 Mos.
HN03 to pH <2
Filter on site 6 Mos.
100 HNO3topH<2 6 Mos.
100 P, G Filter 38 Days
HNO3 to pH <2 (Glass)
13 Days
(Hard
Plastic)
100 P, G HNO3topH<2 38 Days
(Glass)
13 Days
(Hard
Plastic)
-------
TABLE 2 (Continued)
Measurement
Nitrogen
Ammonia
Kjeldahl
Nitrate
Nitrite
NTA
Oil & Grease
-
Organic Carbon
pH
Phenolics
Phosphorus
Ortho-
.phosphate,
Dissolved
Vol.
Req.
(ml) Container Preservative
400 P, G Cool, 4°C
H2SO4 topH<2
500 P, G Cool, 4°C
H2SO4topH<2
100 P, G Cool,4°C
H2SO4 topH<2
50 P, G Cool, 4°C
50 P, G Cool, 4°C
1000 Gonly Cool, 4° C
H2SO4 topH<2
25 P, G Cool, 4°C
H2 SO4 to pH <2
25 P, G Cool, 4°C
Det. on site
500 Gonly Cool, 4° C
H3P04 topH<4
1.0gCuSO4/l
50 P, G Filter on site
Cool, 4°C
Holding
Time(6)
24Hrs.(4>
24Hrs.<4>
24Hrs.(4>
24 Mrs. (4>
24 Mrs.
24Hrs.
24 Mrs.
6Hrs.(3)
24Hrs.
24 Hrs.<4)
-------
TABLE 2 (Continued)
Measurement
Hydrolyzable
Total
Total,
Dissolved
Residue
Filterable
Non-
Filterable
Vol.
Req.
(ml)
50
50
50
100
100
Container Preservative
P, G Cool, 4°C
H2SQ4 topH<2
P, G Cool, 4°C
P, G Filter on site
Cool, 4°C
P, G Cool, 4°C
P, G Cool, 4°C
Holding
Time(6)
24 Hrs.<4>
24Hrs. <4>
24Hrs.<4>
7 Days
. 7 Days
Total
Volatile
100 P, G
Cool, 4°C
100 P, G Cool, 4°C
7Days
7 Days
Settleable Matter 1000 P, G None Req.
24Hrs.
Selenium
Silica
Specific
Conductance
Sulfate
50 P, G HN03 to PH <2 6 Mos.
50 P only Cool, 4°C
100 P, G
50 P, G
Cool, 4°C
Cool, 4°C
xi
7 Days
24Hrs.
7 Days
-------
TABLE 2 (Continued)
Measurement
Vol.
Req.
(ml)
Container Preservative
Holding
Time(6)
Sulfide
50 P,G
2 ml zinc
acetate
24Hrs.
Sulfite
50 P, G
Cool, 4°C
24 Mrs.
'Temperature
1000 P, G
Det. on site
No Holding
Threshold
Odor
200 G only Cool, 4°C
24 Hrs.
Turbidity
100 P, G
Cool, 4°C
7 Days
1. More specific instructions for preservation and sampling are found with each procedure
as detailed in this manual. A general discussion on sampling water and industrial
wastewater may be found in ASTM, Part 23, p. 72-91 (1973).
2. Plastic or Glass
3. If samples cannot be returned to the laboratory in less than 6 hours and holding time
exceeds this limit, the final reported data should indicate the actual holding time.
4. Mercuric chloride may be used as an alternate preservative at a concentration of 40
mg/1, especially if a longer holding time is required. However, the use of mercuric
chloride is discouraged whenever possible.
t
5. If the sample is stabilized by cooling, it should be warmed to 25°C for reading, or
temperature correction made and results reported at 25°C.
6. It has been shown that samples properly preserved may be held for extended periods
beyond the recommended holding time.
xn
-------
ENVIRONMENTAL PROTECTION AGENCY
REGIONAL ANALYTICAL QUALITY CONTROL COORDINATORS
REGION I
Warren H. Oldaker
New England Basin Office
240 Highland Avenue
Needham Heights, MA 02194
(617-223-7337)
REGION II
Gerard F. McKenna
Edison Environmental Lab.
Edison, NJ 08817
(201-548-3427)
REGION III
REGION V
David Payne
Central Regional Lab.
Quality Assurance Officer
1819 W. PershingRoad
Chicago, IL 60609
(312-353-8370)
REGION VI
Dr. Timothy Matzke
1600 Patterson, Suite 1100
Dallas, TX 75201
(214-749-1121)
REGION IX
Dr. Ho L. Young
620 Central Ave., Bldg 1
Alameda, CA 94501
(415-273-7502)
REGION X
Arnold R. Gahler
15345N.E. 36th Street
Redmond, WA 98052
(206-442-0111)
(Ask for: 883-0833)
Orterio Villa
Annapolis Field Office
Annapolis Science Center
Annapolis, MD 21401
(301-597-3311)
(Ask for: 268-5038)
REGION VII
Dr. Harold G. Brown
25 Funston Road
Kansas City, KS 66115
(816-374-4286)
REGION IV
REGION VIII
James H. Finger
Southeast Envr. Res. Lab.
College Station Road
Athens, GA 30601
(404-546-3111)
John R. Tilstra
Denver Federal Center
P.O. Box 25345
Denver, CO 80225
(303-234-3263)
Xlll
-------
TABLE OF CONTENTS
Introduction iv
Sample Preservation vi
EPA Regional Coordinators xiii
Acidity , " 1
Alkalinity
Titrimetric (pH4.5) 3
Automated, Methyl Orange 5
Arsenic 9
Biochemical Oxygen Demand (5 Days, 20°C) 11
Boron (Curcumin Method) 13
Bromide (Titrimetric) 14
Calcium (Titrimetric) 19
Chemical Oxygen Demand
Normal (15 to 2000 mg/1) 20
Low Level (5 to 50 mg/1) . . . 21
High Level for Saline Waters (>250 mg/1) 25
Chloride
Titrimetric , . 29
Automated 31
Chlorine, Total Residual 35
Color
Platinum-Cobalt 36
Spectrophotometric . .' 39
Cyanide
Total 40
Amenable to Chlorination 49
Dissolved Oxygen
Modified Winkler with Full-Bottle Technique 51
Electrode 56
xiv
-------
Fluoride
SPADNS Method with Bellack Distillation 59
Automated Complexone Method 61
Electrode 65
Hardness, Total
Titrimetric 68
Automated 70
Iodide (Titrimetric) 74
Metals (Atomic Absorption Methods) 78
Aluminum 92
Antimony 94
Arsenic (Gaseous Hydride Method) 95
Barium 97
Beryllium 99
Cadmium 101
Calcium 103
Chromium 105
Cobalt 107
Copper 108
Iron 110
Lead 112
Magnesium 114
Manganese 116
Mercury
Manual Cold Vapor Technique (Water) 118
Automated Cold Vapor Technique (Water) 127
Manual Cold Vapor Technique (Sediment) 134
Molybdenum 139
Nickel 141
Potassium 143
Selenium (Gaseous Hydride Method) 145
xv
-------
Metals (Atomic Absorption Methods) Cont'd
Silver 146
Sodium 147
Thallium 149
Tin 150
Titanium 151
Vanadium 153
Zinc 155
Methylene Blue Active Substances (MBAS) 157
Nitrogen
Ammonia
Distillation Procedure 159
Selective Ion Electrode Method 165
Automated Colorimetric Phenate Method 168
Kjeldahl, Total
Manual 175
Automated Phenate Method . . . 182
Automated Selenium Method 190
Nitrate (Brucine) 197
Nitrate-Nitrite
Cadmium Reduction Method 201
Automated Cadmium Reduction Method 207
Nitrite . . 215
NTA
Zinc-Zincon Method 217
Automated Zinc-Zincon Method 220
Oil and Grease
Soxhlet Extraction 226
Separately Funnel Extraction 229
Infrared 232
Organic Carbon (Total and Dissolved) 236
xvi
-------
pH 239
Phenolics
4-AAP Method with Distillation 241
Automated 4-AAP Method with Distillation . 243
Phosphorus
Single Reagent Method 249
Automated Colorimetric Ascorbic Acid Reduction Method 256
Residue
Total, Filterable (Dried at 180°C) 266
Total, Ndn-Filterable 268
Total 270
Volatile 272
Settleable Matter 273
Silica, Dissolved 274
Specific Conductance (/umhos at 25°C) 275
Sulfate
Turbidimetric 277
Automated Chloranilate Method 279
Gravimetric 283
Sulfide (Titrimetric Iodine Method) 284
Sulfite 285
Temperature 286
Threshold Odor (Consistent Method) 287
Turbidity 295
xvii
-------
ACIDITY
STORET NO. 70508
1. Scope and Application
1.1 This method is applicable to surface waters, sewages and industrial wastes,
particularly mine drainage and receiving streams, and other waters containing
ferrous iron or other polyvalent cations in a reduced state.
1.2 The method covers the range from approximately 10 mg/1 acidity to approxi-
mately 1000 mg/1 as CaCO3, using a 50 ml sample.
2. Summary of Method
2.1 The pH of the sample is determined and a measured amount of standard acid is
added, as needed, to lower the pH to 4 or less. Hydrogen peroxide is added, the
solution boiled for several minutes, cooled, and titrated electrometrically with
standard alkali to pH 8.2.
3. Definitions
3.1 This method measures the mineral acidity of a sample plus the acidity resulting
from oxidation and hydrolysis of polyvalent cations, including salts of iron and
aluminum.
4. Interferences
4.1 Suspended matter present in the sample, or precipitates formed during the
titration may cause a sluggish electrode response. This may be offset by allowing a
15-20 second pause between additions of titrant or by slow dropwise addition of
titrant as the endpoint pH is approached.
5. Apparatus
5.1 pH meter, suitable for electrometric titrations.
6. Reagents
6.1 Hydrogen peroxide (H2 02, 30% solution).
6.2 Standard sodium hydroxide, 0.02 N.
6.3 Standard sulfuric acid, 0.02 N.
7. Procedure
7.1 Pipet 50 ml of the sample into a 250 ml beaker.
7.2 Measure the pH of the sample. If the pH is above 4.0 add standard sulfuric acid in
5.0 ml increments to lower the pH to 4.0 or less. If the initial pH of the sample is
less than 4.0, the incremental addition of sulfuric acid is not required.
7.3 Add 5 drops of hydrogen peroxide.
1
-------
7.4 Heat the sample to boiling and continue boiling for 2 to 4 minutes. In some
instances, the concentration of ferrous iron in a sample is such that an additional
amount of hydrogen peroxide and a slightly longer boiling time may be required.
7.5 Cool the sample to room temperature and titrate electrometrically with standard
alkali to pH 8.2.
8. Calculations
(A X B) - (C X D) X 50,000
8.1 Acidity, as mg/1 CaCO3 =- -
ml sample
where :
A = vol. of standard alkali used in titration
B = normality of standard alkali
C = volume of standard acid used to reduce pH to 4 or less
D = normality of standard acid
8.2 If it is desired to report acidity in millequivalents per liter, the reported values as
CaCO3 are divided by 50, as follows:
/, mg/1
Acidity as meq/1 =
9. Precision
9.1 On a round robin conducted by ASTM on 4 acid mine waters, including
concentrations up to 2000 mg/1, the precision was found to be ± 10 mg/1.
1 0. References
1 0. 1 The procedure to be used for this determination can be found in:
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 124, D-1067, Method
E(1973).
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
370, Method 201 (Acidity and Alkalinity) (1971).
-------
ALKALINITY (pH 4.5)
STORET NO. 00410
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The method is suitable for all concentration ranges of alkalinity; however,
appropriate aliquots should be used to avoid a titration volume greater than 50
ml.
1.3 Automated titrimetric analysis is equivalent.
2. Summary of Method
2.1 An unaltered sample is titrated to an electrometrically determined end point of
pH 4.5. The sample must not be filtered, diluted, concentrated, or altered in any
way.
3. Comments
3.1 The sample must be analyzed as soon as practical; preferably, within a few hours.
Do not open sample bottle before analyses.
3.2 Substances, such as salts of weak organic and inorganic acids present in large
amounts, may cause interference in the electrometric pH measurements.
3.3 Oil and grease, by coating the pH electrode, may also interfere, causing sluggish
response.
4. Precision and Accuracy
4.1 Forty analysts in seventeen laboratories analyzed synthetic water samples
containing increments of bicarbonate, with the following results:
Increment as
Alkalinity
mg/liter, CaCO3
8
9
113
119
Precision as
Standard Deviation
mg/liter, CaCO3
1.27
1.14
5.28
5.36
Accuracy as
Bias,
%
+10.61
+22.29
- 8.19
- 7.42
Bias,
rrig/1, CaCO3
+0.85
+2.0
-9.3
-8.8
-------
(FWPCA Method Study 1, Mineral and Physical Analyses)
4.2 In a single laboratory (MDQARL), using surface water samples at an average
concentration of 122 mg CaCO3/l, the standard deviation was ±3.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
52, Method 102, (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 119, D-1067, Method
B, (1973).
5.2 For samples having high concentrations of mineral acids, such as mine wastes and
associated receiving waters, titrate to an electrometric endpoint of pH 3.9, using
the procedure in:
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 123, D-1067, Method
D,(1973).
-------
ALKALINITY
(Automated, Methyl Orange)
STORET NO. 00410
1. Scope and Application
1.1 This automated method is applicable to drinking, surface, and saline waters,
domestic and industrial wastes. The applicable range is 10 to 200 mg/1 as CaCO3.
1.2 This method is not applicable to samples with pH lower than 3.1.
2. Summary of Method
2.1 Methyl orange is used as the indicator in this method because its pH range is in
the same range as the equivalence point for total alkalinity, and it has a distinct
color change that can be easily measured. The methyl orange is dissolved in a
weak buffer at a pH of 3.1, just below the equivalence point, so that any addition
of alkalinity causes a loss of color directly proportional to the amount of
alkalinity.
3. Sample Handling and Preservation
3.1 Sample should be refrigerated at 4°C and run as soon as practical.
4. Interferences
4.1 Sample turbidity and color may interfere with this method. Turbidity must be
removed by filtration prior to analysis. Sample color that absorbs in the
photometric range used will also interfere.
5. Apparatus
5.1 Technicon Auto Analyzer consisting of:
5.1.1 Sampler I.
5.1.2 Manifold.
5.1.3 Proportioning pump.
5.1.4 Colorimeter equipped with 15 mm tubular flow cell and 550 nm filters.
5.1.5 Recorder equipped with range expander.
6. Reagents
6.1 Methyl Orange: Dissolve 0.125 g of methyl orange in 1 liter of distilled water.
6.2 pH 3.1 Buffer: Dissolve 5.1047 g of potassium acid phthalate in distilled water
and add 87.6 ml 0.1 N HC1 and dilute to 1 liter. Stable for one week.
6.3 Methyl Orange-Buffered Indicator: Add 1 liter of pH 3.1 buffer to 200 ml methyl
orange solution and mix well. Stable for 24 hours.
-------
6.4 Stock Solution: Dissolve 1.060 g of anhydrous sodium carbonate (oven-dried at
140°C for 1 hour) in distilled water and dilute to 1000 ml. 1.0 ml = 1.00 mg
CaCO3.
6.4.1 Prepare a series of standards by diluting suitable volumes of stock solution
to 100.0 ml with distilled water. The following dilutions are suggested:
ml of Stock
Solution Cone., mg/1 as CaCO3
1.0 10
2.0 20
4.0 40
6.0 60
8.0 80
10.0 100
18.0 180
20.0 200
7. Procedure
7.1 No advance sample preparation is required. Set up manifold as shown in Figure 1.
7.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening oh colorimeter to obtain stable baseline.
7.3 Place distilled water wash tubes in alternate openings on sampler and set sample
timing at 2.0 minutes.
7.4 Place working standards in sampler in order of decreasing concentration.
Complete filling of sampler tray with unknown samples.
7.5 Switch sample line from distilled water to sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed standards against
known concentrations. Compute concentration of samples by comparing sample
peak heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 15, 57, 154, and 193 mg/1 as CaCO3 the standard deviation was ±0.5.
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 31 and 149 mg/1 as CaCO3 recoveries were 100% and 99%, respectively.
-------
Bibliography
1. Technicon Auto Analyzer Methodology, Bulletin 1261, Technicon Controls, Inc.,
Chauncey, N.Y. (1961).
2. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 52,
Method 102(1971).
-------
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WASH TUBES: ONE
FIGURE 1. ALKALINITY MANIFOLD A A-I
-------
ARSENIC
STORETNO. Total 01002
Inorganic, Dissolved 00995
Inorganic, Total 00997
1. Scope and Application
1.1 The silver diethyldithiocarbamate method determines inorganic arsenic when
present in concentrations at or above 10 /ug/1. The method is applicable to most
fresh and saline waters in the absence of high concentrations of chromium, cobalt,
copper, mercury, molybdenum, nickel, and silver. Domestic and industrial wastes
may also be analyzed after digestion (See 3.3).
1.2 Difficulties may be encountered with certain industrial waste materials containing
volatile substances. High sulfur content of wastes may exceed removal capacity of
the lead acetate scrubber.
2. Summary of Method
2.1 Arsenic in the sample is reduced to arsine, AsH3, in acid solution in a hydrogen
generator. The arsine is passed through a scrubber to remove sulfide and is
absorbed in a solution of silver diethyldithiocarbamate dissolved in pyridine. The
red complex thus formed is measured in a spectrophotometer at 535 nm.
3. Comments
3.1 In analyzing most surface and ground waters, interferences are rarely en-
countered. Industrial waste samples should be spiked with a known amount of
arsenic to establish adequate recovery.
3.2 It is essential that the system be airtight during evolution of the arsine, to avoid
losses.
3.3 If concentration of the sample and/or oxidation of any organic matter is required,
refer to Standard Methods, 13th Edition, Method 104B, p 65, Procedure 4.a
(1971).
3.3.1 Since nitric acid gives a negative interference in this colorimetric test, use
sulfuric acid as a preservative if only inorganic arsenic is being measured.
3.4 1-Ephedrine in chloroform has been found to be a suitable solvent for silver
diethyldithiocarbamate if the analyst finds the odor of pyridine objectionable
[Anal. Chem. 45, 1786(1973)].
-------
4. Precision and Accuracy
4.1 A synthetic unknown sample containing 40 Mg/1, as As, with other metals was
analyzed in 46 laboratories. Relative standard deviation was ±13.8% and relative
error was 0%.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
62, Method 104A(1971).
10
-------
BIOCHEMICAL OXYGEN DEMAND
(5 Days, 20°C)
STORE! NO. 00310
1. Scope and Application
1.1 The biochemical oxygen demand test (BOD) is used for determining the relative
oxygen requirements of municipal and industrial wastewaters. Application of the
test to organic waste discharges allows calculation of the effect of the discharges
on the oxygen resources of the receiving water. Data from BOD tests are used for
the development of engineering criteria for the design of wastewater treatment
plants.
1.2 The BOD test is an empirical bioassay-type procedure which measures the
dissolved oxygen consumed by microbial life while assimilating and oxidizing the
organic matter present. The standard test conditions include dark incubation at
20°C for a specified time period (often 5 days). The actual environmental
conditions of temperature, biological population, water movement, sunlight, and
oxygen concentration cannot be accurately reproduced in the laboratory. Results
obtained must take into account the above factors when relating BOD results to
stream oxygen demands.
2. Summary of Method
2.1 The sample of waste, or an appropriate dilution, is incubated for 5 days at 20°C
in the dark. The reduction in dissolved oxygen concentration during the
incubation period yields a measure of the biochemical oxygen demand.
3. Comments
3.1 Determination of dissolved oxygen in the BOD test may be made by use of either
the Modified Winkler with Full-Bottle Technique or the Probe Method in this
manual.
3.2 Additional information relating to oxygen demanding characteristics of waste-
waters can be gained by applying the Total Organic Carbon and Chemical Oxygen
Demand tests (also found in this manual).
4. Precision and Accuracy
4.1 Eighty-six analysts in fifty-eight laboratories analyzed natural water samples plus
an exact increment of biodegradable organic compounds. At a mean value of 2.1
and 175 mg/1 BOD, the standard deviation was ±0.7 and ±26 mg/1, respectively.
(EPA Method Research Study 3).
4.2 There is no acceptable procedure for determining the accuracy of the BOD test.
11
-------
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
489, Method 219 (1971).
12
-------
BORON
(Curcumin Method)
STORET NO. 01022
1. Scope and Application
1.1 This colorimetric method finds maximum utility for waters whose boron content
is below 1 mg/1.
1.2 The optimum range of the method on undiluted or unconcentrated samples is
0.1-1.0 mg/1 of boron.
2. , Summary of Method
2.1 When a sample of water containing boron is acidified and evaporated in the
presence of curcumin, a red-colored product called rosocyanine is formed. The
rosocyanine is taken up in a suitable solvent, and the red color is compared with
standards either visually or photometrically.
3. Comments
3.1 Nitrate nitrogen concentrations above 20 mg/1 interfere.
3.2 Significantly high results are possible when the total of calcium and magnesium
hardness exceeds 100 mg/1 as CaCO3. Passing the sample through a cation
exchange resin eliminates this problem.
3.3 Close control of such variables as volumes and concentrations of reagents, as well
as time and temperature of drying, must be exercised for maximum accuracy.
3.4 Data to be entered into STORET must be reported as Mg/1.
4. Precision and Accuracy
4.1 A synthetic sample prepared by the Analytical Reference Service, PHS, containing
240 Mg/1 B, 40 Mg/1 As, 250 Mg/1 Be,. 20 Mg/1 Se, and 6 Mg/1 V in distilled water,
was analyzed by the curcumin method with a relative standard deviation of 22.8%
and a relative error of 0% in 30 laboratories.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
69, Method 107A(1971).
13
-------
BROMIDE
(Titrimetric)
STORETNO. 71870
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial waste effluents.
1.2 The concentration range for this method is 2-20 mg bromide/1.
2. Summary of Method
2.1 After pretreatment to remove interferences, the sample is divided into two
aliquots. One aliquot is analyzed for iodide by converting the iodide to iodate
with bromine water and titrating iodometrically with phenylarsine oxide (PAO)
or sodium thiosulfate. The other aliquot is analyzed for iodide plus bromide by
converting these halides to iodate and bromate with calcium hypochlorite and
titrating iodometrically with PAO or sodium thiosulfate. Bromide is then
calculated by difference.
3. Sample Handling and Preservation
3.1 Store at 4°C and analyze as soon as possible.
4. Interferences
4.1 Iron, manganese and organic matter can interfere; however, the calcium oxide
pretreatment removes or reduces these to insignificant concentrations.
4.2 Color interferes with the observation of indicator and bromine-water color
changes. This interference is eliminated by the use of a pH meter instead of a pH
indicator and the use of standardized amounts of oxidant and oxidant-quencher.
5. Reagents
5.1 Acetic Acid Solution (1:8): Mix 100 ml of glacial acetic acid with 800 ml of
distilled water.
5.2 Bromine Water: In a fume hood, add 0.2 ml bromine to 500 ml distilled water.
Stir with a magnetic stirrer and a Teflon-coated stirring bar for several hours or
until the bromine dissolves. Store in a glass-stoppered colored bottle.
5.3 Calcium Carbonate (CaCO3): Powdered
5.4 Calcium Hypochlorite Solution (Ca(OCl)2): Add 35 g of Ca(OCl)2 to approxi-
mately 800 ml of distilled water in a 1 liter volumetric flask. Stir on a magnetic
stirrer for approximately 30 minutes. Dilute to 1 liter and filter. Store in a
glass-stoppered, colored flask.
14
-------
5.5 Calcium Oxide (CaO): Anhydrous, powdered.
5.6 Hydrochloric Acid Solution (1:4): Mix 100 ml of HC1 (sp. gr. 1.19) with 400 ml
of distilled water.
j - :.
5.7 Potassium.Iodide (KI): Crystals, ACS Reagent Grade
5.8 Sodium Acetate Solution (275 g/1): Dissolve 275 g sodium acetate trihydrate
(NaC2H3O2 -3H2O) in distilled water. Dilute to 1 liter and filter.
5.9 Sodium Chloride (NaCl): Crystals, ACS Reagent Grade
5.10 Sodium Formate Solution (500 g/1): Dissolve 50 g sodium formate (NaCHO2) in
hot distilled water and dilute to 100 ml.
5.11 Sodium Molybdate Solution (10 g/1): Dissolve 1 g sodium molybdate (Na2MoO4
2H2O) in distilled water and dilute to 100 ml.
5.12 Sulfuric Acid Solution (1:4): Slowly add 200 ml H2SO4 (sp. gr. 1.84) to 800 ml
of distilled water.
5.13 Phenylarsine Oxide (0.0375N): Hach Chemical Co., or equivalent. Standardize
with 0.0375 N potassium biiodate (5.19, 5.23).
5.14 Phenylarsine Oxide Working Standard (0.0075 N): Transfer 100 ml of com-
mercially available 0.0375 N phenylarsine oxide (5.13) to a 500 ml volumetric
flask and dilute to the mark with distilled water. This solution should be prepared
fresh daily.
5.15 Amylose Indicator: Mallinckrodt Chemical Works or equivalent.
5.16 Sodium Thiosulfate, Stock Solution, 0.75 N: Dissolve 186.5 g Na2S2O3 -5H2O in
boiled and cooled distilled water and dilute to 1 liter. Preserve by adding 5 ml
chloroform.
5.17 Sodium Thiosulfate Standard Titrant, 0.0375 N: Prepare by diluting 50.0 ml of
stock solution (5.16) to 1.0 liter. Preserve by adding 5 ml of chloroform.
Standardize with 0.0375 N potassium biiodate (5.19, or 5.23).
5.18 Sodium Thiosulfate Working Standard (0.0075 N): Transfer 100 ml of sodium
thiosulfate standard titrant (5.17) to a 500 ml volumetric flask and dilute to the
mark with distilled water. This solution should be prepared fresh daily.
5.19 Potassium Biiodate Standard, 0.0375 N: Dissolve 4.387 g potassium biiodate,
previously dried 2 hours at 103°C, in distilled water and dilute to 1.0 liter. Dilute
250 ml to 1.0 liter for 0.0375 N biiodate solution.
5.20 Starch Solution: Prepare an emulsion of 10 g of soluble starch in a mortar or
. beaker with a small quantity of distilled water. Pour this emulsion into 1 liter of
boiling water, allow to boi} a few minutes, and let settle overnight. Use the clear
supernate. This solution may be preserved by the addition of 5 ml per liter of
15
-------
chloroform and storage in a 10°C refrigerator. Commercially available dry,
powdered starch indicators may be used in place of starch solution.
5.21 Nitrogen Gas: Cylinder
5.22 Potassium Fluoride (KF-2H2O): ACS Reagent Grade
5.23 Standardization of 0.0375 N Phenylarsine Oxide and 0.0375 N Sodium
.Thiosulfate; Dissolve approximately 2 g (±1.0 g) KI (5.7) in 100 to 150 ml
distilled water; add 10 ml H2SO4 solution (5.12) followed by 20 ml standard
potassium biiodate solution (5.19). Place in dark for 5 minutes, dilute to 300 ml
and titrate with the phenylarsine oxide (5.13) or sodium thiosulfate (5.17) to a
pale straw color. Add a small scoop of indicator (5.15). Wait until homogeneous
blue color develops and continue the titration drop by drop until the color
disappears. Run in duplicate. Duplicate determinations should agree within ±0.05
ml.
6. Procedure
6.1 Pretreatment
6.1.1 Add a visible excess of CaO (5.5) to 400 ml of sample. Stir or shake
vigorously for approximately 5 minutes. Filter through a dry, moderate-
ly retentive filter paper, discarding the first 75 ml.
6.2 Iodide Determination
6.2.1 Place 100 ml of pretreated sample (6.1) or a fraction thereof diluted to
that volume, into a 150 ml beaker. Add a Teflon-coated stirring bar and
place on a magnetic stirrer. Insert a pH electrode and adjust the pH to
approximately 7 or slightly less by the dropwise addition of H2SO4
solution (5.12).
6.2.2 Transfer the sample to a 250 ml widemouthed conical flask. Wash beaker
with small amounts of distilled water and add washings to the flask. A
250 ml iodine flask would increase accuracy and precision by preventing
possible loss of the iodine generated upon addition of potassium iodide
and sulfuric acid (6.4.1). ..
6.2.3 Add 15 ml sodium acetate solution (5.8) and 5 ml acetic acid solution
v--'
(5.1). Mix well. Add 40 nil bromine water solution (5.2); mix well. Wait
5 minutes.
6.2.4 Add 2 ml sodium formate solution (5.10); mix well. Wait 5 minutes.
6.2.5 Purge space above sample with gentle stream of nitrogen (5.21) for
approximately 30 seconds to remove bromine fumes.
6.2.6 If a precipitate forms (iron), add 0.5 g KF-2H2O (5.22).
16
-------
6.2.7 A distilled water blank must be run with each set of samples because of
iodide in reagents. If the blank is consistently shown to be zero for a
particular "lot" of chemicals, it can be ignored.
6.2.8 Proceed to step (6.4).
6.3 Bromide Plus Iodide Determination
6.3.1 Place 100 ml of pretreated sample (6.1) or a fraction thereof diluted to
that volume, in a 150 ml beaker. Add 5 g NaCl and stir to dissolve.
Neutralize by dropwise addition of HC1 solution (5.6) as in (6.2.1).
Transfer as in (6.2.2).
6.3.2 Add 20 ml of calcium hypochlorite solution (5.4). Add 1 ml of HC1
solution (5.6) and add approximately 0.2 g calcium carbonate (5.3).
6.3.3 Heat to boiling on a hot plate; maintain boiling for 8 minutes.
6.3.4 Remove from hot plate and carefully add 4 ml sodium formate solution
(5.10). Caution: TOO RAPID ADDITION MAY CAUSE FOAMING.
Wash down sides with distilled water.
. 6.3.5 Return to hot plate and maintain boiling conditions for an additional 8
minutes. Occasionally wash down sides with distilled water if residue is
deposited from boiling action.
6.3.6 Remove from hot plate. Wash down sides and allow to cool.
6.3.7 If a precipitate forms (iron), add 0.5 g KF-2H2O (5.22).
6.3.8 Add 3 drops sodium molybdate solution (5.11).
6.3.9 A distilled water blank must be run with each set of samples because of
iodide, iodate, bromide, and/or bromate in reagents.
6.3.10 Proceed to step (6.4).
6.4 Titration
6.4.1 Dissolve approximately 1 g potassium iodide (5.7) in sample from (6.2.8
or 6.3.10). Add 10 ml of H2SO4 solution (5.12) and place in dark for 5
minutes.
6.4.2 Titrate with standardized phenylarsine oxide working standard (5.14) or
sodium thiosulfate working standard (5.18), adding indicator (5.15, or
5.20) as end point is approached (light straw color). Titrate to colorless
solution. Disregard returning blue color.
7. Calculations
7.1 Principle: Iodide is determined by the titration of the sample as oxidized in (6.2):
bromide plus iodide is determined by the titration of the sample as oxidized in
(6.3). The amount of bromide is then determined by difference. The number of
17
-------
equivalents of iodine produced a constant of 13,320 as shown in the equation in
(7.2). Experimental data is entered in the appropriate place and the equation is
solved for mg/1 bromide.
7.2 Equation
. . AXB\ /DXE
Br(mg/l)= 13,320
K
where
A = the number of ml of PAO needed to titrate the sample for bromide plus
iodide (with the number of ml of PAO needed to titrate the blank
subtracted).
B = the normality of the PAO needed to titrate the sample for bromide plus
iodide
C = the volume of sample taken (100 ml or a fraction thereof) to be titrated
for bromide phis iodide.
D = the number of ml of PAO needed to titrate the sample for iodide
(with the number of ml of PAO needed to titrate the blank subtracted).
The blank for the iodide titration is often zero.
E = the normality of the PAO used to titrate the sample for iodide.
F = the volume of sample taken (100 ml or a fraction thereof) to be titrated
for iodide.
8. Precision and Accuracy
8.1 In a single laboratory (MDQARL), using a mixed domestic and industrial waste
effluent, at concentrations of 0.3, 2.8, 5.3, 10.3 and 20.3 mg/1 of bromide, the
standard deviations were ±0.13, ±0.37, ±0.38, ±0.44 and ±0.42 mg/1, respectively.
8.2 In a single laboratory (MDQARL), using a mixed domestic and industrial waste
effluent, at concentrations of 2.8, 5.3, 10.3 and 20.3 mg/1 of bromide, recoveries
were 96, 83, 97 and 99%, respectively.
Bibliography
1. ASTM Standards, Part 23, Water; Atmospheric Analysis, p 331-333, Method D1246-C
(1973).
18
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CALCIUM
STORET NO. Calcium (mg/1 CaCO3 ) 00910
Calcium, Total (rtig/1 Ca) 00916
1. Scope and Application
1.1 This method is applicable to drinking and surface waters, domestic and industrial
wastes.
1.2 The lower detection limit of this method is approximately 0.5 mg/1 as CaCO3 ; the
upper limit can be extended to all concentrations by sample dilution. It is
recommended that a sample aliquot containing not more than 25 mg CaCO3 be
used.
2. Summary of Method
2.1 Calcium ion is sequestered upon the addition of disodium dihydrogen ethylene-
diamine tetraacetate (EDTA). The titration end point is detected by means of an
indicator which combines with calcium only.
3. Interferences
3.1 Strontium and barium interfere and alkalinity in excess of 30 mg/1 may cause an
indistinct end point. Magnesium interference is reduced or eliminated by raising
the pH between 12-13 to precipitate magnesium hydroxide.
4. Precision and Accuracy
4.1 A synthetic unknown sample containing 108 mg/1 Ca, 82 mg/1 Mg, 3.1 mg/1 K,
19.9 mg/1 Na, 241 mg/1 chloride, 1.1 mg/1 nitrate N, 250pg/l nitrite N, 259 mg/1
sulfate, and 42.5 mg/1 total alkalinity in distilled water was determined by this
method with a relative standard deviation of 9.2% and a relative error of 1.9% in 44
laboratories.
5. Reference
5.1 The procedure to be used for this determination is found in: Standard Methods
for the Examination of Water and Wastewater, 13th Edition, p 84, Method 1 IOC
(1971).
19
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CHEMICAL OXYGEN DEMAND
STORE! NO. 00340
1. Scope and Application
1.1 The Chemical Oxygen Demand (COD) method determines the quantity of oxygen
required to oxidize the organic matter in a waste sample, under specific
conditions of oxidizing agent, temperature, and time.
1.2 Since the test utilizes a rigorous chemical oxidation rather than a biological
process, the result has no defineable relationship to the Biochemical Oxygen
Demand (BOD) of the waste. The test result should be considered as an
independent measurement of organic matter in the sample, rather than as a
substitute for the BOD test.
1.3 The method can be applied to domestic and industrial waste samples having an
organic carbon concentration greater than 15 mg/1. For lower concentrations of
carbon such as in surface water samples, the Low Level Modification should be
used. When the chloride concentration of the sample exceeds 2000 mg/1, the
modification for saline waters is required.
2. Summary of Method
2.1 Organic substances in the sample are oxidized by potassium dichromate in 50%
sulfuric acid solution at reflux temperature. Silver sulfate is used as a catalyst and
mercuric sulfate is added to remove chloride interference. The excess dichromate
is titrated with standard ferrous ammonium sulfate, using orthophenanthroline
ferrous complex as an indicator.
3. . Comments
3.1 To reduce loss of volatile organics, the flask should be cooled during addition of
the sulfuric acid solution.
4. Precision and Accuracy
4.1 Eighty-six analysts in fifty-eight laboratories analyzed a distilled water solution
containing oxidizable organic material equivalent to 270 mg/1 COD. The standard
deviation was ±17.76 mg/1 COD with an accuracy as percent relative error (bias)
of -4.7%. (EPA Method Research Study 3).
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
495, Method 220 (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 470, Method D
1252-67(1973).
20
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CHEMICAL OXYGEN DEMAND
(Low Level)
STORET NO. 00335
1. Scope and Application
1.1 The scope of this modification of the Chemical Oxygen Demand (COD) test is the
same as for the high level test. It is applicable to the analysis of surface waters,
domestic and industrial wastes with low demand characteristics.
1.2 This method (low level) is applicable for samples having a COD in the range of
5-50 mg/1 COD.
2. Summary of Method
2.1 Organic and oxidizable inorganic substances in an aqueous sample are oxidized by
potassium dichromate solution in 50 percent (by volume) sulfuric acid in
solution. The excess dichromate is titrated with standard ferrous ammonium
sulfate using orthophenanthroline ferrous complex (ferroin) as an indicator.
3. Sampling and Preservation
3.1 Collect the samples in glass bottles, if possible. Use of plastic containers is
permissible if it is known that no organic contaminants are present in the
containers.
3.2 Biologically active samples should be tested as soon as possible. Samples
containing settleable material should be well mixed, preferably homogenized, to
permit removal of representative aliquots.
3.3 Samples may be preserved with sulfuric acid at a rate of 2 ml of cone. H2SO4 per
liter of sample.
4. Interferences
4.1 Traces of organic material either from the glassware or atmosphere may cause a
gross, positive error.
4.1.1 Extreme care should be exercised to avoid inclusion of organic materials
in the distilled water used for reagent preparation or sample dilution.
4.1.2 Glassware used in the test should be conditioned by running blank
procedures to eliminate traces of organic material.
4.2 Volatile materials may be lost when the sample temperature rises during the
sulfuric acid addition step.
4.3 Chlorides are quantitatively oxidized by dichromate and represent a positive
interference. Mercuric sulfate is added to the digestion flask to complex the
21
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chlorides, thereby effectively eliminating the interference on all but brine and
estuarine samples.
5. Apparatus
5.1 Reflux apparatus: Glassware should consist of a 500 ml Erlenmeyer flask or a 300
ml round bottom flask made of heat-resistant glass connected to a 12 inch Allihn
condenser by means of a ground glass joint. Any equivalent reflux apparatus may be
substituted provided that a ground-glass connection is used between the flask and
;the condenser.
6.1 Distilled water: Special precautions should be taken to insure that distilled water
used in this test be low in organic matter.
6.2 Standard potassium dichromate solution (0.025 N): Dissolve 12.259 g K2Cr2O7,
primary standard grade, previously dried at 103°C for two hours, in distilled
water and dilute to 1000 ml. Mix this solution thoroughly then dilute 100.0 ml to
1000 ml with distilled water.
6.3 Sulfuric acid reagent: Cone. H2SO4 containing 23.5 g silver sulfate, Ag2SO4, per
9 Ib. bottle (one to two days required for dissolution).
6.4 Standard ferrous ammonium sulfate (0.025 N): Dissolve 98 g of Fe(NH4)2
(SO4)2-6H2O in distilled water. Add 20 ml of cone. H2SO4 (6.8), cool and
dilute to 1 liter. Dilute 100 ml of this solution to 1 liter with distilled water. This
solution must be standardized daily against K2Cr2O7 solution.
6.4.1 Standardization: To 15 ml of distilled water add 10.0 ml of 0.025 N
K2Cr2O7 (6.2) solution. Add 20 ml of H2SO4 (6.8) and cool. Titrate with
ferrous ammonium sulfate (6.4) using 1 drop of ferroin indicator (6.6).
The color change is sharp, going from blue-green to reddish-brown.
(ml K2Cr2O7) (0.025)
Normality =-
mlFe(NH4)2 (SO4)2
6.5 Mercuric sulfate : Powdered HgSO4.
6.6 Phenanthroline ferrous sulfate (ferroin) indicator solution: Dissolve 1.48 g of 1-10
/
(ortho)phenanthroline monohydrate, together with 0.70 g of FeSO4-7H2O in
100 ml of water. This indicator may be purchased already prepared.
6.7 Silver sulfate : Powdered Ag2 SO4.
6.8 Sulfuric acid (sp. gr. 1.84) : Concentrated H2SO4.
7. Procedure
7.1 Place several boiling stones in the reflux flask, followed by 1 g of HgSO4 (6.5).
Add 5.0 ml cone. H2SO4 (6.8); swirl until mercuric sulfate has dissolved. Place
22
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reflux flask in an ice bath and slowly add, with swirling, 25.0 ml of 0.025 N
K2Cr2O7 (6.2). Now add 70 ml of sulfuric acid-silver sulfate solution (6.3) to the
cooled reflux flask, again using slow addition with swirling motion.
7.2 With the reflux flask still in the ice bath, place 50.0 ml of sample or an aliquot
diluted to 50.0 ml into the reflux flask.
Caution: Care must be taken to assure that the contents of the flask are well
mixed. If not, superheating may result, and the mixture may be blown out of the
open end of the condenser. Attach the flask to the condenser and start the
cooling water.
7.3 Apply heat to the flask and reflux for 2 hours. For some waste waters, the 2-hour
reflux period is not necessary. The time required to give the maximum oxidation
for a wastewater of constant or known composition may be determined and a
shorter period of refluxing may be permissible.
7.4 Allow the flask to cool and wash down the condenser with about 25 ml of
distilled water. If a round bottom flask has been used, transfer the mixture to a
500 ml Erlenmeyer flask, washing out the reflux flask 3 or 4 times with distilled
water. Dilute the acid solution to about 300 ml with distilled water and allow the
solution to cool to about .room temperature. Add 8 to 10 drops of ferroin
indicator (6.6) to the solution and titrate the excess dichromate with 0.025 N
ferrous ammonium sulfate (6.4) solution to the end point. The color change will
be sharp, changing from a blue-green to a reddish hue.
7.5 Blank Simultaneously run a blank determination following the details given in
(7.1) and (7.2), but using low COD water in place of sample.
8. Calculation
8.1 Calculate the COD in the sample in mg/1 as follows:
(A-B)N X 8000
COD, mg/liter =
where
A = milliliters of Fe(NH4 )2 (SO4 )2 solution required for titration of the
blank,
B = milliliters of Fe(NH4 )2 (SO4 )2 solution required for titration of the
sample,
N = normality of the Fe(NH4 )2 (SO4 )2 solution, and
S = milliliters of sample used for the test.
23
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9. Precision and Accuracy
9.1 Eighty-six analysts in fifty-eight laboratories analyzed a distilled water solution
containing oxidizable organic material equivalent to 12.3 mg/1 COD. The
standard deviation was ±4.15 mg/1 COD with an accuracy as percent relative error
(bias) of 0.3%. (EPA Method Research Study 3.)
24
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CHEMICAL OXYGEN DEMAND
(High Level for Saline Waters)
STORET NO. 00340
1. Scope and Application
1.1 When the chloride level exceeds 1000 mg/1 the minimum accepted value for the
COD will be 250 mg/1. COD levels which fall below this value are highly
questionable because of the high chloride correction which must be made.
i
2. Summary of Method
2.1 Organic and oxidizable inorganic substances in an aqueous sample are oxidized by
potassium dichromate solution in 50 percent (by volume) sulfuric acid solution.
The excess dichromate is titrated with standard ferrous ammonium sulfate using
orthophenanthroline ferrous complex (ferroin) as an indicator.
3. Sample Handling and Preservation
3.1 Collect the samples in glass bottles, if possible. Use of plastic containers is
permissible if it is known that no organic contaminants are present in the
containers.
3.2 Biologically active samples should be tested as soon as possible. Samples
containing settleable material should be well mixed, preferably homogenized, to
permit removal of representative aliquots,
3.3 Samples are preserved by the addition of 2 ml of cone. H2 SO4 per liter of sample.
4. Interferences
4.1 Traces of organic material either from the glassware or atmosphere may cause a
gross, positive error.
4.1.1 Extreme care should be exercised to avoid inclusion of organic materials
in the distilled water used for reagent preparation or sample dilution.
4.1.2 Glassware used in the test should be conditioned by running blank
procedures to eliminate traces of organic material.
4.2 Volatile materials may be lost when the sample temperature rises during the
sulfuric acid addition step.
4.3 Chlorides are quantitatively oxidized by dichromate and represent a positive
interference. Mercuric sulfate is added to the digestion flask to complex the
chlorides, thereby effectively eliminating the interference on all but brine
samples.
25
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5. Apparatus
5.1 Reflux apparatus: Glassware should consist of a 500 ml Erlenmeyer flask or a 300
ml round bottom flask made of heat-resistant glass connected to a 12 inch Allihn
condenser by means of a ground glass joint. Any equivalent reflux apparatus may
be substituted provided that a ground-glass connection is used between the flask
and the condenser.
6. Reagents
6.1 Standard potassium dichromate solution, (0.25 N): Dissolve 12.2588 g of
K2Cr2O7, primary standard grade, previously dried for 2 hours at 103°C in water
and dilute to 1000ml.
6.2 Sulfuric acid reagent: Cone. H2SO4 containing 23.5 g silver sulfate, Ag2SO4, per
9 Ib. bottle (1 to 2 days required for dissolution).
6.3 Standard ferrous ammonium sulfate, 0.250 N; Dissolve 98 g of Fe(NH4 )2 (SO4 )2
6H2O in distilled water. Add 20 ml of cone. H2SO4, (6.7), cool and dilute to 1
liter. This solution must be standardized against the standard potassium
dichromate solution (6.1) daily.
6.3.1 Standardization: Dilute 25.0 ml of standard dichromate solution (6.1) to
about 250 ml with distilled water. Add 75 ml cone, sulfuric acid (6.7).
Cool, then titrate with ferrous ammonium sulfate titrant (6.3), using 10
drops of ferroin indicator (6.5).
(mlK2Cr207)(0.25)
Normality =-
mlFe(NH4)2 (SO4)2
6.4 Mercuric sulfate : Powdered HgSO4.
6.5 Phenanthroline ferrous sulfate (ferroin) indicator solution: Dissolve 1.48 g of
l-10-(ortho)-phenanthroline monohydrate, together with 0.70 g of FeSO4
7H2 O in 100 ml of water. This indicator may be purchased already prepared.
6.6 Silver sulfate : Powdered Ag2 SO4.
6.7 Sulfuric acid (sp. gr. 1.84) : Concentrated H2SO4.
7. Procedure
7.1 Pipet a 50.0 ml aliquot of sample not to exceed 800 mg/1 of COD into a 500 ml,
flat bottom, Erlenmeyer flask. Add 25.0 ml of 0.25 N K2Cr2O7 (6.1), then 5 ml
of cone. H2SO4 (6.7). Add HgSO4 (6.4) in the ratio of 10 mg to 1 mg chloride,
based upon the mg of chloride in the sample aliquot. Swirl until all the mercuric
sulfate has dissolved. Carefully add 70 ml of sulfuric acid-silver sulfate solution
26
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(6.2) and gently swirl until the solution is thoroughly mixed. Glass beads should
be added to the reflux mixture to prevent bumping, which can be severe and
dangerous.
Caution: The reflux mixture must be thoroughly mixed before heat is applied. If
this is not done, local heating occurs in the bottom of the flask, and the mixture
may be blown out of the condenser.
7.1.1 If volatile organics are present in the sample, use an Allihn condenser and
add the sulfuric acid-silver sulfate solution through the condenser, while
cooling the flask, to reduce loss by volatilization.
7.2 Attach the flask to the condenser and reflux the mixture for two hours.
^ > ,
7.3 Cool, and wash down the interior of the condenser with 25 ml of distilled water.
Disconnect the condenser and wash the flask and condenser joint with 25 ml of
distilled water so that the total volume is 350 ml. Cool to room temperature.
7.4 Titrate with standard ferrous ammonium sulfate (6.3) using 10 drops of ferroin
(6.5) indicator. (This amount must not vary from blank, sample and standardiza-
tion). The color change is sharp, going from blue-green to reddish-brown and
should be taken as the end point although the blue-green color may reappear
within minutes.
7.5 Run a blank, using 50 ml of distilled water in place of the sample together with all
reagents,and subsequent treatment.
7.6 For COD values greater than 800 mg/1, a smaller aliquot of sample should be
taken; however, the volume should be readjusted to 50 ml with distilled water
having a chloride concentration equal to the sample.
7.7 Chloride correction <' >: Prepare a standard curve of COD versus mg/1 of chloride,
using sodium chloride solutions of varying concentrations following exactly the
procedure outlined. The chloride interval, as a minimum should be 4000 mg/lup
to 20,000 mg/1 chloride. Lesser intervals of greater concentrations must be run as
per the requirements of the data, but in no case must extrapolation be used.
8. Calculation
[(A-B)CX 8,000] -SOD
8.1 mg/1 COD = X 1.2
ml sample
Where:
COD = chemical oxygen demand from dichromate
A = ml Fe (NH4)2 (SO4)2 for blank;
B= ml Fe(NH4)2 (SO4)2 for sample;
27
-------
C = normality of Fe (NH4 )2 (SO4 )2;
D = chloride correction from curve (step 7.7)
1.2 = compensation factor to account for the extent of chloride oxidation which is
dissimilar in systems containing organic and non-organic material.
9. Precision and Accuracy.
9.1 Precision and accuracy data are not available at this time.
Bibliography
1. Burns, E. R., Marshall, C, Journal WPCF, Vol. 37, p 1716-1721 (1965).
28
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CHLORIDE
STORET NO. 00940
/
_1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The method is suitable for all concentration ranges of chloride con tent; however,
in order to avoid large titration volumes, use a sample aliquot containing not more
than 10 to 20 mg Cl per 50 ml.
1.3 Automated titration may be used.
2. Summary of Method
2.1 Dilute mercuric nitrate solution is added to an acidified sample in the presence of
mixed diphenylcarbazone-bromophenol blue indicator. The end point of the
titration is the formation of the blue-violet mercury diphenylcarbazone complex.
3. Comments
3.1 Anions and cations at concentrations normally found in surface waters do not
interfere.
3.2 Sulfites interfere. If presence is suspected, oxidize by treating 50 ml of sample
with 0.5 to 1 mlofH2O2.
4. Precision and Accuracy
4.1 Forty-two analysts in eighteen laboratories analyzed synthetic water samples
containing exact increments of chloride, with the following results:
Increment as
Chloride
mg/liter
17
18
91
97
382
398
Precision as
Standard Deviation
mg/liter
1.54
1.32
2.92
3.16
11.70
11.80
Accuracy as
Bias,
%
+2.16
+3.50
+0.11
-0.51
-0.61
-1.19
Bias,
mg/liter
+0.4
+0.6
+0.1
-0.5
-2.3
-4.7
(FWPCA Method Study 1, Mineral and Physical Analyses)
29
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4.2 In a single laboratory (MDQARL), using surface water samples at an average
concentration of 34 mg Cl/1, the standard deviation was ±1.0.
Reference
5.1 The procedure to be used for this determination is found in:
ASTM Standards, Part. 23, Water; Atmospheric Analysis, p 273, Method 512-67,
Referee Method A (1973).
30
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CHLORIDE
(Automated)
STORET NO. 00940
1. Scope and Application
1.1 This automated method is applicable to drinking, surface, and saline waters,
domestic and industrial wastes. The applicable range is 1 to 250 mg Cl/1.
Approximately 15 samples per hour can be analyzed.
2. Summary of Method
.2.1 Thiocyanate ion (SCN) is liberated from mercuric thiocyanate, through sequestra-
tion of mercury by chloride ion to form un-ionized mercuric chloride. In the
presence of ferric ion, the liberated SCN forms highly colored ferric thiocyanate,
in concentration proportional to the original chloride concentration.
3. Sample Handling and Preservation
3.1 No special requirements.
4. Interferences
4.1 No significant interferences.
5. Apparatus
5.1 Technicon AutoAnalyzer consisting of:
5.1.1 Sampler I.
5.1.2 Continuous filter.
5.1.3 Manifold.
5.1.4 Proportioning pump.
5.1.5 Colorimeter equipped with 15 mm tubular flow cell and 480 nm filters.
5.1.6 Recorder.
6. Reagents
6.1 Ferric Ammonium Sulfate: Dissolve 60 g of FeNH4(SO4)2 * 12H2O in
approximately 500 ml distilled water. Add 355 ml of cone. HNO3 and dilute to 1
liter with distilled water. Filter.
6.2 Saturated Mercuric Thiocyanate: Dissolve 5 g of Hg(SCN)2 in 1 liter of distilled
water. Decant and filter a portion of the saturated supernatant liquid to use as the
reagent and refill the bottle with distilled water.
6.3 Stock Solution (0.0141 N NaCl): Dissolve 0.8241 g of pre-dried (140°C) NaCl in
distilled water. Dilute to 1 liter in a volumetric flask. 1 ml = 0.5 mg Cl.
31
-------
6.3.1 Prepare a series of standards by diluting suitable volumes of stock
solution to 100.0 ml with distilled water. The following dilutions are
suggested:
ml of Stock Solution Cone., mg/1
1.0 5.0
2.0 10.0
4.0 20.0
8.0 40.0
15.0 75.0
20.0 100.0
30.0 150.0
40.0 . 200.0
50.0 250.0
7. Procedure
7.1 No advance sample preparation is required. Set up manifold as shown in Figure 1.
For water samples known to be consistently low in chloride content, it is
advisable to use only one distilled water intake line.
7.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain stable baseline.
7.3 Place distilled water wash tubes in alternate openings in sampler and set sample
timing at 2.0 minutes.
7.4 Place working standards in sampler in order of decreasing concentrations.
Complete filling of sampler tray with unknown samples.
7.5 Switch sample line from distilled water to sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed standards against
known concentrations. Compute concentration of samples by comparing sample
peak heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 1, 100, and 250 mgCl/1, the standard deviation was ±0.3.
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 10 and 100 mg Cl/1, recoveries were 97% and 104%, respectively.
-32
-------
Bibliography
1. J. E. O'Brien, "Automatic Analysis of Chlorides in Sewage," Waste Engr., 33, 670-672
(Dec. 1962).
33
-------
OJ
SMALL
MIXING
COILS
Ism)
WASTE
COLORIMETER
15MM TUBULAR f/c
480 «M FILTERS
BLUE
P
PROPORTIONING
PUMP
CONTINUOUS FILTER
I.60 Fe NH4(S04)2
Hg
2.50
WASTE
IX
.rrn.nc.
"ECP"OE"
SAMPLING TIME: 2.0 MINUTES
WASH TUBES: ONE
FIGURE 1. CHLORIDE MANIFOLD AA-I
-------
CHLORINE, Total Residua!
STORETNO. 50060
1. Scope and Application
1.1 The Amperometric Titration method is applicable to all types of waters and
wastes that do not contain a substantial amount of organic matter. This method
cannot be used for samples containing above 5 mg/1 total residual chlorine.
2. Summary of Method
2.1 Phenylarsine oxide is titrated into a buffered sample contained in an ampero-
metric titration cell until the generation of current ceases. Potassium iodide is
added when chlorine is present as a chloramine.
2.2 In the iodometric titration, chlorine liberates free iodine from potassium iodide
solutions when its pH is 8 or less. The liberated iodine is titrated with a standard
solution of sodium thiosulfate or phenylarsine oxide with starch as an indicator.
3. Interferences
3.1 Samples containing significant amounts of organic matter interfere with the
amperometric titration and the iodometric method must be used.
3.2 The amperometric titration is not subject to interference from color, turbidity,
iron, manganese, or nitrite nitrogen.
4. Sample Handling and Preservation
4.1 Chlorine determinations must be started immediately after sampling, avoiding
excessive light and agitation. Samples to be analyzed for chlorine cannot be
stored.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and wastewater, 13th Edition, p
382, Method 204A (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 280, Method
01253-68(1973).
35
-------
COLOR
(Platinum-Cobalt)
STORE! NO. 00080
1. Scope and Application
1.1 The Platinum-Cobalt method is useful for measuring color of water derived from
naturally occurring materials, i.e., vegetable residues such as leaves, barks, roots,
humus and peat materials. The method is not applicable to color measurement on
waters containing highly colored industrial wastes.
NOTE 1: The Spectrophotometric and Tristimulus methods are useful for
detecting specific color problems. The use of these methods, however, is laborious
and unless determination of the hue, puri.ty, and luminance is desired, they are of
limited value.
2. Summary of Method
2.1 Color is measured by visual comparison of the sample with platinum-cobalt
standards. One unit of color is that produced by 1 mg/1 platinum in the form of
the chloroplatinate ion.
3. Interferences
3.1 Since very slight amounts of turbidity interfere with the determination, samples
showing visible turbidity should be clarified by centrifugation.
3.2 Method is pH dependent.
4. Sample Handling and Preservation
4.1 Representative samples shall be taken in scrupulously clean glassware.
4.2 Since biological activity may change the color characteristics of a sample, the
determination should be made as soon as possible. Refrigeration at 4°C is
recommended.
5. Apparatus
5.1 Nessler tubes : Matched, tall form, 50 ml capacity.
6. Reagents
6.1 Standard chloroplatinate solution: Dissolve 1.246 g potassium chlorplatinate,
K2PtCl6, (equivalent to 0.500 g metallic Pt) and 1 g crystalline cobaltous
chloride, CoCl2 6H2O, in distilled water containing 100 ml of cone. HC1. Dilute
to 1000 ml with distilled water. This standard solution is equivalent to 500 color
units.
36
-------
7. Preparation of Standards
7.1 Prepare standards in increments from 5 to 70 units. The following series is
suggested:
ml of Standard Solution
Diluted to 50.0 ml Color in
with Distilled Water Chloroplatinate Units
0.0 0
0.5 . 5
1.0 10
1.5 15
2.0 20
2.5 25
3.0 30
3.5 35
4.0 40
X4:5 45
5.0 50
6.0 60
7.0 70
7.2 Protect these standards against evaporation and contamination by use of clean,
inert stoppers.
NOTE 2: The standards also must be protected against the absorption of
ammonia since an increase in color will result.
8. Procedure
8.1 Apparent color: Observe the color of the sample by filling a matched Nessler tube
to the 50 ml mark with the water and compare with standards. This comparison is
made by looking vertically downward through the tubes toward a white or
specular surface placed at such an angle that light is reflected upward through the
columns of liquid. If turbidity has not been removed by the procedure given in
(8.2), report the color as "apparent color". If the color exceeds 70 units, dilute
the sample with distilled water in known proportions until the color is within the
range of the standards.
37
-------
8.2 True color: Remove turbidity by centrifuging the sample until the supernatant is
\
clear. The time required will depend upon the nature of the sample, the speed of
the motor, and the radius of the centrifuge, but rarely will more than one hour be
necessary. Compare the centrifuged sample with distilled water to insure that
turbidity has been removed. If the sample is clear, then compare with standards as
given in (8.1).
9. Calculation
9.1 Calculate the color units by means o.f the following equation:
AX 50
Color units =
where:
A = estimated color of diluted sample.
V = ml sample taken for dilution.
9.2 Report the results in whole numbers as follows:
Color Units Record to Nearest
1-50 . 1
51-100 . 5
101-250 10
251-500 20
10. Precision and Accuracy
10.1 Precision and accuracy data are not available at this time.
11. Reference
11.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
160, Method 118(1971).
38
-------
COLOR
(Spectrophotometric)
STORET NO. 00080
1. Scope and Application ' .
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes. It must be used for industrial wastes that cannot be determined
by the Platinum-Cobalt method.
2. Summary of Method
2.1 Color characteristics are measured at pH 7.6 .and at the original pH by obtaining
the visible absorption spectrum of the sample on a spectrophotometer. The
percent transmission at certain selected wavelengths is used to calculate the
results.
2.2 The results are expressed in terms of dominant wavelength, hue, luminance, and
purity.
3. Interferences
3.1 Since very slight amounts of turbidity interfere with the determination, samples
must be filtered before analysis.
4. Sample Handling and Preservation
4.1 Since biological activity may change the color characteristics of a sample, the
determination should be made as soon as possible. Refrigeration at 4°C is
recommended.
5. Reference
5.1 The procedure to be used for this determinationis found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition p
391, Method 206A (1971).
39
-------
CYANIDE, Total
STORE! NO. 00720
1. Scope and Application
1.1 This method is applicable to the '"determination of cyanide in drinking, surface,
and saline waters, domestic and industrial wastes.
1.2 The titration procedure using silver nitrate with p-dimethylamino-benzal-
rhodanine indicator is used for measuring concentrations of cyanide exceeding 1
mg/1 (0.2 mg/200 ml of absorbing liquid).
1.3 The colorimetric procedure is used for concentrations below 1 mg/1 of cyanide
and is sensitive to about 0.02 mg/1.
2. Summary of Method
2.1 The cyanide as hydrocyanic acid (HCN) is released from cyanide complexes by
means of a reflux-distillation operation and absorbed in a scrubber containing
sodium hydroxide solution. The cyanide ion in the absorbing solution is then
determined by volumetric titration or colorimetrically. -
2.2 In the colorimetric measurement the cyanide is converted to cyanogen chloride,
CNC1, by reaction with chloramine-T at a pH less than 8 without hydrolyzing to
the cyanate. After the reaction is complete, color is formed on the addition of
pyridine-pyrazolone or pyridine-barbituric acid reagent. The absorbance is read at
620 nm when using pyridine-pyrazolone or 578 nm for pyridine-barbituric acid.
To obtain colors of comparable intensity, it is essential to have .the same salt
content in both the sample and the standards.
2.3 The titrimetric measurement uses a standard solution of silver nitrate to titrate
cyanide in the presence of a silver sensitive indicator.
3. Definitions
3.1 Cyanide is defined as cyanide ion and complex cyanides converted to hydrocyanic
acid (HCN) by reaction in a reflux system of a mineral acid in the presence of
cuprous ion.
4. Sample Handling and Preservation
4.1 The sample should be collected in plastic bottles of 1 liter or larger size. All
bottles must be thoroughly cleansed and thoroughly rinsed to remove soluble
material from containers.
4.2 Samples must be preserved with 2 ml of 10 N sodium hydroxide per liter of
sample (pH>l 2) at the time of collection.
40
-------
4.3 Samples should be analyzed as rapidly as possible after collection. If storage is
required, the samples should be stored in a refrigerator or in an ice chest filled
with water and ice to maintain temperature at 4°C.
4.4 Oxidizing agents such as chlorine decompose most of the cyanides. Test a drop of
the sample with potassium iodide-starch test paper (Kl-starch paper); a blue color
indicates the need for treatment. Add ascorbic acid, a few crystals at a time, until
a drop of sample produces no color on the indicator paper. Then add an
additional 0.6 g of ascorbic acid for each liter of sample volume.
5. Interferences
5.1 Interferences are eliminated or reduced by using the distillation procedure
described in Procedure (8.1 through 8.5).
5.2 Sulfides adversely affect the colorimetric and titration procedures. If a drop of
the sample on lead acetate test paper indicates the presence of sulfides, treat 25
ml more of the stabilized sample (pH>12) than that required for the cyanide
determination with powdered cadmium carbonate. Yellow cadmium sulfide
precipitates if the sample contains sulfide. Repeat this operation until a drop of
the treated sample solution does not darken the lead acetate test paper. Filter the
solution through a dry filter paper into a dry beaker, and from the filtrate,
measure the sample to be used for analysis. Avoid a large excess of cadmium and a
long contact time in order to minimize a loss by complexation or occlusion of
cyanide on the precipitated material.
5.3 Fatty acids will distill and form soaps under the alkaline titration conditions,
making the end point almost impossible to detect.
5.3.1 Acidify the sample with acetic acid (1+9) to pH 6.0 to 7.0.
Caution: This operation must be performed in the hood and the sample
left there until it can be made alkaline again after the extraction has been
performed.
5.3.2 Extract with iso-octane, hexane, or chloroform (preference in order
named) with a solvent volume equal to 20% of the sample volume. One
extraction is usually adequate to reduce the fatty acids below the
interference level. Avoid multiple extractions or a long contact time at
low pH in order to keep the loss of HCN at a minimum. When the
extraction is completed, immediately raise the pH of the sample to above
1 2 with NaOH solution.
6. Apparatus
6.1 Reflux distillation apparatus such as shown in Figure 1 or Figure 2. The boiling
41
-------
flask should be of J liter size with inlet tube and provision for condenser. The gas
absorber may be a Fisher-MiHigan scrubber.
6.2 Microburet, 5.0 ml (for titration).
6.3 Spectrophotometer suitable for measurements at 578 nm or 620 nm with a 1.0
cm cell or larger.
7. Reagents
7.1 Sodium hydroxide solution: Dissolve 50 g of NaOH in distilled water, and dilute
to 1 liter with distilled water.
7.2 Cadmium carbonate: powdered.
7.3 Ascorbic acid: crystals.
7.4 Cuprous Chloride Reagent: Weigh 20 g of finely powdered Cu2Cl2 into an 800 ml
beaker. Wash twice, by decantation, with 250 ml portions of dilute sulfuric acid
(H2SO4, 1 + 49) and then twice with water. Add about 250 ml of water and then
hydrochloric acid (HC1, sp gr 1.19) in 1/2 ml portions until the salt dissolves (See
Note 1). Dilute to 1 liter with distilled water and store in a tightly stoppered
bottle containing a few lengths of pure copper wire or rod extending from the
bottom to the mouth of the bottle (See Note 2).
Note 1: The reagent should be clear; dark discoloration indicates the presence of
cupric salts.
Note 2: If it is desired to use a reagent bottle of smaller volume, it should be kept
completely filled and tightly stoppered. Refill it from the stock solution after
each use.
7.5 Sulfuric acid: concentrated.
7.6 Sodium dihydrogenphosphate, 1 M: Dissolve 138 g of NaH2PO4 -H2O in 1 liter of
distilled water. Refrigerate this solution.
7.7 Stock cyanide solution: Dissolve 2.51 g of KCN and 2 g KOH in 1 liter of distilled
water. Standardize with 0.0192 N AgNO3. Dilute to appropriate concentration so
that 1 ml = 1 mg CN.
7.8 Standard cyanide solution, intermediate: Dilute 10.0 ml of stock (1 ml = 1 mg
CN) to 1000 ml with distilled water (1 ml = lOjug).
7.9 Standard cyanide solution: Prepare fresh daily by diluting 100.0 ml of
intermediate cyanide solution to 1000 ml with distilled water and store in a glass
stoppered bottle. 1 ml = 1.0/ig CN (1.0 mg/1).
7.10 Standard silver nitrate solution, 0.0192 N: Prepare by crushing approximately 5 g
AgNO3 crystals and drying to constant weight at 40°C. Weigh out 3.2647 g of
dried AgNO3, dissolve in distilled water, and dilute to 1000 ml (1 ml = mg CN).
42
-------
7.11 Rhodanine indicator: Dissolve 20 nig of p-dimethyl-amino-benzalrhodanine in
100 ml of acetone.
7.12 Chloramine T solution: Dissolve 1.0 g of white, water soluble Chlofamine T in 100
ml of distilled water and refrigerate until ready to use. Prepare fresh weekly.
7.13 Color Reagent One of the following may be used:
7.13.1 Pyridine-Barbituric Acid Reagent: Place 15 g of barbituric acid in a
250 ml volumetric flask and add just enough distilled water to wash
the sides of the flask and wet the barbituric acid. Add 75 ml of
pyridine and mix. Add 15 ml of HC1 (sp gr 1.19), mix, and cool to
room temperature. Dilute to 250 ml with distilled water and mix.
This reagent is stable for approximately six months if stored in a
cool, dark plate.
7.13.2 Pyridine-pyrazolohe solution:
7.13.2.1 3-Methyl-l-phenyl-2-pyrazolin-5-one reagent, saturated solu-
tion. Add 0.25 g of 3-methyl-l-phenyl-2-pyrazolin-5-one to
50 ml of distilled water, heat to 60° C with stirring. Cool to
roorh temperature.
7.13.2.2 3,3'Dimethyl-l, l'-diphenyl-[4,4'-bi-2 pyrazoline] -5,5'dione
(bispyrazolone). Dissolve 0.01 g of bispyrazolone in 10 ml of
pyridine.
7.13.2.3 Pour solution (7.13.2.1) through nonacid-washed filter paper.
Collect the filtrate. Through the same filter paper pour
solution (7.13.2.2) collecting the filtrate in the same contain-
er as filtrate from (7.13.2.1). Mix until the filtrates are
homogeneous. The mixed reagent develops a pink color but
this does not affect the color production with cyanide if used
within 24 hours of preparation.
8. Procedure
8.1 Place 500 ml of sample, or an aliquot diluted to 500 nil in the i liter boiling flask.
Add 50 ml of sodium hydroxide (7.1) to the absorbing tube and dilute if
necessary with distilled water to obtain an adequate depth of liquid in the
absorber. Connect the boiling flask, condenser, absorber and trap in the train.
8.2 Start a slow stream of air entering the boiling flask by adjusting the vacuum
source. Adjust the vacuum so that approximately one bubble of air per second
enters the boiling flask through the air inlet tube.
43
-------
Caution: The bubble rate will not remain constant after the reagents have been
added and while heat is being applied to the flask. It will be necessary to readjust
the air rate occasionally to prevent the solution in the boiling flask from backing
up into the air inlet tube.
8.3 Slowly add 25 ml cone, sulfuric acid (7.5) through the air inlet tube. Rinse the
tube with distilled water and allow the airflow to mix the flask contents for 3
min. Pour 10 ml of Cu2 C12 reagent (7.4) into the air inlet and wash down with a
stream of water.
8.4 Heat the solution to boiling, taking care to prevent the solution from backing up
into and overflowing from the air inlet tube. Reflux for one hour. Turn off heat
and continue the airflow for at least 15 minutes. After cooling the boiling flask,
disconnect absorber and close off the vacuum source.
8.5 Drain the solution from the absorber into a volumetric flask and bring up to
volume with distilled water washings from the absorber tube.
8.6 Withdraw 50 ml of the solution from the volumetric flask and transfer to a 100
ml volumetric flask. Add 15 ml of sodium phosphate solution (7.6) and 2.0 ml of
Chloramine T solution (7.12) and mix. Immediately add 5.0 ml pyridine-
barbituric acid solution (7.13.1), or pyridine-pyrazolone solution (7.13.2.3), mix
and bring to mark with distilled water and mix again.
8.7 For pyridine-pyrazolone solution allow 40 minutes for color development then
read absorbance at 620 nm in a 1 cm cell. When using pyridine-barbituric acid,
allow 8 minutes for color development then read absorbance at 578 nm in a 1.0
cm cell within 15 minutes.
8.8 Prepare a series of standards by diluting suitable volumes of standard solution to
500.0 ml with distilled water as follows:
ml of Standard Solution Cone., When Diluted to
(1.0=ljugCN) 500ml,mg/lCN
0 (Blank) 0
5.0 0.01
10.0 0.02
20.0 0.04
50.0 0.10
100.0 0.20
150.0 0.30
200.0 0.40
44
-------
8.8.1 Standards must be treated in the same manner as the samples, as outlined
in (8.1) through (8.7) above.
8.8.2 Prepare a standard curve by plotting absorbance of standard vs. cyanide
concentrations.
8.8.3 Subsequently, at least two standards (a high and a low) should be treated
as in (8.8.1) to verify standard curve. If results are not comparable
(±20%), a complete new standard curve must be prepared.
8.8.4 To check the efficiency of the sample distillation, add an increment of
cyanide from either the intermediate standard (7.8) or the working
standard (7.9) to insure a level of 20^g/l or a significant increase in
absorbance value. Proceed with the analysis as in Procedure (8.8.1) using
the same flask and system from which the previous sample was just
distilled.
8.9 Alternatively, if the sample contains more than 1 mg of CN transfer the distillate,
or a suitable aliquot diluted to 250 ml, to a 500 ml Erlenmeyer flask. Add 10-12
drops of the benzalrhodanine indicator.
8.10 Titrate with standard silver nitrate to the first change in color from yellow to
brownish-pink. Titrate a distilled water blank using the same amount of sodium
hydroxide and indicator as in the sample.
8.11 The analyst should familiarize himself with the end point of the titration and the
amount of indicator to be used before actually titrating the samples. A 5 or 10 ml
rnicroburet may be conveniently used to obtain a more precise titration.
9. Calculation
9.1 Using the colorimetric procedure, calculate concentration of CN, mg/1, directly
from prepared standard curve compensating for sample dilution if less than 500
ml was used for distillation.
9.2 Using the titrimetrrc procedure, calculate concentration of CN as follows:
(A-B) 1000 250
CN, mg/1 = X
ml original sample ml of aliquot titrated
where:
A = volume of AgNO3 for titration of sample.
B = volume of AgNO3 for titration of blank.
10. Precision and Accuracy
10.1 In a single laboratory (MDQARL), using mixed industrial and domestic waste
samples at concentrations of 0.06, 0.13, 0.28 and 0.62 mg/1 CN, the standard
deviations were ±0.005, ±0.007, ±0.031, and ±0.094, respectively.
45
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10.2 In a single laboratory (MDQARL), using mixed industrial and domestic waste
samples at concentrations of 0.28 and 0.62 mg/1 CN, recoveries were 85% and
102%, respectively.
Bibliography
1. Bark, L. S., and Higson, H. G. "Investigation of Reagents for the Colorimetric
Determination of Small Amounts of Cyanide". Talanta, 2:471-479 (1964).
2. Elly, C. T. "Recovery of Cyanides by Modified Serfass Distillation". Journal Water
Pollution Control Federation, 40:848-856 (1968).
3. ASTM Standards, Part 23, Water: Atmospheric Analysis, p 498, Method D2036-72
Referee Method A (1973).
46
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ALLIHN CONDENSER
AIR INLET
-CONNECTING TUBING
ONE LITER
BOILING FLASK
SUCTION
FIGURE 1
CYANIDE DISTILLATION APPARATUS
47
-------
COOLING WATER
INLET TUBEv
HEATER-
SCREW CLAMP
J
&
TO LOW VACUUM
SOURCE
- ABSORBER
" DISTILLING FLASK
O
FIGURE 2
CYANIDE DISTILLATION APPARATUS
48
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CYANIDES, Amenable to Chlorination
STORET NO. 00722
1. Scope and Application
1.1 This method is applicable to the determination of cyanides amenable to
chlorination in drinking, surface, and saline waters, and domestic and industrial
wastes.
1.2 The titration procedure is used for measuring concentrations of cyanide exceeding
1 mg/1 after removal of the cyanides amenable to chlorination. Below this level
the colorimetric determination is used.
2. Summary of Method
2.1 A portion of the sample is chlorinated at a pH>l 1 to decompose the cyanide.
Cyanide levels in the chlorinated sample are then determined by the method for
Cyanide, Total, in this manual. Cyanides amenable to chlorination are then
calculated by difference.
3. Reagents
3.1 Calcium Hypochlorite solution: Dissolve 5 g of calcium hypochlorite (Ca(OCl)2)
in 100 ml of distilled water.
3.2 Sodium Hydroxide solution: Dissolve 50 g of sodium hydroxide (NaOH) in
distilled water and dilute to ! liter.
3.3 Ascorbic acid: crystals.
3.4 Potassium Iodide - starch test paper.
4. Procedure
4.1 Two sample aliquots are required to determine cyanides amenable to chlorination.
To one 500 ml aliquot or a volume diluted to 500 ml, add calcium hypochlorite
solution (3.1) dropwise while agitating and maintaining the pH between 11 and
12 with sodium hydroxide (3.2).
Caution: The initial reaction product of alkaline chlorination is the very toxic gas
cyanogen chloride; therefore, it is recommended that this reaction be performed
in a hood. For convenience, the sample may be agitated in a. 1 liter beaker by
means of a magnetic stirring device.
4.2 Test for residual chlorine with Kl-starch paper (3.4) and maintain this excess for
one hour, continuing agitation. A distinct blue color on the test paper indicates a
sufficient chlorine level. If necessary, add additional hypochlorite solution.
49
-------
4.3 After one hour, add 0.5 g portions of ascorbic acid (3.3) until Kl-starch paper
shows no residual chlorine. Add an additional 0.5 g of ascorbic acid to insure the
presence of excess reducing agent.
4.4 Test for total cyanide in both the chlorinated and unchlorinated aliquots as in the
method Cyanide, Total, in this manual.
5. Calculation
5.1 Calculate the cyanide amenable to chlorination as follows:
CN, mg/1 = A-B
where:
A = mg/1 total cyanide in unchlorinated aliquot
B = mg/1 total in chlorinated aliquot
Bibliography
1. ASTM Standards, Part 23, Water; Atmospheric Analysis, p 503, Method B, D2036-72
(1973).
50
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DISSOLVED OXYGEN
(Modified Winkler With Full-Bottle Technique)
STORET NO. 00300
1. Scope and Application
1.1 This method is applicable for use with most wastewaters and streams that contain
nitrate nitrogen and not more than 1 mg/1 of ferrous iron. Other reducing or
oxidizing materials should be absent. If 1 ml of fluoride solution is added before
acidifying the sample and there is no delay in titration, the method is also
applicable in the presence of 100-200 mg/1 ferric iron.
1.2 The Dissolved Oxygen (DO) Probe technique gives comparable results on all
sample types.
1.3 The azide modification is not applicable under the following conditions: (a)
samples containing sulfite, thiosulfate, polythionate, appreciable quantities of free
chlorine or hypochlorite; (b) samples high in suspended solids; (c) samples
containing organic substances which are readily oxidized in a highly alkaline
solution, or which are oxidized by free iodine in an acid solution; (d) untreated
domestic sewage; (e) biological floes; and (f) where sample color interferes with
endpoint detection. In instances where the azide modification is not applicable,
the DO probe should be used.
2. Summary of Method
2.1 The sample is treated with manganous sulfate, potassium hydroxide, and
potassium iodide (the latter two reagents combined in one solution) and finally
sulfuric acid. The initial precipitate of manganous hydroxide, Mn(OH)2, combines
with the dissolved oxygen in the sample to form a brown precipitate, manganic
hydroxide, MnO(OH)2. Upon acidification, the manganic .hydroxide forms
manganic sulfate which acts as an oxidizing agent to release free iodine from the
potassium iodine. The iodine, which is stoichiometrically equivalent to the
dissolved oxygen in the sample is then titrated with sodium thiosulfate or
phenylarsine oxide (PAO).
3. Interferences
3.1 There are a number of interferences to the dissolved oxygen test, including
oxidizing and reducing agents, nitrate ion, ferrous iron, and organic matter.
3.2 Various modifications of the original Winkler procedure for dissolved oxygen have
been developed to compensate for or eliminate interferences. The Alsterberg
51
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modification is commonly used to successfully eliminate the nitrite interference,
the Rideal-Stewart modification is designed to eliminate ferrous iron interference,
and the Theriault procedure is used to compensate for high concentration of
organic materials.
3.3 Most of the common interferences in the Winkler procedure may be overcome by
use of the dissolved oxygen probe.
4. Sample Handling and Preservation
4.1 Where possible, collect the sample in a 300 ml BOD incubation bottle. Special
precautions are required to avoid entrainment or solution of atmospheric oxygen
or loss of dissolved oxygen.
4.2 Where samples are collected from shallow depths (less than 5 feet), use of an
APHA-type sampler is recommended. Use of a Kemmerer type sampler is
recommended for samples collected from depths of greater than 5 feet.
4.3 When a Kemmerer sampler is used, the BOD sample bottle should be filled to
^
overflowing. (Overflow for approximately 10 seconds). Outlet tube of Kemmerer
should be inserted to bottom of BOD bottle. Care must be taken to prevent
turbulence and the formation of bubbles when filling bottle.
4.4 At time of sampling, the sample temperature should be recorded as precisely as
required.
4.5 Do not delay the determination of dissolved oxygen in samples having an
appreciable iodine demand or containing ferrous iron. If samples must be
preserved either method (4.5.1) or (4.5.2) below, may be employed.
4.5.1 Add 2 ml of manganous sulfate solution (6.1) and then 2 ml of alkaline
iodide-azide solution (6.2) to the sample contained in the BOD bottle.
Both reagents must be added well below the surface of the liquid.
Stopper the bottle immediately and mix the contents thoroughly. The
sample should be stored at the temperature of the collection water, or
water sealed and kept at a temperature of 10 to 20°C, in the dark.
Complete the procedure by adding 2 ml H2SO4 (see 7.1) at time of
analysis.
4.5.2 Add 0.7 ml of cone. H2SO4 (6.3) and 1 ml sodium azide solution (2 g
NaN3 in 100 ml distilled water) to the sample in the BOD bottle. Store
sample as in (4.5.1). Complete the procedure using 2 ml of manganous
sulfate solution (6.1), 3 ml alkaline iodide-azide solution (6.2), and 2 ml
of cone. H2 SO4 (6.3) at time of analysis.
4.6 If either preservation technique is employed, complete the analysis within 4-8
hours after sampling.
52
-------
5. Apparatus
5.1 Sample bottles - 300 ml ±3 ml capacity BOD incubation bottles with tapered
ground glass pointed stoppers and flared mouths.
5.2 Pipets with elongated tips capable of delivering 2.0 ml ±0.10 ml of reagent.
6. Reagents
6.1 Manganous sulfate solution: Dissolve 480 g manganous sulfate (MnSO4 -4H2O) in
distilled water and dilute to 1 liter.
6.1.1 Alternatively, use 400 g of MnSO4 -2H2O or 364 g of MnSO4 -H2O per
liter. When uncertainty exists regarding the water of crystallization, a
solution of equivalent strength may be obtained by adjusting the specific
gravity of the solution to 1.270 at 20° C.
6.2 Alkaline iodide-azide solution: Dissolve 500 g of sodium hydroxide (NaOH) or
700 g of potassium hydroxide (KOH) and 135 g of sodium iodide (Nal) or 150 g
of potassium iodide (KI) in distilled water and dilute to 1 liter. To this solution
add 10 g of sodium azide (NaN3) dissolved in 40 ml of distilled water.
6.3 Sulfuric acid: concentrated.
6.4 Starch solution: Prepare an emulsion of 10 g soluble starch in a mortar or beaker
with a small quantity of distilled water. Pour this emulsion into 1 liter of boiling
water, allow to boil a few minutes, and let settle overnight. Use the clear
supernate. This solution may be preserved by the addition of 5 ml per liter of
chloroform and storage in a 10°C refrigerator.
6.4.1 Dry, powdered starch indicators such as "thyodene" may be used in
place of starch solution.
6.5 Potassium fluoride solution: Dissolve 40 g KF«2H2O in distilled water and dilute
to 100ml.
6.6 Sodium thiosulfate, stock solution, 0.75 N: Dissolve 186.15 g Na2S2O3 -5H2O in
boiled and cooled distilled water and dilute to 1 liter. Preserve by adding 5 ml
chloroform.
6.7 Sodium thiosulfate standard titrant, 0.0375 N: Prepare by diluting 50.0 ml of
stock solution to 1 liter. Preserve by adding 5 ml of chloroform. Standard sodium
thiosulfate, exactly 0.0375 N is equivalent to 0.300 mg of DO per 1.00 ml.
Standardize with 0.0375 N potassium biiodate.
6.8 Potassium biiodate standard, 0.0375 N: For stock solution, dissolve 4.873 g of
potassium biiodate, previously dried 2 hours at 103°C, in 1000 ml of distilled
water. To prepare working standard, dilute 250 ml to 1000 ml for 0.0375 N
biiodate solution.
53
-------
6.9 Standardization of 0.0375 N sodium thidsulfate: Dissolve approximately 2 g
(±1:0 g) KI in 100 to 150 ml distilled water; add 10 ml of 10% H2SO4 followed
by 20.0 ml standard potassium biiodate (6.8). Place in dark for 5 minutes, dilute
to 300 ml, and titrate with the standard sodium thiosulfate (6.7) to a pale straw
coldr. Add 1-2 ml starch solution and continue the titration drop by drop until
the blue color disappears, kun in duplicate. Duplicate determinations should
agree within ±0.05 ml.
6.10 As an alternative to the sodium thiosulfate, phenylarsine oxide (PAO) may be
used. This is available, already standardized, from commercial sources.
7. Procedure
7.1 To the sample collected in the BOD incubation bottle, add 2 ml of the manganous
sulfate solution (6.1) followed by 2 ml of the alkaline iodide-azide solution (6.2),
well below the surface of the liquid; stopper with care to exclude air bubbles, and
mix well by inverting the bottle several times. When the precipitate settles, leaving
a clear supernatant above the manganese hydroxide floe, shake again. When
settling has produced at least 200 ml of clear supernant, carefully remove the
stopper and immediately add 2 ml of cone. H2SO4 (6.3)(sulfamic acid packets, 3
g may be substituted for H2SO4-)** > by allowing the acid to run down the neck of
the bottle, re-stopper, and mix by gentle inversion until the iodine is uniformly
distributed throughout the bottle. Complete the analysis within 45 minutes.
7.2 Transfer the entire bottle contents by inversion into a 500 ml wide mouth flask
and titrate with 0.0375 N thiosulfate solution (6.7) (0.0375 N phenyarsine oxide
(PAO) may be substituted as titrant) to a pale straw color. Add 1-2 ml of starch
solution (6.4) or 0.1 g of powdered indicator and continue to titrate to the first
disappearance of the blue color.
7.3 If ferric iron is present (100 to 200 ppm), add 1.0 ml of KF (6.5) solution before
acidification.
7.4 Occasionally, a dark brown or black precipitate persists in the bottle after
acidication. This precipitate will dissolve if the solution is kept for a few minutes
longer than usual or, if particularly persistent, a few more drops of H2 SO4 will
effect dissolution.
8. Calculation
8.1 Each rill of 0.0375 sodium thiosulfate (or PAO) titrant is equivalent to 1 mg DO
when the entire bottle contents are titrated.
8.2 If the results are desired in milliliters of oxygen gas per liter at 0°C and 760 mm
pressure, multiply mg/1 DO by 0.698.
54
-------
8.3 To express the results as percent saturation at 760 mm atmospheric pressure, the
solubility data in Table 218 (WhippJe & Whipple Table, p 480, Standard Methods,
13th Edition) may be used. Equations for correcting the solubilities to barometric
pressures other than mean sea level are given below the table.
8.4 The solubility of DO in distilled water at any barometric pressure, p (mm Hg),
temperature, T°C, and saturated vapor pressure, p. (mm Hg), for the given T, may
be calculated between the temperature of 0° and 30°C by:
(P-M) X 0.678
ml/1 DO =-
35+ T
and between 30° and 50°C by:
(P-/*) X 0.827
ml/1 DO=-
49+ T
9. Precision and Accuracy
9.1 Exact data are unavailable on the precision and accuracy of this technique;
however, reproducibility is approximately 0.2 ppm of DO at the 7.5 ppm level
due to equipment tolerances and uncompensated displacement errors.
Bibliography
1. Kroner, R. C., Longbottom, J. E., Gorman, R. A., "A Comparison of Various Reagents
Proposed for Use in the Winkler Procedure for Dissolved Oxygen", PHS Water Pollution
Surveillance System Applications and Development, Report #12, Water Quality
Section, Basic Data Branch, July 1964.
55
-------
DISSOLVED OXYGEN
(Electrode)
STORE! NO. 00299
1. Scope and Application
1.1 The probe method for dissolved oxygen is recommended for those samples
containing materials which interfere with the modified Winkler procedure such as
sulfite, thiosulfate, polythionate, mercaptans, free chlorine or hypochlorite,
organic substances readily hydrolyzed in alkaline solutions, free iodine, intense
color or turbidity, biological floes, etc.
1.2 The probe method is recommended as a substitute for the modified Winkler
procedure in monitoring of streams, lakes, outfalls, etc., where it is desired to
obtain a continuous record of the dissolved oxygen content of the water under
observation.
1.3 The probe method may be used as a substitute for the modified Winkler
procedure in BOD determinations where it is desired to perform nondestructive
DO measurements on a sample.
1.4 The probe method may be used under any circumstances as a substitute for the
modified Winkler procedure provided that the probe itself is standardized against
the Winkler method on samples free of interfering materials.
1.5 The electronic readout meter for the output from dissolved oxygen probes is
normally calibrated in convenient scale (0 to 10, 0 to 15, 0 to 20 mg/1 for
example) with a sensitivity of approximately 0.05 mg/liter.
2. Summary of Method
2.1 The most common instrumental probes for determination of dissolved oxygen in
water are dependent upon electrochemical reactions. Under steady-state condi-
tions, the current or potential can be correlated with DO concentrations.
Interfacial dynamics at the probe-sample interface are a factor in probe response
and a significant" degree of interfacial turbulence is necessary. For precision
performance, turbulence should be constant.
3. Sample Handling and Preservation
3.1 See 4ri, 4.2, 4.3, 4.4 under Modified Winkler Method.
4. Interferences
4.1 Dissolved organic materials are not known to interfere in the output from
dissolved oxygen probes.
56
-------
4.2 Dissolved inorganic salts are a factor in the performance of dissolved oxygen
probe.
4.2.1 Probes with membranes respond to partial pressure of oxygen which in
turn is a function of dissolved inorganic salts. Conversion factors for
seawater and brackish waters may be calculated from dissolved oxygen
saturation versus salinity data. Conversion factors for specific inorganic
salts may be developed experimentally. Broad variations in the kinds and
concentrations of salts in samples can make the use of a membrane probe
difficult.
4.2.2 The thallium probe requires the presence of salts in concentrations which
provide a minimum conductivity of approximately 200 micromhos.
4.3 Reactive compounds can interfere with the output or the performance of
dissolved oxygen probes.
4.3.1 Reactive gases which pass through the membrane probes may interfere.
For example, chlorine will depolarize the cathode and cause a high
probe-output. Long-term exposures to chlorine will coat the anode with
the chloride of the anode metal and eventually desensitize the probe.
Alkaline samples in which free chlorine does not exist will not interfere.
Hydrogen sulfide will interfere with membrane probes if the applied
potential is greater than the half-wave potential of the sulfide ion. If the
applied potential is less than the half-wave potential, an interfering
reaction will not occur, but coating of the anode with the sulfide of the
anode metal can take place.
4.3.2 Sulfur compounds (hydrogen sulfide, sulfur dioxide and mercaptans, for
example) cause interfering outputs from the thallium probe. Halogens do
not interfere with the thallium probe.
4.4 At low dissolved oxygen concentrations, pH variation below pH 5 and above pH 9
interfere with the performance of the thallium probe (approximately ±0.05 mg/1
DO per pH unit). The performance of membranes is not affected by pH changes.
4.5 Dissolved oxygen probes are temperature sensitive, and temperature compensa-
tion is normally provided by the manufacturer. The thallium probe has a
temperature coefficient of 1.0 mv/°C. Membrane probes have a temperature
coefficient of 4 to 6 percent/°C dependent upon the membrane employed.
5. Apparatus
5,1 No specific probe or accessory is especially recommended as superior. However,
probes which have been evaluated or are in use and found to be reliable are the
57
-------
Weston & Stack DO Analyzer Model 30, the Yellow Springs Instrument (YSI)
Model 54, and the Beckman Fieldlab Oxygen Analyzer.
6. Calibration
Follow manufacturer instructions.
7. Procedure
Follow manufacturer instructions.
8. Calculation
Follow manufacturer instructions.
9. Precision and Accuracy
Manufacturer's specification claim 0.1 mg/1 repeatability with ±1% accuracy.
58
-------
FLUORIDE, Total
(SPADNS Method with Bellack Distillation)
STORET NO. Total 00951
Dissolved 00950
1. Scope and Application
1.1 This method is applicable to the measurement of fluoride in drinking, surface, and
saline waters, domestic and industrial wastes.
1.2 The method covers the range from 0.1 to about 2.5 mg/1 F.
2. Summary of Method
2.1 Following distillation to remove interferences, the sample is treated with the
SPADNS reagent. The loss of color resulting from the reaction of fluoride with
the zirconyl-SPADNS dye is a function of the fluoride concentration.
3. Comments
3.1 The SPADNS reagent is more tolerant of interfering materials than other accepted
fluoride reagents. Reference to Table 121(1), p 169, Standard Methods for the
Examination of Waters and Wastewaters, 13th Edition, will help the analyst
decide if distillation is required. The addition of the highly colored SPADNS
reagent must be done with utmost accuracy because the fluoride concentration is
measured as a difference of absorbance in the blank and the sample. A small error
in reagent addition is the most prominent source of error in this test.
3.2 Care must be taken to avoid overheating the flask above the level of the solution.
This is done by maintaining an even flame entirely under the boiling flask.
4. Precision and Accuracy
4.1 On a sample containing 0.83 mg/1 F with no interferences, 53 analysts using the
Bellack distillation and the SPADNS reagent obtained a mean of 0.81 mg/1 F
with a standard deviation of ±0.089 mg/1.
4:2 On a sample containing 0.57 mg/1 F (with 200 mg/1 SO4 and 10 mg/1 Al as
interferences) 53 analysts using the Bellack distillation obtained a mean of 0.60
mg F/l with a standard deviation of ±0.103 mg/1.
4.3 On a sample containing 0.68 mg/1 F (with 200 mg/1 SO4 2 mg/1 Al and 2.5 mg/1
[Na(PO3)6 ] as interferences), 53 analysts using the Bellack distillation obtained a
mean of 0.72 mg/1 F with a standard deviation of ±0.092 mg/1.
(Analytical Reference Service, Sample 111-B water, Fluoride, August, 1961.)
59
-------
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods-for the Examination of Water and Wastewaters, p 171-172
(Method No. 121 A, Preliminary Distillation Step) and p 174-176 (Method 121 C,
SPADNS) 13th Edition, (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 312, Method D
1179-72, (1973).
60
-------
FLUORIDE
(Automated Complexone Method)
STORET NO. 00950
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes. The applicable range of the method is 0.05 to 1.5 mg F/l.
Twelve samples per hour can be analyzed.
1.2 For Total or Total Dissolved Fluoride, the Bellack Distillation must be performed
on the samples prior to analysis by the complexone method.
2. Summary of Method
2.1 Fluoride ion reacts with the red cerous chelate of alizarin complexone. It is unlike
other fluoride procedures in that a positive color is developed as contrasted to a
bleaching action in previous methods.
3. Sample Handling and Preservation
3.1 No special requirements.
4. Interferences
4.1 Method is free from most anionic and cationic interferences, except aluminum,
which forms an extremely stable fluoro compound, A1F6 ~3. This is overcome by
treatment with 8-hydroxyquinoline to complex the aluminum and by subsequent
extraction with chloroform. At aluminum levels below 0.2 mg/1, the extraction
procedure is not required.
5. Apparatus
5.1 Technicon AutoAnalyzer Unit consisting of:
5.1.1 Sampler I.
5.1.2 Manifold.
5.1.3 Proportioning pump.
5.1.4 Continuous filter.
5.1.5 Colorimeter equipped with 15 mm tubular flow cell and 650 nm filters.
5.1.6 Recorder equipped with range expander.
6. Reagents
6.1 Sodium acetate solution: Dissolve 272 g (2 moles) of sodium acetate in distilled
water and dilute to 1 liter.
6.2 Acetic acid-8-hydroxyquinoline solution: Dissolve 6 g of 8-hydroxyquinoline in
34 ml of cone, acetic acid, and dilute to 1 liter with distilled water.
61
-------
6.3 Chloroform: Analytical reagent grade.
6.4 Ammonium acetate solution (6.7%): Dissolve 67 g of ammonium acetate in
distilled water and dilute to 1 liter.
6.5 Hydrochloric acid (2 N): Dilute 172 ml of cone. HC1 to 1 liter.
6.6 Lanthanum alizarin fluoride blue solution*1): Dissolve 0.18 g of alizarin fluoride
blue in a solution containing 0.5 ml of cone, ammonium hydroxide and 15 ml of
6.7% ammonium acetate (6.4). Add a solution that contains 41 g of anhydrous
sodium carbonate and. 70 ml of glacial acetic acid in 300 ml of distilled water.
Add 250 ml of acetone. Dissolve 0.2 g of lanthanum oxide in 12.5 ml of 2 N
hydrochloric acid (6.5) and mix with above solution. Dilute to 1 liter.
6.7 Stock solution: Dissolve 2.210 g of sodium fluoride in 100 ml of distilled water
and dilute to 1 liter in a volumetric flask. 1.0 ml = 1.0 mg F.
6.8 Standard Solution: Dilute 10.0 ml of stock solution to 1 liter in a volumetric
flask. 1.0 ml =0.01 mg F.
6.8.1 Using standard solution, prepare the following standards in 100 ml
volumetric flasks:
mgF/1 ml Standard Solution/100 ml
0.05 0.5
0.10 1.0
0.20 2.0
0.40 4.0
0.60 6.0
0.80 8.0
1.00 10.0
1.20 12.0
1.50 15.0
7. Procedure
7.1 Set up manifold as shown in Figure 1.
7.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain stable baseline.
7.3 Place distilled water wash tubes in alternate openings in Sampler and set sample
timing at 2.5 minutes.
62
-------
7.4 Arrange fluoride standards in Sampler in order of decreasing concentration.
Complete loading of Sampler tray with unknown samples.
7.5 Switch sample line from distilled water to Sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed fluoride standards
against concentration values. Compute concentration of samples by comparing
sample peak heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.06, 0.15, and 1.08 mg F/l, the standard deviation was ±0.018.
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.14 and 1.25 mg F/l, recoveries were 89% and 102%, respectively.
Bibliography
1. J. T. Baker Laboratory Chemical No. Jl 12 or equivalent.
2. Greerihaigh, R., and Riley, J. P., "The Determination of Fluorides in Natural Waters,
with Particular Reference to Sea Water". Anal. Chim. Acta, 25, 179 (1961).
3. Chan, K. M., and Riley, J. P., "The Automatic Determination of Fluoride in Sea Water
and Other Natural Waters". Anal. Chim. Acta, 35, 365 (1966).
63
-------
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FIGURE 1. FLUORIDE MANIFOLD AA-I
-------
FLUORIDE
(Electrode)
STORE! NO: Total 00951
Dissolved 00950
1. Scope and Application
1.1 This method is applicable to the measurement of fluoride in drinking, surface, and
saline waters, domestic and industrial wastes.
1.2 Concentration of fluoride from 0.1 up to 1000 mg/liter may be measured.
1.3 For Total or Total Dissolved Fluoride, the Bellack distillation must be performed
on the samples prior to electrode analysis.
2. Summary of Method
2.1 The fluoride is determined potentiometrically using a selective ion fluoride
electrode in conjunction with a standard single junction sleeve-type reference
electrode and a pH meter having an expanded millivolt scale or a selective ion
meter having a direct concentration scale for fluoride.
2.2 The fluoride electrode consists of a lanthanum fluoride crystal across which a
potential is developed by fluoride ions. The cell may be represented by Ag/Ag Cl,
Q- (0.3), F- (0.001) LaF/test solution/SCE/.
3. Interferences
3.1 Extremes of^pH interfere; sample pH should be between 5 and 9. Polyvalent
cations of Si + 4, Fe + 3 and Al + 3 interfere by forming complexes with fluoride.
The degree of interference depends upon the concentration of the complexing
cations, the concentration of fluoride and the pH of the sample. The addition of a
pH 5.0 buffer (described below) containing a strong, chelating agent preferentially
complexes aluminum (the most common interference), silicon, and iron and
eliminates the pH problem.
4. Sampling Handling and Preservation
4.1 No special requirements.
5; Apparatus
5.1 Electrometer, (pH meter), with expanded mv scale, or a selective ion meter such as
the Orion 400 Series.
5.2 Fluoride Ion Activity Electrode, such as Orion No. 94-09O ).
5.3 Reference electrode, single junction, sleeve-type, such as Orion No. 90-01,
Beckman No. 40454, or Corning No. 476010.
5.4 Magnetic Mixer, Teflon-coated stirring bar.
65
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6. Reagents
6.1 Buffer solution, pH 5.0-5.5: To approximately 500 ml of distilled water in a 1
liter beaker add 57 ml of glacial acetic acid, 58 g of sodium chloride and 2 g of
CDTA<2). Stir to dissolve and cool to room temperature. Adjust pH of solution to
between 5.0 and 5.5 with 5 N sodium hydroxide (about 150 ml will be required).
Transfer solution to a 1 liter volumetric flask and dilute to the mark with distilled
water. For work with brines, additional NaCl should be added to raise the
chloride level to twice the highest expected level of chloride in the sample.
6.2 Sodium fluoride, stock solution: 1.0 ml = 0.1 mg F. Dissolve 0.2210 g of sodium
fluoride in distilled water and dilute to 1 liter in a volumetric flask. Store in
chemical-resistant glass or polyethylene.
6.3 Sodium fluoride, standard solution: 1.0 ml = 0.01 mg F. Dilute 100.0 ml.of
sodium fluoride stock solution (6.2) to 1000 ml with distilled water.
7. Calibration
7.1 Prepare a series of standards using the fluoride standard solution (6.3) in the range
of 0 to 2.00 mg/1 by diluting appropriate volumes to 50.0 ml. The following series
may be used:
Milimeters of Standard Concentration when Diluted
(1.0 ml = 0.01 mg/F) to 50 ml, mg F/liter
0.00 ^ 0.00
1.00 0.20
2.00 0.40
3.00 0.60
4.00 0.80
5.00 1.00
6.00 1.20
- 8.00 ' 1.60
10.00 2.00
7.2 Calibration of Electrometer: Proceed as described in (8.1). Using semilogarithmic
graph paper, plot the concentration of fluoride in mg/liter on the log axis vs. the
electrode potential developed in the standard on the linear axis, starting with the
lowest concentration at the bottom of the scale. Calibration of a selective ion
meter: Follow the directions of the manufacturer for the operation of the
instrument.
66
-------
8. Procedure
8.1 Place 50.0 ml of sample or standard solution and 50.0 ml of buffer (See Note) in
a 150 ml beaker. Place on a magnetic stirrer and mix at medium speed. Immerse
the electrodes in the solution and observe the meter reading while mixing. The
electrodes must remain in the solution for at least three minutes or until the
reading has stabilized. At concentrations under 0.5 mg/liter F, it may require as
long as five minutes to reach a stable meter reading; higher concentrations
stabilize more quickly. If a pH meter is used, record the potential measurement
for each unknown sample and convert the potential reading to the fluoride ion
concentration of the unknown using the standard curve. If a selective ion meter is
used, read the fluoride level in the unknown sample directly in mg/1 on the
fluoride scale.
.NOTE: For industrial waste samples, this amount of buffer may not be adequate.
Analyst should check pH first. If highly basic (>11), add 1 N HC1 to adjust pH to
8.3.
9. Precision and Accuracy
9.1 A synthetic sample prepared by the Analytical Reference Service, PHS, containing
0.85 mg/1 fluoride and no interferences was analyzed by 111 analysts; a mean of
0.84 mg/1 with a standard deviation of ±0.03 was obtained.
9.2 On the same study, a synthetic sample containing 0.75 mg/1 fluoride, 2.5 mg/1
polyphosphate and 300 mg/1 alkalinity, was analyzed by the same 111 analysts; a
mean of 0.75 mg/1 fluoride with a standard deviation of ±0.036 was obtained.
Bibliography
1. Patent No. 3,431,182 (March 4, 1969).
2. CDTA is the abbreviated designation of 1, 2-cyclohexylene dinitrilo tetraacetic acid,
(Mathieson, Coleman & Bell, Cat. No. P8661) or cyclohexane diamine tetraacetic acid
(Merck-Titriplex IV or Baker Cat. No. G083).
3. Standard Methods for the Examination of Water and Wastewaters, p 171, Method No.
121A, Preliminary Distillation Step (Bellack), 13th Edition, 1971.
67
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HARDNESS, Total
STORET NO. 00900
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The method is suitable for all concentration ranges of hardness; however, in order
to avoid large titration volumes, use a sample aliquot containing not more than 25
mg CaCO3.
1.3 Automated titration may be used.
2. Summary of Method
2.1 Calcium and magnesium ions in the sample are sequestered upon the addition of
disodium ethylenediamine tetraacetate (Na2EDTA). The end point of the
reaction is detected by means of Calmagite Indicator, which has a red color in the
presence of calcium and magnesium and a blue color when the cations are
sequestered.
3. Comments
3.1 Excessive amounts of heavy -metals can interfere. This is usually overcome by
complexing the metals with cyanide.
3.1.1 Routine addition of sodium cyanide solution (Caution: deadly poison)
to prevent potential metallic interference is recommended.
4. Precision and Accuracy
4.1 Forty-three analysts in nineteen laboratories analyzed six synthetic water
samples containing exact increments of calcium and magnesium salts, with the
following results:
Increment as
Total Hardness
mg/liter, CaCO3
31
33
182
194
417
444
Precision as
Standard Deviation
mg/liter, CaCO3
2.87
2.52
4.87
2.98
9.65
8.73
Accuracy as
Bias, Bias,
% mg/liter, CaCO3
-0.87
-0.73
-0.19
-1.04
-3.35
-3.23
-0.003
-0.24
-0.4
-2.0
-13.0
-14.3
68
-------
(FWPCA Method Study 1, Mineral and Physical Analyses)
4.2 In a single laboratory (MDQARL), using surface water samples at an average con-
centration of 194 mg CaCO3/l, the standard deviation was ±3.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
179, Method 122B (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 168, Method
Dl 126-67 (1973).
69
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HARDNESS, Total
(Automated)
STORET NO. 00900
1. Scope and Application
1.1 This automated method is applicable to drinking, surface, and saline waters. The
applicable range is 10 to 400 mg/1 as CaCO3. Approximately 12 samples per hour
can be analyzed.
2. Summary of Method
2.1 The magnesium EDTA exchanges magnesium on an equivalent basis for any
calcium and/or other cations to form a more stable EDTA chelate than
magnesium. The free magnesium reacts with calmagite at a pH of 10 to give a
red-violet complex. Thus, by measuring only magnesium concentration in the
final reaction stream, an accurate measurement of total hardness is possible.
3. Sample Handling and Preservation
3.1 No special requirements.
4. Interferences
4.1 No significant interferences.
5. Apparatus
5.1 Technicon Auto Analyzer consisting of:
5.1.1 Sampler I.
5.1.2 Continuous Filter.
5.1.3 Manifold.
5.1.4 Proportioning Pump.
5.1.5 Colorimeter equipped with 15 mm tubular flow cell and 520 nm filters.
5.1.6 Recorder equipped with range expander.
6. Reagents
6.1 Buffer: Dissolve 67.6 g NH4C1 in 572 ml of N^OH and dilute to 1 liter with
distilled water.
6.2 Calmagite Indicator: Dissolve 0.25 g in 500 ml of distilled water by stirring
approximately 30 minutes on a magnetic stirrer. Filter.
6.3 Magnesium ethylenediamine-tetraacetate (MgEDTA): Dissolve 0.2 g of MgEDTA
in 1 liter of distilled water.
6.4 Stock Solution: Weigh 1.000 g of calcium carbonate (pre-dried at 105°C) into
500 ml Erlenmeyer flask; add 1:1 HC1 until all CaCO3 has dissolved. Add 200 ml
70
-------
of distilled water and boil for a few minutes. Cool, add a few drops of methyl red
indicator, and adjust to the orange color with 3N NH4OH and dilute to 1000ml
with distilled water. 1.0 ml = 1.0 mg CaCO3.
6.4.1 Dilute each of the following volumes of stock solutions to 250 ml in a
volumetric flask for appropriate standards:
Stock Solution, ml CaCO3, mg/1
2.5 10.0
5.0 20.0
10.0 40.0
15.0 60.0
25.0 100.0
35.0 140.0
50.0 200.0
75.0 300.0
100.0 400.0
7. Procedure
7.1 Set up manifold as shown in Figure 1.
7.2 Allow both colorimeter and recorder to warm up for 30:minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain stable baseline.
7.3 Place distilled water wash tubes in alternate openings in Sampler and set sample
timing at 2.5 minutes.
7.4 Arrange working standards-in Sampler in order of decreasing concentrations.
Complete loading of Sampler tray with unknown samples.
7.5 Switch sample line from distilled water to Sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed standards against
concentration values. Compute concentration of samples by comparing sample
peak heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 19, 120, 385, and 366 mg/1 as CaCO3, the standard deviations were ±1.5, ±1.5,
±4.5, and ±5.0, respectively.
71
-------
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 39 and 296 mg/1 as CaCO3, recoveries were 89% and 93%, respectively.
Bibliography
1. Technicon Auto Analyzer Methodology, Bulletin No. 2, Technicon Controls, Inc.,
Chauncey, New York (July 1960).
2. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 179,
Method 122B(1971).
72
-------
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FIGURE 1. HARDNESS MANIFOLD AA-I
-------
IODIDE
(Titrimetric)
STORETNO. 71865
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, sewage and
industrial waste effluents.
1.2 The concentration range for this method is 2-20 mg/1 of iodide.
2. Summary of Method
2.1 After pretreatment to remove interferences, the sample is analyzed for iodide by
converting the iodide to iodate with bromine water and titrating with phenylar-
sine oxide (PAO) or sodium thiosulfate.
3. Sample Handling and Preservation
3.1 Store at 4°C and analyze as soon as possible.
4. Interferences
4.1 Iron, manganese and organic matter can interfere; however, the calcium oxide
pretreatment removes or reduces these to insignificant concentrations.
4.2 Color interferes with the observation of indicator and bromine-water color
changes. This interference is eliminated by the use of a pH meter instead of a pH
indicator and the use of standardized amounts of bromine water and sodium
formate solution instead of observing the light yellow color changes.
5. Reagents
5.1 Acetic Acid Solution (1:8): Mix 100 ml of glacial acetic acid with 800 ml of
distilled water.
5.2 Bromine Water: In a fume hood, add 0.2 ml bromine to 500 ml distilled water.
Stir with a magnetic stirrer and a Teflon-coated stirring bar for several hours or
until the bromine dissolves. Store in a glass-stoppered colored bottle.
5.3 Calcium Oxide (CaO): Anhydrous, powdered.
5.4 Potassium Iodide (KI): Crystals, ACS Reagent Grade.
5.5 Sodium Acetate Solution (275 g/1): Dissolve 275 g of sodium acetate trihydrate
(NaC2 H3 O2 -3H2 O) in distilled water. Dilute to 1 liter and filter.
5.6 Sodium Formate Solution (500 g/1): Dissolve 50 g of sodium formate (NaCHO2)
in hot distilled water and dilute to 100 ml.
5.7 Nitrogen Gas: cylinder.
5.8 Sulfuric Acid Solution (1:4): Slowly add 200 ml of H2SO4 (sp. gr. 1.84) to 800
ml of distilled water.
74
-------
5.9 Phenylarsine Oxide (0.0375 N): Hach Chemical Co. or equivalent. Standardize
with 0.0375 N potassium biiodate (5.15, 5.18).
5.10 Phenylarsine Oxide Working Standard (0.0075 N): Transfer 100 ml of commer-
cially available 0.0375 N phenylarsine oxide (5.9) to a 500 ml volumetric flask
and dilute to the mark with distilled water. This solution should be prepared fresh
daily.
5.11 Amylose Indicator: Mallinckrodt Chemical Works or equivalent.
5.12 Sodium Thiosulfate, Stock Solution, 0.75 N: Dissolve 186.15 g (Na2S2 O3
. 5H2O) in boiled and cooled distilled water and dilute to 1.0 liter. Preserve by
adding 5 ml chloroform.
5.13 Sodium Thiosulfate Standard Titrant, 0.0375 N: Prepare by diluting 50.0 ml of
stock solution to 1.0 liter. Preserve by adding 5 ml of chloroform. Standardize
with 0.0375 N potassium biiodate (5.15, 5.18).
5.14 Sodium Thiosulfate Working Standard (0.0075 N): Transfer 100 ml of sodium
thiosulfate standard titrant (5.13) to a 500 ml volumetric flask and dilute to the
mark with distilled water. This solution should be prepared fresh daily.
5.15 Potassium Biiodate Standard, 0.0375 N: Dissolve 4.387 g potassium biiodate,
previously dried 2 hours at 103°C, in distilled water and dilute to 1.0 liter. Dilute
250 ml to 1.0 liter for 0.0375 N biiodate solution.
5.16 Starch Solution: Prepare an emulsion of 10 g of soluble starch iri a mortar or
beaker with a small quantity of distilled water. Pour this emulsion into 1 liter of
boiling water, allow to boil a few minutes, and let settle overnight. Use the clear
supernate. This solution may be preserved by the addition of 5 ml per liter of
chloroform and storage in a 10°C refrigerator. Commercially available, powdered
starch indicators may be used in place of starch solution.
5.17 Potassium Fluoride (KF-2H2O): ACS Reagent Grade
5.18 Standardization of 0.0375 N Phenylarsine Oxide and 0.0375 N sodium
thiosulfate: Dissolve approximately 2 g (±1.0 g) KI (5.4) in 100 to 150 ml
distilled water; add 10 ml H2SO4 solution (5.8) followed by 20 ml standard
potassium biiodate solution (5.15). Place in dark for 5 minutes, dilute to 300 ml
and titrate with phenylarsine oxide (5.9) or sodium thiosulfate standard titrant
(5.13) to a pale straw color. Add a small scoop of indicator (5.11). Wait until
homogeneous color develops and continue the titration drop by drop until the
blue color disappears. Run in duplicate. Duplicate determinations should agree
within ±0.05 ml.
75
-------
6. Procedure
6.1 Pretreatment
6.1.1 Add a visible excess of CaO (5.3) to 400 ml of sample. Stir or shake
vigorously for approximately 5 minutes. Filter through a dry, moderate-
ly retentive filter paper, discarding the first 75 ml.
6.2 Iodide Determination
6.2.1 Place 100 ml of pretreated sample (6.1) or a fraction thereof diluted to
that volume, into a 150 ml beaker. Add a Teflon-coated stirring bar and
place on a magnetic stirrer. Insert a pH electrode and adjust the pH to
approximately 7 or slightly less by the dropwise addition of H2SO4
solution (5.8).
6.2.2 Transfer the sample to a 250 ml wide-mouthed conical flask. Wash
beaker with small amounts of distilled water and add washings to the
flask.
NOTE: A 250 ml iodine flask would increase accuracy and precision by
preventing possible loss of the iodine generated upon addition of
potassium iodide and sulfuric acid (6.3.1).
6.2.3 Add 15 ml sodium acetate solution (5.5) and 5 ml acetic acid solution
(5.1). Mix well. Add 40 ml bromine water solution (5.2); mix well. Wait
5 minutes.
6.2.4 Add 2 ml sodium formate solution (5.6); mix well. Wait 5 minutes.
6.2.5 Purge the space above the sample with a gentle stream of nitrogen (5.7)
for approximately 30 seconds to remove bromine fumes.
6.2.6 If a precipitate forms (iron), add 0.5 g KF-2H2O (5.17).
6.2.7 A distilled water blank must be run with each set of samples because of
iodide in reagents. If a blank is consistently shown to be zero for a
particular "lot" of chemicals it can then be ignored.
6.3 Titration
6.3.1 Dissolve approximately 1 g potassium iodide (5.4) in sample. Add 10 ml
of H2 SO4 solution (5.8) and place in dark for 5 minutes.
6.3.2 Titrate with phenylarsine oxide working standard (5.10) or sodium
thiosulfate working standard solution (5.14) adding indicator (5.11 or
5.15) as end point is approached (light straw color). Titrate to colorless
solution, Disregard returning blue color.
7. Calculations r- -,
I-(mg/l)=21,150 U^ 1
76
-------
ml = the number of ml of PAO need to titrate the sample.
N = the normality of the PAO used to titrate the sample.
V = the volume of sample taken (100 ml or a fraction thereof)
21,150 was calculated from the number of equivalents of iodine produced when the
potassium iodide was added and from the rearrangement of the equation to produce
the value in terms of mg/1.
8. Precision and Accuracy
8.1 In a single laboratory (MDQARL), using a mixed domestic and industrial waste
effluent, at concentrations of 1.6, 4.1, 6.6, 11.6 and 21.6 mg/1 of iodide, the
standard deviations were ±0.23, ±0.17, ±0.10, ±0.06 and ±0.50 mg/1, respective-
ly.
8.2 In a single laboratory (MDQARL), using a mixed domestic and industrial waste
effluent at concentrations of 4.1, 6.6, 11.6 and 21.6 mg/1 of iodide, recoveries
were 80, 97, 97, and 92%, respectively.
Bibliography
1. ASTM Standards, Part 23, Water; Atmospheric Analysis, p 331-333, Method D1246 C
(1973).
77
-------
METALS
(Atomic Absorption Methods)
1. Scope and Application
1.1 Metals in solution may be readily determined 'by atomic absorption spectroscopy.
The method is simple, rapid, and applicable to a large number of metals in
drinking, surface, and saline waters, and domestic and industrial wastes. While
drinking waters may be analyzed directly, domestic and industrial wastes require
processing to solubilize suspended material. Sludges, sediments and other solid
type samples may also be analyzed after proper pretreatment.
1.2 Detection limits, sensitivity and optimum ranges of the metals will vary with the
various makes and models of satisfactory atomic absorption spectrophotometers.
The data shown in Table 1, however, provide some indication of the actual
concentration ranges measurable with conventional atomization. In the majority
of instances the concentration range shown in the table may be extended much
lower with scale expansion and conversely extended upwards by using a less
sensitive wavelength or by rotating the burner 90 degrees. Detection limits may
also be extended through concentration of the sample, through solvent extraction
techniques and/or the use of the so called furnace techniques. The latter includes
the heated graphite atomizer, the carbon rod and the tantalum strip accessories.
When using furnace techniques, however, the analyst should be cautioned as to
possible chemical reactions occurring at elevated temperatures which may result
in either suppression or enhancement of the analysis element. Methods of
standard addition are mandatory with these furnace techniques to insure valid
data.
1.3 Where conventional, atomic absorption techniques do not provide adequate
sensitivity, reference is made to colorimetric or specialized procedures. Examples
of these specialized techniques would be the gaseous hydride method for arsenic
and selenium and the cold vapor technique for mercury.
1.4 Atomic absorption procedures are provided as the methods of choice; however,
other instrumental methods have also been shown to be capable of producing
precise and accurate analytical data. These instrumental techniques include
emission spectroscopy, X-ray fluorescence, spark source mass spectroscopy, and
anodic stripping to name but a few. The analyst should be cautioned that these
methods are highly specialized techniques requiring a high degree of skill to
interpret results and obtain valid data. (
78
-------
TABLE 1
Atomic Absorption Concentration Ranges With
Conventional Atomization***
Metal
Aluminum
Antimony
Arsenic*
Barium
Beryllium
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Mot-/^ <-<>**
""eTCUijr
Molybdenum
Nickel
Potassium
Selenium*
Silver
Sodium
Thallium
Tin
Titanium
Vanadium
Zinc
Detection
Limit
mg/1
0.1
0.2
0.002
0.03
0.005
0.002
0.003
0.02
0.03
0.01
0.02
0.05
0.0005
0.01
0.0002
0.1
0.02
0.005
0.002
0.01
0.002
0.1
0.8
0.3
0.2
0.005
Sensitivity
mg/1
1
0.5
0.4
0.025
0.025
0.08
0.1
0.2
0.1
0.12
0.5
0.007
0.05
0.3
0.15
0.04
0.06
0.015
0.5
4
2
0.8
O.Q2
Optimum
Concentration
Range
mg/1
5
1
0.002
1
0.05
0.05
0.2
0.2
0.5
Q.2
0.3
1
0.02
0.1
0.0002 -
0.5
0.3
0.1
0.002
0.1
0.03
1
10
5
1
0.05
100
40
0.02
20
2
2
20
10
10
10
10
20
2
10
0.01
20
10
2
0.02
4
1
20
200
100
100
2
*Gaseous hydride method.
**Cold vapor technique.
***The concentrations shown above are not contrived values
conventional aspiration on any satisfactory atomic absorption
79
and should be obtainable with
spectrophotometer.
-------
2. Summary of Method
2.1 Atomic absorption spectroscopy is similar to flame emission photometry in that a
sample is atomized and aspirated into a flame. Flame photometry, however,
measures the amount of light emitted, whereas, in atomic absorption spectro-
photometry a light beam is directed through the flame into a monochromator,
and onto a detector that measures the amount of light absorbed. In many
instances absorption is more sensitive because it depends upon the presence of
free unexcited atoms and generally the ratio of unexited to excited atoms at a
given moment is very high. Since the wavelength of the light beam is characteristic
of only the metal being determined, the light energy absorbed by the flame is a
measure of the concentration of that metal in the sample. This principle is the
basis of atomic absorption spectroscopy.
2.2 Although methods have been reported for the analysis of solids by atomic
absorption spectroscopy (Spectrochim Acta, 24B 53, 1969) the technique
generally is limited to metals in solution or solubilized through some form of
sample processing.
2.2.1 Preliminary treatment of wastewater and/or industrial effluents is usually
necessary because of the complexity and variability of the sample
matrix. Suspended material must be solubilized through some form of
digestion. This may vary because of the metals to be determined but
generally will include a wet digestion with nitric acid.
2.2.2 In those instances where complete characterization of a sample is
desired, the suspended material must be analyzed separately. This may
be accomplished by filtration and acid digestion of the suspended
material. Metallic constituents in this acid digest are subsequently
determined and the sum of the dissolved plus suspended concentrations
will then provide the total concentrations present. The sample should be
filtered as soon as possible after collection and the filtrate acidified
immediately.
2.2.3 The total sample may also be treated with acid before filtration to
measure what may be termed "extractable" concentrations.
3. Definition of Terms
3.1 Sensitivity: The concentration in milligrams of metal per liter that produces an
absorption of 1%.
3.2 Detection Limit: The concentration that produces absorption equivalent to twice
the magnitude of the fluctuation in the background (zero absorption).
80
-------
3.3 Dissolved Metals: Those constituents (metals) which will pass through a 0.45 \i
membrane filter.
3.4 Suspended Metals: Those constituents (metals) which are retained by a 0.45 /x
membrane filter.
3.5 Total Metals: The concentration of metals determined on an unfiltered sample
following vigorous digestion (Section 4.1.3), or the sum of the concentrations of
metals in both the dissolved and suspended fractions.
3.6 Extractable Metals: The concentration of metals in an unfiltered sample following
treatment with hot dilute mineral acid (Section 4.1.4).
4. Sample Handling and Preservation
4.1 For the determination of trace metals, contamination and loss are of prime
concern. Dust in the laboratory environment, impurities in reagents and
impurities on laboratory apparatus which the sample contacts are all sources of
potential contamination. For liquid samples, containers can introduce either
positive or negative errors in the measurement of trace metals by (a) contributing
contaminants through leaching or surface desorption and (b) by depleting
concentrations through adsorption. Thus the collection and treatment of the
sample prior to analysis requires particular attention. The sample bottle should be
thoroughly washed with detergent and tap water; rinsed with 1:1 nitric acid, tap
water, 1:1 hydrochloric acid, tap water and finally deionized distilled water in
that order.
NOTE 1: Chromic acid may be useful to remove organic deposits from glassware;
however, the analyst should be cautioned that the glassware must be thoroughly
rinsed with water to remove the last traces of chromium. This is especially
important if chromium is to be included in the analytical scheme. Chromic acid
f
should not be used with plastic bottles.
Before collection of the sample a decision must be made as to the type of data
desired, i.e., dissolved, suspended, total or extractable.
4.1.1 For the determination of dissolved constituents the sample must be
filtered through a 0.45 fi membrane filter as soon as practical after
collection. (Glass or plastic filtering apparatus are recommended to avoid
possible contamination.) Use the first 50-100 ml to rinse the filter flask.
Discard this portion and collect the required volume of filtrate. Acidify
the filtrate with 1:1 redistilled HNO3 to a pH of 2. Normally, 3 ml of
(1:1) acid per liter should be sufficient to preserve the sample (See Note
81
-------
2). Analyses performed on a sample so treated shall be reported as
"dissolved" concentrations.
NOTE 2: It has been suggested (International Biological Program,
Symposium on Analytical Methods, Amsterdam, Oct. 1966) that
additional acid, as much as 25 ml of cone. HCl/liter, may be required to
stabilize certain types of highly buffered samples if they are to be stored
for any length of time. Therefore, special precautions should be observed
for preservation and storage of unusual samples intended for metal
analysis.
4.1.2 For the determination of suspended metals a representative volume of
unpreserved sample must be filtered through a 0.45 n membrane filter.
When considerable suspended material is present, as little as 100 ml of a
well mixed sample is filtered.
Record the volume filtered and transfer the membrane filter containing
the insoluble material to a 250 ml Griffin beaker and add 3 ml cone.
redistilled HNO3. Cover the beaker with a watch glass and heat gently.
The warm acid will soon dissolve the membrane. Increase the tempera-
ture of the hot plate and digest the material. When the acid has
evaporated, cool the beaker and watch glass and add another 3 ml of
cone, redistilled HNO 3.
Cover and continue heating until the digestion is complete, generally
indicated by a light colored residue. Add distilled 1:1 HC1 (2 ml) to the
dry residue and again warm the beaker gently to dissolve the material.
Wash down the watch glass and beaker walls with deionized distilled
water and filter the sample to remove silicates and other insoluble
material that could clog the atomizer. Adjust the volume to some
predetermined value based on the expected concentrations of metals
present. This volume will vary depending on the metal to be determined.
The sample is now ready for analysis. Concentrations so determined shall
be reported as "suspended".
4.1.3 For the determination of total metals the sample is acidified with 1:1
redistilled HNO3 to a pH of 2 at the time of collection. The sample is
not filtered before processing. Choose a volume of sample appropriate
for the expected level of metals. If much suspended material is present,
as little as 50-100 ml of well mixed sample will most probably be
82
-------
sufficient. (The sample volume required may also vary proportionally
with the number of metals to be determined).
Transfer a representative aliquot of the well mixed sample to a Griffin
beaker and add 3 ml of cone, redistilled HNO3. Place the beaker on a hot
plate and evaporate to dryness cautiously, making certain that the
sample does not boil. Cool the beaker and add another 3 ml portion of
cone, redistilled HNO3. Cover the beaker with a watch glass and return
to the hot plate. Increase the temperature of the hot plate so that a
gentle reflux action occurs. Continue heating, adding additional acid as
necessary, until the digestion is complete (generally indicated by a light
colored residue). Add sufficient distilled 1:1 HC1 and again warm the
beaker to dissolve the residue. Wash down the beaker walls and watch
glass with distilled water and filter the sample to remove silicates and
other insoluble material that could clog the atomizer. Adjust the volume
to some predetermined value based on the expected metal concentra-
tions. The sample is now ready for analysis. Concentrations so
determined shall be reported as "total" (See Note 3). STORET
parameter numbers for reporting this type of data have been assigned
and are given for each metal.
NOTE 3: Certain metals such as titanium, silver, mercury, and arsenic
require modification of the digestion procedure and the individual sheets
for these metals should be consulted.
4.1.4 To determine metals soluble in hot, dilute, HC1 HNO3, acidify the
entire sample at the time of collection with cone, redistilled HNO3, 5
ml/1. At the time of analysis a 100 ml aliquot of well mixed sample is
transferred to a beaker or flask. Five ml of distilled HC1 (1:1) is added
and the sample heated for 15 minutes at 95°C on a steam bath or hot
plate. After this treatment the sample is filtered and the volume adjusted
to 100 ml. The sample is then ready for analysis.
The data so obtained are significant in terms of "total" metals in the
sample, with the reservation that something less than "total" is probably
measured. Concentrations of metal found, especially in heavily silted
samples, will be substantially higher than data obtained on only the
soluble fraction. STORET parameter numbers for the storage of this
/
type data are not available at this time.
83
-------
5. Interferences
5.1 The most troublesome type of interference in atomic absorption spectrophoto-
metry is usually termed "chemical" and is caused by lack of absorption of atoms
bound in molecular combination in the flame. This phenomenon can occur when
the flame is not sufficiently hot to dissociate the molecule, as in the case of
phosphate interference with magnesium, or because the dissociated atom is
immediately oxidized to a compound that will not dissociate further at the
temperature of the flame. The addition of lanthanum will overcome the
phosphate interference in the magnesium, calcium and barium determinations.
Similarly, silica interference in the determination of manganese can be eliminated
by the addition of calcium.
5.1.1 Chemical interferences may also be eliminated by separating the metal
from the interfering material. While complexing agents are primarily
employed to increase the sensitivity of the analysis, they may also be
used to eliminate or reduce interferences.
5.2 The presence of high dissolved solids in the sample may result in an interference
from non-atomic absorbance such as light scattering. If background correction is
not available, a non-absorbing wavelength should be checked. Preferably, high
solids type samples should be extracted (See 5.1.1 and 9.2).
5.3 lonization interferences occur where the flame temperature is sufficiently high to
generate the removal of an electron from a neutral atom, giving a positively
charged ion. This type of interference can generally be controlled by the addition,
to both standard and sample solutions, of a large excess of an easily ionized
element.
5.4 Spectral interference can occur when an absorbing wavelength of an element
present in the sample but not being determined falls within the width of the
absorption line of the element of interest. The results of the determination will
then be erroneously high, due to the contribution of the interfering element to
the atomic absorption signal. Spectral interference may sometimes be reduced by
narrowing the slit width.
6. Apparatus
6.1 Atomic absorption spectrophotometer: Any commercial atomic absorption
/
instrument having an energy source, an atomizer burner system, a mono-
chroma tor, and a detector is suitable.
6.2 Burner: The burner recommended by the particular instrument manufacturer
should be used. For certain elements the nitrous oxide burner is required.
84
-------
6.3 Separatory flasks: 250 ml, or larger, for extraction with organic solvents,
6.4 Glassware: All glassware, including sample bottles, should be washed with
detergent, rinsed with tap water, 1:1 nitric acid, tap water, 1:1 hydrochloric acid,
tap water and deionized distilled water in that order. (jSee Note 1 under (4.1)
concerning the use of chromic acid.]
6.5 Borosilicate glass distillation apparatus.
7. Reagents
7.1 Deionized distilled water: Prepare by passing distilled water through a mixed bed
of cation and anion exchange resins. Use deionized distilled water for the
preparation of all reagents, calibration standards, and as dilution water.
7.2 Nitric acid (cone.): If metal impurities are found to be present, distill reagent
grade nitric acid in a borosilicate glass distillation apparatus. Prepare a 1:1
dilution with deionized distilled water.
Caution: Distillation should be performed in hood with protective sash in place.
7.3 Hydrochloric acid (1:1): Prepare a 1:1 solution of reagent grade hydrochloric acid
and deionized distilled water. If metal impurities are found to be present, distill
this mixture from a borosilicate glass distillation apparatus.
7.4 Stock metal solutions: Prepare as directed in (8.1) and under the individual metal
procedures. Commercially available stock standard solutions may also be used.
7.5 Standard metal solutions: Prepare a series of standards of the metal by dilution of
the appropriate stock metal solution to cover the concentration range desired.
7.6 Fuel and oxidant: Commercial grade acetylene is generally acceptable. Air may be
supplied from a compressed air line, a laboratory compressor, or from a cylinder
of compressed air.. Reagent grade nitrous oxide is also required for certain
determinations.
7.7 Special reagents for the extraction procedure.
7.7.1 Pyrrolidine dithiocarbamic acid (PDCA): Prepare by adding 18 ml of
analytical reagent grade pyrrolidine to 500 ml of chloroform in a liter
flask. Cool and add 15 ml of carbon disiilfide in small portions and with
swirling. Dilute to 1 liter with chloroform. The solution can be used for
several months if stored in a brown bottle in a refrigerator.
7.7.2 Ammonium hydroxide, 2N: Dilute 3 ml cone. NH4OH to 100 ml with
deionized distilled water.
7.7.3 Bromphenol blue indicator.
7.7.4 HC1: Dilute 2 ml redistilled HC1 to 40ml with deionized distilled water.
85
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8. Preparation of Standards and Calibration
8.1 Stock solutions are prepared from high purity metals, oxides or nonhygroscopic
reagent grade salts using redistilled nitric or hydrochloric acids. Sulfuric or
phosphoric acids should be avoided as they produce an adverse effect on many
elements. The stock solutions are prepared at concentrations of 1000 mg of the
metal per liter.
8.2 Standard solutions are prepared by diluting the stock metal solutions at the time
of analysis. For best results, calibration standards should be prepared fresh each
time an analysis is to be made and discarded after use. Prepare a blank and
calibration standards in graduated amounts in the appropriate range. The
calibration standards should be prepared using the same type of acid (HC1, HNO3
or H2 SO4) and at the same concentration as will result in the samples following
processing. As filtered water samples are preserved with 1:1 redistilled HNQ3 (3
ml per liter), calibration standards for these analyses should be similarly prepared
with HNO3. Samples processed for suspended metals (4.1.2) or total metals
(4.1.3) should be analyzed using calibration standards prepared in HC1. Beginning
with the blank and working toward the highest standard, aspirate the solutions
and record the readings. Repeat the operation with both the calibration standards
and the samples a sufficient number of times to secure a reliable average reading
for each solution.
8.3 Where the sample matrix is so complex that viscosity, surface tension and
components cannot be accurately matched with standards, the method of
standard addition must be used. This technique relies on the addition of small,
known amounts of the analysis element to portions of the sample the
absorbance difference between those and the original solution giving the slope of
the calibration curve. The method of standard addition is described in greater
detail in (8.5).
8.4 For those instruments which do not read out directly in concentration, a
calibration curve is prepared to cover the appropriate concentration range.
Usually, this means the preparation of standards which produce an absorption of
0 to 80 percent. The correct method is to convert the percent absorption readings
to absorbance and plot that value against concentration. The following relation-
ship is used to convert absorption values to absorbance:
absorbance = log (100/%T) = 2 - log % T
where % T = 100 - % absorption
86
-------
As the curves are frequently nonlinear, especially at high absorption values, the
number of standards should be increased in that portion of the curve.
8.5 Standard Addition Method: In this method, equal volumes of sample are added to
a deionized distilled water blank and to three standards containing different
known amounts of the test element. The volume of the blank and the standards
must be the same. The absorbance of each solution is determined and then
plotted on the vertical axis of a graph, with the concentrations of the known
standards plotted on the horizontal axis. When the resulting line is extrapolated
back to zero absorbance, the point of interception of the abscissa is the
concentration of the unknown. The abscissa on the left of the ordinate is scaled
the same as on the right side, but in the opposite direction from the ordinate. An
example of a plot so obtained is shown in Fig. 1.
87
-------
0
o
s
.0
Zero
Absorponce
Concentration
I Cone, of
Sample
Addn 0
No Addn
Addn I
Addn of 50%
of Expected
Amount
Addn 2
Addn of 100%
of Expected
Amount
Addn 3
Addn of 150%
of Expected
Amount
FIGURE 1. STANDARD ADDITION PLOT
88
-------
9. General Procedure for Analysis by Atomic Absorption
9.1 Differences between the various makes and models of satisfactory atomic
absorption spectrophotometers prevent the formulation of detailed instructions
applicable to every instrument. The analyst should follow the manufacturer's
operating instructions for his particular instrument. In general, after choosing the
proper hollow cathode lamp for the analysis, the lamp should be allowed to warm
up for a minimum of 15 minutes. During this period, align the instrument,
position the monochromator at the correct wavelength, select the proper
monochromator slit width, and adjust the hollow cathode current according to
the manufacturer's recommendation. Subsequently, light the flame and regulate
the flow of fuel and oxidant, adjust the burner and nebulizer flow rate for
maximum percent absorption and stability, and balance the photometer. Run a
series of standards of the element under analysis and construct working curves by
plotting the concentrations of the standards against the absorbance. For those
instruments which read directly in concentration set the curve corrector to read
out the proper concentration. Aspirate the samples and determine the concentra-
tions either directly or from the calibration curve. For best results run standards
each time a sample or series of samples are run.
9.2 Special Extraction Procedure: When the concentration of the metal is not
sufficiently high to determine directly, or when considerable dissolved solids are
present in the sample, certain of the metals may be chelated and extracted with
organic solvents. Ammonium pyrrolidine dithiocarbamate (APDC) in methyl
isobutyl ketone (MIBK) is widely used for this purpose and is particularly useful
for zinc, cadmium, iron, manganese, copper, silver, lead and chromium*6.
Tri-valent chromium does not react with APDC unless it has first been converted
to the hexavalent form [Atomic Absorption Newsletter 6, p 128 (1967)].
Aluminum, beryllium, barium and strontium also do not react with APDC. While
the APDC-MIBK chelating-solvent system can be used satisfactorily, it is possible
to experience difficulties. Also, when multiple metals are to be determined either
larger sample volumes must be extracted or individual extractions made for each
metal being determined. The acid form of APDC-pyrrolidine dithiocarbamic acid
prepared directly in chloroform as described by Lakanen, [Atomic Absorption
Newsletter 5, p 17 (1966)], has been found to be most advantageous. It is
very stable and may be stored in a brown bottle in the refrigerator for months.
Because chloroform is used as the solvent, it may not be aspirated into the flame.
The following procedure is suggested.
89
-------
9.2.1 Extraction Procedure with pyrrolidine dithiocarbamic acid (PDCA) in
chloroform.
a. Transfer 200 ml of sample into a 250 ml separately funnel,
add 2 drops bromphenol blue indicator solution (7.7.3) and
mix.
b. Prepare a blank and sufficient standards in the same manner
and adjust the volume of each to approximately 200 ml
with deiomzed distilled water. All of the metals to be
determined may be combined into single solutions at the
appropriate concentration levels.
c. Adjust the pH by addition of 2N NH4OH solution (7.7.2)
until a blue color persists. Add HC1 (7.7.4) dropwise until
the blue color just disappears; then add 2.0 ml HC1 (7.7.4)
in excess. The pH at this point should be 2.3. (The pH
adjustment may be made with a pH meter instead of using
indicator).
d. Add 5 ml of PDCA-chloroform reagent (7.7.1) and shake
vigorously for 2 minutes. Allow the phases to separate and
drain the chloroform layer into a 100 ml beaker.
e. Add a second portion of 5 ml PDCA-chloroform reagent
(7.7.1) and shake vigorously for 2 minutes. Allow the
phases to separate and combine the chloroform phase with
that obtained in step (d).
f. Determine the pH of the aqueous phase and adjust to 4.5.
g. Repeat step (d) again combining the solvent extracts.
h. Readjust the pH to 5.5, extract, readjust to 6.5 and extract
a fifth time. Combine all extracts and evaporate to dryness
pn a steam bath.
i. Hold the beaker at a 45 degree angle, and slowly add 2 ml
of cone, distilled nitric acid, rotating the beaker to effect
thorough contact of the acid with the residue.
j. Place the beaker on a low temperature hotplate and
evaporate just to dryness.
k. Add 2 ml of nitric acid (1:1) to the beaker and heat for 1
minute. Cool, quantitatively transfer the solution to a 10 ml
volumetric flask and bring to volume with distilled water.
The sample is now ready for analysis.
90
-------
9.3 General-purpose electrically heated devices (flarrieless atomization) have recently
been introduced as a means of extending detection limits. These techniques are
generally acceptable but the analyst should be cautioned as to possible
suppression or enhancement effects. With flameless atomization, background
correction becomes of high importance. This is because certain samples, when
atomized, may absorb or scatter light from the hollow cathode lamp. It can be
caused by the presence of gaseous molecular species,.salt particles, or smoke in
the sample beam. If no correction is made, sample absorbance will be greater than
it should be, and the analytical result will be erroneously high.
10. Calculation
10.1 Direct determination of liquid samples: Read the metal value in mg/1 from the
calibration curve or directly from the readout system of the instrument.
10.1.1 If dilution of sample was required:
mg/1 metal in sample = (mg/1 of metal in the diluted aliquot) X D
/ ml of \ /ml of deionized \
I I + I )
\ aliquot / \ distilled water /
where D =
ml of aliquot
10.2 For samples containing particulates:
AXB
mg/1 metal in sample =
C
where:
A = mg/1 of metal in processed sample
B = final volume of processed sample in ml
C = volume of sample aliquot processed in ml
10.3 For solid samples: Report all concentrations as mg/kg dry weight.
10.3.1 Dry sample
(mg/1 of constituent \ / volume of prepared \
in prepared sample/ \ sample in ml /
mg/kg =
weight of dry sample in g
10.3.2 Wet sample
/mg/1 of constituent^ /volume of prepared\
\in prepared sample / \ sample in ml /
mg/kg =
(weight of wet sample in g) X (% solids)
91
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ALUMINUM
(Standard Conditions)
STORET NO. Total 01105
Optimum Concentration Range: 5-100 mg/1 using a wavelength of 309.2 nm
Sensitivity: 1.0 mg/1
Detection Limit: 0.1 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.000 gram of aluminum metal (analytical
reagent grade). Add 15 ml of cone. HC1 to the metal in a covered beaker and
warm gently. When solution is complete, transfer quantitatively to a 1 liter
volumetric flask and make up to volume with deionized distilled water. 1 ml = 1
mgAl( 1000 mg/1).
2. Potassium Chloride Solution: Dissolve 95 g potassium chloride (KC1) in deionized
distilled water and make up to 1 liter.
3. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3 ) and at the same concentration as the samples for analysis.
To each 100 ml of standard and sample alike add 2.0 ml potassium chloride
solution.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Aluminum hollow cathode lamp
2. Wavelength: 309.2 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous oxide
5. Type of flame: Fuel rich
Interferences
1. Aluminum is partially ionized in the nitrous oxide-acetylene flame. This problem
may be controlled by the addition of an alkali metal (potassium, 1000 Mg/ml) to
both sample and standard solutions.
92
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Notes
1. The following lines may also be used:
308.2 nm Relative Sensitivity 1
396.2 nm Relative Sensitivity 2
394.4 nm Relative Sensitivity 2.5 .
2. Data-to be entered into STORET must be reported as ng/\.
3. For concentrations of aluminum below 0.3 mg/1, the Eriochrome cyanine R
method may be used (Standard Methods, 13th Edition, p 57).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for aluminum were as follows:
Number
of Labs
38
38
37
37
22
21
True values
Mg/Hter
1205
1004
500
625
35
15
Mean Value
jug/liter
1281
1003
463
582
96
109
Standard
Deviation
jug/liter
299
391
202
272
108
168
Accuracy as
% Bias
6.3
-0.1
-7.4
-6.8
175
626
93
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ANTIMONY
(Standard Conditions)
STORETNO. Total 01097
Optimum Concentration Range: 1-40 mg/1 using a wavelength of 217.6 nm
Sensitivity: 0.5 mg/1
Detection Limit: 0.2 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 2.7426 g of antimony potassium tartrate
(analytical reagent grade) and dissolve in deionized distilled water. Dilute to 1
liter with deionized distilled water. 1 ml = 1 mg Sb (1000 mg/1).
2. Prepare dilutions .of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Antimony hollow cathode lamp
2. Wavelength: 217.6 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Fuel lean
Interferences
1. In the presence of lead (1000 mg/1), a spectral interference may occur at the 217.6
nm resonance line. In this case the 231.1 nm antimony line should be used.
2. Increasing acid concentrations decrease antimony absorption. To avoid this effect,
the acid concentration in the samples and in the standards should be matched.
Notes
1. Data to be entered into STORET must be reported as Aig/1.
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste effluent
at concentrations of 5.0 and 15 mg Sb/1, the standard deviations were ±0.08 and
±0.1, respectively. Recoveries at these levels were 96% and 97%, respectively.
94
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ARSENIC
(Gaseous Hydride Method)
STORET NO. Total 01002
1. Scope and Application
1.1 The gaseous hydride method determines inorganic arsenic when present in
concentrations at or above 2 jug/1. The method is applicable to most fresh and
saline waters in the absence of high concentrations of chromium, cobalt, copper,
mercury, molybdenum, nickel and silver.
2. Summary of Method
2.1 Arsenic in the sample is first reduced to the trivalent form using SnCl2 and
converted to arsine, AsH3, using zinc metal. The gaseous hydride is swept into an
argon-hydrogen flame of an atomic absorption spectrophotometer. The working
range of the method is 2-20 jug/1. The 193.7 nm wavelength line is used.
3. Comments
3.1 In analyzing most surface and ground waters, interferences are rarely en-
countered. Industrial waste samples should be spiked with a known amount of
arsenic to establish adequate recovery.
3.2 Organic forms of arsenic must be converted to inorganic compounds and organic
matter must be oxidized before beginning the analysis. The oxidation procedure
given in Standard Methods, 13th Edition, Method 104B, p 65, Procedure 4.a has
been found suitable.
3.3 The silver diethyldithiocarbamate colorimetric procedure may also be used
(Standard Methods, 13th Edition, p 62) with the digestion described in (3.2).
1-Ephedrine in chloroform has been found to be a suitable solvent for silver
diethyldithiocarbamate if the analyst finds the odor of pyridine objectionable
[Anal. Chem. 45, 1786(1973)].
3.4 Data to be entered into STORET must be reported as /ug/1.
4. Precision and Accuracy
4.1 Ten replicate solutions of o-arsenilic acid at the 5, 10 and 20 /ug/1 level were
analyzed by a single laboratory (Caldwell, et. al.). Standard deviations were ±0.3,
±0.9 and ±1.1 with recoveries of 94, 93 and 85%, respectively.
95
-------
Bibliography
1. Caldwell, J. S., Lishka, R. J., and McFarren, E. F., "Evaluation of a Low Cost Arsenic
and Selenium Determination at Microgram per Liter Levels", JAWWA., vol 65, p 731,
Nov., 1973.
96
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BARIUM
(Standard Conditions)
STORET NO. Total 01007
Optimum Concentration Range: 1-20 mg/1 using a wavelength of 553.6 nm
Sensitivity: 0.4 mg/1
Detection Limit: 0.03 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.7787 g barium chloride (BaCl2-2H2O, analytical
reagent grade) in deionized distilled water and dilute to 1 liter.
1 ml = 1 mg Ba (1000 mg/1).
2. Potassium chloride solution: Dissolve 95 g potassium chloride, KC1, in deionized
distilled water and make up to 1 liter.
3. Prepare dilutions of the stock barium solution to be used as calibration standards
at the time of analysis. To each 100 ml of standard and sample alike add 2.0 ml
potassium chloride solution. The calibration standards should be prepared using
the same type of acid (HC1 or HNO3) and at the same concentration as the
samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Barium hollow cathode lamp
2. Wavelength: 553.6 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous oxide
5. Type of flame: Fuel rich
Interferences
1. The use of a nitrous oxide-acetylene flame virtually eliminates chemical
interference; however, barium is easily ionized in this flame and potassium must
be added (1000 mg/1) to standards and samples alike to control this effect.
2. If the nitrous oxide flame is not available and acetylene-air is used, phosphate,
silicon and aluminum will severely depress the barium absorbance. This may be
overcome by the addition of 2000 mg/1 lanthanum.
97
-------
Notes
1. Data to be entered into STORET must be reported as jug/1.
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.40 and 2.0 mg Ba/1, the standard deviations were
±0.043 and ±0.13, respectively. Recoveries at these levels were 94% and 113%,
respectively.
98
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BERYLLIUM
(Standard Conditions)
STORETNO. Total 01012
Optimum Concentration Range: 0.05-2 mg/1 using a wavelength of 234.9 nm
Sensitivity: 0.025 mg/1
Detection Limit: 0.005 mg/1
Preparation of Standard Solution
1. Stock solution: Dissolve 11.6586 g beryllium sulfate,BeSO4,in deionized distilled
water containing 2 ml cone, nitric acid and dilute to 1 liter. 1 ml = 1 mg Be (1000
mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Beryllium hollow cathode lamp
2. Wavelength: 234.9 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous oxide
5. Type of flame: Fuel rich
Interferences:
1. Sodium and silicon at concentrations in excess of 1000 mg/1 have been found to.
severely depress the beryllium absorbance.
2. Bicarbonate ion is reported to interfere; however, its effect is eliminated when
samples are acidified to a pH of 1.5.
3. Aluminum at concentrations of >500 jug/1 is reported to depress the sensitivity of
beryllium [Spectrochim Acta 22, 1325 (1966)].
Notes
1. Data to be entered into STORET must be reported as //g/1.
2. The "aluminon method" may also be used (Standard Methods, 13th Edition, p
67).
99
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Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.01, 0.05 and 0.25 mg Be/1, the standard deviations
were ±0.001, ±0.001 and ±0.002, respectively. Recoveries at these levels were
100%, 98% and 97%, respectively.
100
-------
CADMIUM
(Standard Conditions)
STORET NO. Total 01027
Optimum Concentration Range: 0.05-2 mg/1 using a wavelength of 228.8 nm
Sensitivity: 0.025 mg/1
Detection Limit: 0.002 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 2.282 g of cadmium sulfate (CdSO4-8H2O,
analytical reagent grade) and dissolve in deionized distilled water. 1 ml = 1 mg Cd
(1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Cadmium hollow cathode lamp
2. Wavelength: 228.S nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. For levels of cadmium below 20 M§/1> the extraction procedure is recommended.
2. Data to be entered into STORET must be reported as jug/1-
3. The dithizone colorimetric procedure may be used (Standard Methods, 13th
Edition, p 422).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for cadmium were as follows:
101
-------
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs jug/liter jug/liter Mg/liter % Bias
74 71 70 21 -2.2
73 78 74 18 -5.7
63 14 16.8 11.0 19.8
68 18 18.3 10.3 1.9
55 1.4 3.3 5.0 135
51 2.8 2.9 2.8 4.7
102
-------
CALCIUM
(Standard Conditions)
STORET NO. Total 00916
Optimum Concentration Range: 0.2-20 mg/1 using a wavelength of 422.7 niii
Sensitivity: 0.08 mg/1
Detection Limit: 0.003 mg/1
Preparation of Standard Solution
1. Stock Solution: Suspend 1.250 g of CaCO3 (analytical reagent grade), dried at
180°C for 1 hour before weighing, in deionized distilled water and dissolve
cautiously with a minimum of dilute HC1. Dilute to 1000 ml with deionized
distilled water. 1 ml = 0.5 mg Ca (500 mg/1).
2. Lanthanum chloride solution: Dissolve 29 g of La2O3, slowly and in small
portions, in 250 ml cone. HC1 (Caution: Reaction is violent) and dilute to 500 ml
with deionized distilled water.
. 3. Prepare dilutions of the stock calcium solutions to be used as calibration
standards at the time of analysis. To each calibration standard solution, add 1.0
mi of LaCl3 solution for each 10 ml of volume of working standard, ie., 20 ml
working standard + 2 ml LaCl3 = 22 ml.
Sample Preparation
1. For the analysis of total calcium in domestic and industrial effluents, the
procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
2. For ambient waters, a representative aliquot of a well-mixed sample may be used
directly for analysis. If suspended solids are present in sufficient amounts to clog
the nebulizer, the sample may be allowed to settle and the supernatant liquid
analyzed directly.
Instrumental Parameters (General)
1. Calcium hollow cathode lamp
2. Wavelength: 422.7 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Reducing
103
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Notes
1. Phosphate, sulfate and aluminum interfere but are masked by the addition of
lanthanum. Since low calcium values result if the pH of the sample is above 7,
both standards and samples are prepared in dilute hydrochloric acid solution.
Concentrations of magnesium greater than 1000 mg/1 also cause low calcium
values. Concentrations of up to 500 mg/1 each of sodium, potassium and nitrate
cause no interference.
2. Anionic chemical interferences can be expected if lanthanum is not used in
samples and standards.
3. The nitrous oxide-acetylene flame will provide two to five times greater sensitivity
and freedom from chemical interferences. lonization interferences should be
controlled by adding a large amount of alkali to the sample and standards. The
analysis appears to be free from chemical suppressions in the nitrous oxide-
acetylene flame.
4. The 239.9 nm line may also be used. This line has a sensitivity of 20 mg/1.
5. Data to be entered into STORET must be reported as mg/1.
6. The EDTA titrimetric method may be used (Standard Methods, 13th Edition, p
84).
Precision and Accuracy
1. In a single laboratory (MDQARL), using distilled water at concentrations of 9.0
and 36 mg/1, the standard deviations were ±0.3 and ±0.6, respectively. Recoveries
at both these levels were 99%.
104
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CHROMIUM
(Standard Conditions)
STORET NO. Total 01034
Optimum Concentration Range: 0.2-10 mg/1 using a wavelength of 357.9 nm
Sensitivity: 0.1 mg/1
Detection Limit: 0.02 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.923 g of chromium trioxide (CrO3, reagent grade) in
deionized distilled water. When solution is complete, acidify with redistilled
HNO3 and dilute to 1 liter with deionized distilled water. 1 ml = 1 mgCr(1000
mg/1).
2, Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
i. Chromium hollow cathode lamp
2. Wavelength: 357.9 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Slightly fuel rich
Notes
1. The following wavelengths may also be used:
360.5 nm Relative Sensitivity 1.2
359.3 nm Relative Sensitivity 1.4
425.4 nm Relative Sensitivity 2
427.5 nm Relative Sensitivity 3
428.9 nm Relative Sensitivity 4
2. The nitrous oxide-acetylene flame provides greater sensitivity and freedom from
chemical interference.
105
-------
3. The absorption of chromium is suppressed by iron and nickel. If the analysis is
performed in a lean flame the interference can be lessened but the sensitivity will
also be reduced. The interference does not exist in a nitrous oxide-acetylene
flame.
4. For levels of chromium below 50 jug/1, the extraction procedure is recommended.
Only hexavalent chromium will react with APDC, thus, to measure trivalent
chromium an oxidation step must be in eluded. [Atomic Absorption Newsletter, 6,
p 128(1967)].
5. Data to be entered into STORET must be reported as jug/1.
6. The diphenylcarbazide colorimetric procedure may be used (Standard Methods,
13th Edition, p 426).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for chromium were as follows:
Number
of Labs
74
76
72
70
47
47
True Values
Mg/liter
370
407
74
93
7.4
15.0
Mean Value
Mg/liter
353
380
72
84
10.2
16.0
Standard
Deviation
Mg/liter
105
128
29
35
7.8
9.0
Accuracy as
%Bias
-4.5
-6.5
-3.1
-10.2
37.7
6.8
106
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COBALT
(Standard Conditions)
STORE! NO.Total 01037
Optimum Concentration Range: 0.5-10 mg/1 using a wavelength of 240.7 nm
Sensitivity: 0.2 mg/1
Detection Limit: 0.03 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 4.307 g of cobaltous chloride, CoCl2'6H2O (analytical
reagent grade), in deionized distilled water. Add 10 ml of concentrated nitric acid
and dilute to 1. liter with deionized distilled water. 1 ml = 1 mg Co (1000 mg/1).
2. Prepare dilutions of the stock cobalt solution to be used as calibration standards
at the time of analysis. The calibration standards should be prepared using the
same type of acid (HC1 or HNO3) and at the same concentration as the samples
for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Cobalt hollow cathode lamp
2. Wavelength: 240.7 nm
3. Fuel: Acetylene'
4. Oxidant: Air
5. Type of Flame: Stoichiometric
Notes
1. For levels of cobalt below 50 jug/1, the extraction procedure is recommended.
2. Data to be entered into STORET must be reported as Mg/1-
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.20, 1.0 and 5.0 mg Co/1, the standard deviations
were ±0.013, ±0.1 and ±0.05, respectively. Recoveries at these levels were 98%,
98% and 97%, respectively.
107
-------
COPPER
(Standard Conditions)
STORETNO. Total 01042
Optimum Concentration Range: 0.2-10 mg/1 using a wavelength of 324.7 nm
Sensitivity: 0.1 mg/1
Detection Limit: 0.01 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.00 g of electrolyte copper (analytical reagent
grade). Dissolve in 5 ml redistilled HNO3 and make up to 1 liter with deionized
distilled water. Final concentration is 1 mg Cu per ml (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Copper hollow cathode lamp
2. Wavelength: 324.7 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. For levels of copper below 20 jug/1, the extraction procedure is recommended.
2. Copper atoms are distributed over a wider area in laminar flow-flames than that
normally found. Consequently, the burner parameters are not as critical as for
most other elemental determinations.
3. Because of the spectral intensity of the 324.7 nm line, the P.M. tube may become
saturated. If this situation occurs the current should be decreased.
4. Numerous absorption lines are available for the determination of copper. By
selecting a suitable absorption wavelength, copper samples may be analyzed over a
very wide range of concentration. The following lines may be used:
327.4 nm Relative Sensitivity 2
108
-------
217.8 nm Relative Sensitivity 4
216.5 nm Relative Sensitivity 7
218.1 nm Relative Sensitivity 9
222.5 nm Relative Sensitivity 20
249.2 nm Relative Sensitivity 90
5. Data to be entered into STORET must be reported as Mg/1-
6. The 2,9-dimethyl-l, 10-phenanthroline colorimetric method may be used (Stand-
ard Methods, 13th Edition, p. 430).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural -vater
samples. The statistical results for copper were as follows:
Number
of Labs
91
92
86
84
66
66
True Values
Mg/liter
302
332
60
75
7.5
12.0
Mean Value
jug/liter
305
324
64
76
9.7
13.9
Standard
Deviation
jug/liter
56
56
23
22
6.1
9.7
Accuracy as
%Bias
0.9
-2.4
7.0
1.3
29.7
15.5
109
-------
IRON
(Standard Conditions)
STORETNO. Total 01045
Optimum Concentration Range: 0.3-10 mg/1 using a wavelength of 248.3 nm
Sensitivity: 0.12 mg/1
Detection Limit: 0.02 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.000 g of pure iron wire (analytical reagent
grade) and dissolve in 5 ml redistilled HNO3, warming if necessary. When solution
is complete make up to 1 liter with deionized distilled water. 1 ml = 1 mg Fe
(1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Iron hollow cathode lamp
2. Wavelength: 248.3 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. The following lines may also be used:
248.8 nm Relative Sensitivity 2
271.9 nm Relative Sensitivity 4
302.1 nm Relative Sensitivity 5
252.7 nm Relative Sensitivity 6
372.0 nm Relative Sensitivity 10
386.0 nm Relative Sensitivity 20
344.1'nm Relative Sensitivity 30
2. Absorption is strongly dependent upon the lamp current.
110
-------
3. Better signal-to-noise can be obtained from a neon-filled hollow cathode lamp
than an argon filled lamp.
4. Data to be reported into STORET must be reported as ^g/1.
5. The 1,10-phenanthroline colorimetric method may be used (Standard Methods,
13th Edition, p 433).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for iron were as follows:
Number
of Labs
82
85
78
79
57
C/l
«/-r
True Values
Mg/liter
840
700
350
438
24
10
Mean Value
jug/liter
855
680
348
435
58
48
Standard
Deviation
Mg/liter
173
178
131
183
69
69
Accuracy as
% Bias
1.8
-2.8
-0.5
-0.7
141
382
111
-------
LEAD
(Standard Conditions)
STORE! NO. Total 01051
Optimum Concentration Range: 1-20 mg/1 using a wavelength of 283.3 nm
Sensitivity: 0.5 mg/1
Detection Limit: 0.05 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.599 g of lead nitrate, Pb(NO3)2 (analytical
reagent grade), and dissolve in deionized distilled water. When solution is
complete acidify with 10 ml redistilled HNO3 and dilute to 1 liter with deionized
distilled water. 1 ml = 1 mg Pb (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Lead hollow cathode lamp
2. Wavelength: 283.3 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Slightly oxidizing
Notes
1. The analysis of this metal is exceptionally sensitive to turbulence and absorption
bands in the flame. Therefore, some care should be taken to position the light
beam in the most stable, center portion of the flame. To do this, first adjust the
burner to maximize the absorbance reading with a lead standard. Then, aspirate a
water blank and make minute adjustments in the burner alignment to minimize
the signal.
2. For levels of lead below 100 Mg/1, the extraction procedure is recommended. The
optimum pH for the extraction of lead is 2.8.
112
-------
3. The following lines may also be used:
217.0 nm Relative Sensitivity 0.4
261.4 nm Relative Sensitivity 30
4. Data to be entered into STORET must be reported as
5. The dithizone cqlorimetric method may be used (Standard Methods, 13th
Edition, p 436).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for lead were as follows:
Number
of Labs
True Values
Mg/liter
Mean Value
Mg/liter
Standard
Deviation
Mg/liter
Accuracy as
%Bias
74
74
64
64
61
60
367
334
101
84
37
25
377
340
101
85
41
31
128
111
46
40
25
22
2.9
1.8
-0.2
1.1
9.6
25.7
113
-------
MAGNESIUM
(Standard Conditions)
STORET NO. Total 00927
Optimum Concentration Range: 0.02-2 mg/1 using a wavelength of 285.2 nm
Sensitivity: 0.007 mg/1
Detection Limit: 0.0005 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 0.829 g of magnesium oxide, MgO (analytical reagent
grade), in 10 ml of redistilled HNO3 and dilute to 1 liter with deionized distilled
water. 1 ml = 0.50 mg Mg (500 mg/1).
2. Lanthanum chloride solution: Dissolve 29 g of La2O3, slowly and in small
portions in 250 ml cone. HC1, (Caution: Reaction is violent) and dilute to 500 ml
with deionized distilled water.
3. Prepare dilutions of the stock magnesium solution to be used as calibration
standards at the time of analysis. To each calibration standard solution, add 1.0
ml of LaCl3 solution for each 10 ml of volume of working standard, ie., 20 ml
working standard + 2 ml LaCl3 = 22 ml.
Sample Preparation
1. For the analysis of total magnesium in domestic and industrial effluents, the
procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
2. For ambient waters, a representative aliquot of a well-mixed sample may be used
directly for analysis. If suspended solids are present in sufficient amounts to clog
the nebulizer, the sample may be allowed to settle and the supernatant liquid
analyzed directly.
3. Samples should be preserved with (1:1) nitric acid to a pH of 2 at the time of
collection.
Instrumental Parameters (General)
1. Magnesium hollow cathode lamp
2. Wavelength: 285.2 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Reducing
114
-------
Notes
1. Analytical sensitivity decreases with increased lamp current.
2. The interference caused by aluminum at concentrations greater than 2 mg/1 is
masked by addition of lanthanum. Sodium, potassium and calcium cause no
interference at concentrations less than 400 mg/1.
3. Because of the spectral intensity of the 285.2 nm line, the P.M. tube may become
saturated. If this situation occurs, the current should be decreased.
4. The following line may also be used:
202.5 nm Relative Sensitivity 25
5. To cover the range of magnesium values normally observed in surface waters
(0.1-20 mg/1), it is suggested that the burner be rotated 55°.
6. Data to be entered into STORET must be reported as mg/1.
7. The gravimetric method may be used (Standard Methods, 13th Edition, p 201).
Precision and Accuracy
1. In a single laboratory (MDQARL), using a distilled water sample at concentrations
of 2.1 and 8.2 mg/1, the standard deviations were ±0.1 and ±0.2, respectively.
Recoveries at both of these levels were 100%.
115
-------
MANGANESE
(Standard Conditions)
STORETNO. Total 01055
Optimum Concentration Range: 0.1-10 mg/1 using a wavelength of 279.5 nm
Sensitivity: 0.05 mg/1
Detection Limit: 0.01 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.000 g of manganese metal (analytical reagent
grade) and dissolve in 10 ml of redistilled HNO3. When solution is complete dilute
to 1 liter with 1%(V/V)HC1. 1 ml= 1 mg Mn (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Manganese hollow cathode lamp
2. Wavelength: 279.5 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. For levels of manganese below 25 Mg/1, the extraction procedure is recommended.
The extraction is carried out at a pH of 4.5 to 5. The manganese chelate is very
unstable and the analysis must be made without delay to prevent its re-solution in
the aqueous phase.
2. Analytical sensitivity is somewhat dependent on lamp current.
3. The following line may also be used:
403.1 nm Relative Sensitivity 10.
4. Data to be entered into STORET must be reported as Mg/1-
116
-------
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum,
cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for manganese were as follows:
Number
of Labs
True Values
Mg/Hter
Mean Value
Mg/Hter
Standard
Deviation
jug/liter
Accuracy as
%Bias
77
78
71
70
55
55
426
469
84
106
11
17
432
474
86
104
21
21
70
97
26
31
27
20
1.5
1.2
2.1
-2.1
93
22
117
-------
MERCURY IN WATER
(Manual Cold Vapor Technique)
STORET NO. Total 71900
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 In addition to inorganic forms of mercury, organic mercurials may also be
present. These organo-mercury compounds will not respond to the flameless
atomic absorption technique unless they are first broken down and converted to
mercuric ions. Potassium permanganate oxidizes many of these compounds, but
recent studies have shown that a number of organic mercurials, including phenyl
mercuric acetate and methyl mercuric chloride, are only partially oxidized by this
reagent. Potassium persulfate has been found to give approximately 100%
recovery when used as the oxidant with these compounds. Therefore, a persulfate
oxidation step following the addition of the permanganate has been included to
insure that organomercury compounds, if present, will be oxidized to the
mercuric ion before measurement. A heat step is required for methyl mercuric
chloride when present in or spiked to a natural system. For distilled water the
heat step is not necessary.
1.3 The range of the method may be varied through instrument and/or recorder
expansion. Using a 100 ml sample, a detection limit of 0.2 jug Hg/1 can be
achieved; concentrations below this level should be reported as <0.2 (see
Appendix 11.2).
2. Summary of Method
2.1 The flameless AA procedure is a physical method based on the absorption of
radiation at 253.7 nm by mercury vapor. The mercury is reduced to the elemental
state and aerated from solution in a closed system. The mercury vapor passes
through a cell positioned in the light path of an atomic absorption spectro-
photometer. Absorbance (peak height) is measured as a function of mercury
concentration and recorded in the usual manner.
3. Sample Handling and Preservation
3.1 Until more conclusive data are obtained, samples should be preserved by
acidification with nitric acid to a pH of 2 or lower immediately at the time of
collection. If only dissolved mercury is to be determined, the sample should be
118
-------
filtered through an all glass apparatus before the acid is added. For total mercury
the filtration is omitted.
4. Interference
4.1 Possible interference from sulfide is eliminated by the addition of potassium
permanganate. Concentrations as high as 20 mg/1 of sulfide as sodium sulfide do
not interfere with the recovery of added inorganic mercury from distilled water.
4.2 Copper has also been reported to interfere; however, copper concentrations as
high as 10 mg/1 had no effect on recovery of mercury from spiked samples.
4.3 Sea waters, brines and industrial effluents high in chlorides require additional
permanganate (as11 much as 25 ml). During the oxidation step, chlorides are
converted to free chlorine which will also absorb radiation at 253 nm. Care must
be taken to assure that free chlorine is absent before the mercury is reduced and
swept into the cell. This may be accomplished by using an excess of
hydroxylamine sulfate reagent (25 ml). In addition, the dead air space in the BOD
bottle must be purged before the addition of stannous sulfate. Both inorganic and
organic mercury spikes have been quantitatively recovered from sea water using
this technique.
4.4 Interference from certain volatile organic materials which will absorb at this
wavelength is also possible. A preliminary run without reagents should determine
if this type of interference is present (see Appendix 11.1).
5. Apparatus
5.1 Atomic Absorption Spectrophotometer: (See Note 1) Any atomic absorption unit
having an open sample presentation area in which to mount the absorption cell is
suitable. Instrument settings recommended by the particular manufacturer should
be followed. Note 1: Instruments designed specifically for the measurement of
mercury using the cold vapor technique are commercially available and may be
substituted for the atomic absorption Spectrophotometer.
5.2 Mercury Hollow Cathode Lamp: Westinghouse WL-22847, argon filled, or
equivalent.
5.3 Recorder: Any multi-range variable speed recorder that is compatible with the UV
detection system is suitable.
5.4 Absorption Cell: Standard Spectrophotometer cells 10 cm long, having quartz end
windows may be used. Suitable cells may be constructed from plexiglass tubing,
1" O.D. X 4-1/2". The ends are ground perpendicular to the longitudinal axis and
quartz windows (1" diameter X 1/16" thickness) are cemented in place. Gas inlet
and outlet ports (also of plexiglass but 1/4" O.D.) are attached approximately
119
-------
1/2" from each end. The cell is strapped to a burner for support and aligned in the
light beam by use of two 2" by 2" cards. One inch diameter holes are cut in the
middle of each card; the cards are then placed over each end of the cell. The cell is
then positioned and adjusted vertically and horizontally to give the maximum
transmittance.
5.5 Air Pump: Any peristaltic pump capable of delivering 1 liter of air per minute
may be used. A Masterflex pump with electronic speed control has been found to
be satisfactory.
5.6 Flowmeter: Capable of measuring an air flow of 1 liter per minute.
5.7 Aeration Tubing: A straight glass frit having a coarse porosity. Tygon tubing is
used for passage of the mercury vapor from the sample bottle to the absorption
cell and return.
5.8 Drying Tube: 6" X 3/4" diameter tube containing 20 g of magnesium perchlorate
(see Note 2). The apparatus is assembled as shown in Figure 1.
NOTE 2: In place of the magnesium perchlorate drying tube, a small reading lamp
with 60W bulb may be used to prevent condensation of moisture inside the cell.
The lamp is positioned to shine on the absorption cell maintaining the air
temperature in the cell about 10°C above ambient.
6. Reagents
6.1 Sulfuric Acid, Cone: Reagent grade.
6.1.1 Sulfuric acid, 0.5 N: Dilute 14.0 ml of cone, sulfuric acid to 1.0 liter.
6.2 Nitric Acid, Cone: Reagent grade of low mercury content (See Note 3).
NOTE 3: If a high reagent blank is obtained, it may be necessary to distill the
nitric acid.
6.3 Stannous Sulfate: Add 25 g stannous sulfate to 250 ml of 0.5 N sulfuric acid. This
mixture is a suspension and should be stirred continuously during use. (Stannous
chloride may be used in place of stannous sulfate.)
6.4 Sodium Chloride-Hydroxylamine Sulfate Solution: Dissolve 12 g of sodium
chloride and 12 g of hydroxylamine sulfate in distilled water and dilute to 100.0
ml. (Hydroxylamine hydrochloride may be used in place of hydroxylamine
sulfate.)
6.5 Potassium Permanganate: 5% solution, w/v. Dissolve 5 g of potassium perman-
ganate in 100 ml of distilled water.
6.6 Potassium Persulfate: 5% solution, w/v. Dissolve 5 g of potassium persulfate in
100 ml of distilled water.
120
-------
6.7 Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in 75 ml of
distilled water. Add 10 ml of cone, nitric acid and adjust the volume to 100.0 ml.
1 ml = 1 mg Hg.
6.8 Working Mercury Solution: Make successive dilutions of the stock mercury
solution to obtain a working standard containing 0.1 //g per ml. This working
standard and the dilutions of the stock mercury solution should be prepared fresh
daily. Acidity of the working standard should be maintained at 0.15% nitric acid.
This acid should be added to the flask as needed before the addition of the
aliquot.
7. Calibration
7.1 .Transfer 0, 0.5, 1.0, 2.0, 5.0 and 10.0 ml aliquots of the working mercury
solution containing 0 to 1.0 ng of mercury to a series of 300 ml BOD bottles. Add
enough distilled water to each bottle to make a total volume of 100 ml. Mix
thoroughly and add 5 ml of cone, sulfuric acid (6.1) and 2.5 ml of cone, nitric
acid (6.2) to each bottle. Add 15 ml of KMnO4 (6.5) solution to each bottle and
allow to stand at least 15 minutes. Add 8 ml of potassium persulfate (6.6) to each
bottle and heat for 2 hours in a water bath maintained at 95°C. Cool and add 6
ml of sodium chloride-hydroxylamine sulfate solution (6.4) to reduce the excess
permanganate. When the solution has been decolorized wait 30 seconds, add 5 ml
of the stannous sulfate solution (6.3) and immediately attach the bottle to the
aeration apparatus forming a closed system. At this point the sample is allowed to
stand quietly without manual agitation. The circulating pump, which has
previously been adjusted to a rate of 1 liter per minute, is allowed to run
continuously (See Note 4). The absorbance will increase and reach maximum
within 30 seconds. As soon as the recorder pen levels off, approximately 1
minute, open the bypass valve and continue the aeration until the absorbance
returns to its minimum value (see Note 5). Close the bypass valve, remove the
stopper and frit from the BOD bottle and continue the aeration. Proceed with the
standards and construct a standard curve by plotting peak height versus
micrograms of mercury.
NOTE 4: An open system where the mercury vapor is passed through the
absorption cell only once may be used instead of the closed system.
NOTE 5: Because of the toxic nature of mercury vapor precaution must be taken
to avoid its inhalation. Therefore, a bypass has been included in the system to
either vent the mercury vapor into an exhaust hood or pass the vapor through
some absorbing media, such as:
121
-------
a) equal volumes of 0.1 M KMnO4 and 10% H2 SO4
b) 0.25% iodine in a 3% KI solution
A specially treated charcoal that will adsorb mercury vapor is also available from
Barnebey and Cheney, E. 8th Ave. and N. Cassidy St., Columbus, Ohio 43219,
Cat. #580-13 or #580-22.
122
-------
I
<*^
c
n -t
U ^
AIR PUMP
^ r^^~~~^^^n /^
^"" 1 1 >
'7 DESICCANT ,, ,'
1 1
ABSORPTION
]^ -BUBBLER CELL
J
c
I
"^
I-*
SAMPLE SOLUTION
IN BOD BOTTLE
SCRUBBER
CONTAINING
A MERCURY
ABSORBING
MEDIA
FIGURE 1. APPARATUS FOR FLAMELESS
MERCURY DETERMINATION
123
-------
8. Procedure
8.1 Transfer 100 ml or an aliquot diluted to 100 ml, containing not more than 1.0/ig
of mercury, to a 300 ml BOD bottle. Add 5 ml of sulfuric acid (6.1) and 2.5 ml
of cone, nitric acid (6.2) mixing after each addition. Add 15 ml of potassium
permanganate solution (6.5) to each sample bottle. For sewage samples additional
permanganate may be required. Shake and add additional portions of potassium
permanganate solution, if necessary, until the purple color persists for at least 15
minutes. Add 8 ml of potassium persulfate (6.6) to each bottle and heat for 2
hours in a water bath at 95°C. Cool and add 6 ml of sodium chloride-hydroxyla-
mine sulfate (6.4) to reduce the excess permanganate. After a delay of at least 30
seconds add 5 ml of stannous sulfate (6.3) and immediately attach the bottle to
the aeration apparatus. Continue as described under Calibration.
9. Calculation
9.1 Determine the peak height of the unknown from the chart and read the mercury
value from the standard curve.
9.2 Calculate the mercury concentration in the sample by the formula:
("8H£in)(
\ aliquot / \vol. of aliquot in ml.
9.3 Report mercury concentrations as follows: Below 0.2 p.g/1, <0.2; between 1 and
10 /zg/1, one decimal; above 10 ptg/1, whole numbers.
10. Precision and Accuracy
10.1 In a single laboratory (MDQARL), using an Ohio River composite sample with
a background mercury concentration of 0.35 jug/1, spiked with concentrations
of 1, 3 and 4 jug/1, the standard deviations were ±0.14, ±0.10 and ±0.08,
respectively. Standard deviation at the 0.35 level was ±0.16. Percent recoveries
at the three levels were 89, 87, and 87%, respectively.
10.2 In a joint EPA/ASTM interlaboratory study of the cold vapor technique for
total mercury in water, increments of organic and inorganic mercury were
added to natural waters. Recoveries were determined by difference. A
statistical summary of this study follows:
124
-------
Number
of Labs
76
80
82
77
82
79
79
78
True Values
Mg/liter
0.21
0.27
0.51
0.60
3.4
4.1
8.8
9.6
Mean Value
jug/liter
0.349
0.414
0.674
0.709
3.41
3.81
8.77
9.10
Standard
Deviation
jug/liter
0.276
0.279
0.541
0.390
1.49
1.12
3.69
3.57
Accuracy as
%Bias
66
53
32
18
0.34
-7.1
-0.4
-5.2
11. Appendix
11.1 While the possibility of absorption from certain organic substances actually
being present in the sample does exist, the MDQAR Laboratory has not
encountered such samples. This is mentioned only to caution the analyst of the
possibility. A simple correction that may be used is as follows: If an
interference has been found to be present (4.4), the sample should be analyzed
both by using the regular procedure and again under oxidizing conditions only,
that is without the reducing reagents. The true mercury value can then be
obtained by subtracting the two values.
11.2 If additional sensitivity is required, a 200 ml sample with recorder expansion
may be used provided the instrument does not produce undue noise. Using a
Coleman MAS-50 with a drying tube of magnesium perchlorate and a variable
recorder, 2 mv was set to read full scale. With these conditions, and distilled
water solutions of mercuric chloride at concentrations of 0.15, 0.10, 0.05 and
0.025 ptg/1 the standard deviations were ±0.027, ±0.006, ±0.01 and ±0.004.
Percent recoveries at these levels were 107, 83, 84 and 96%, respectively.
11.3 Directions for the disposal of mercury-containing wastes are given in ASTM
Standards, Part 23, Water and Atmospheric Analysis, p 352, Method D3223
(1973).
125
-------
Bibliography
1. Kopp, J. F., Longbottom, M. C. and Lobring, L. B., "Cold Vapor Method for
Determining Mercury", AWWA, vol 64, p. 20, Jan., 1972.
2. ASTM Standards, Part 23, Water; Atmospheric Analysis, p 346, Method D-3223
(1973).
126
-------
MERCURY IN WATER
(Automated Cold Vapor Technique)
STORET NO. 71900
1. Scope and Application
1.1 This method is applicable to surface waters. It may be applicable to saline waters,
wastewaters, effluents, and domestic sewages providing potential interferences are
not present (See Interference 4).
1.2 The working range is 0.2 to 20.0 Mg Hg/1.
2. Summary of Method
2.1 The flameless AA procedure is a physical method based on the absorption of
radiation at 253.7 nm by mercury vapor. The mercury is reduced to the elemental
state and aerated from solution. The mercury vapor passes through a cell
positioned in the light path of an atomic absorption spectrophotometer.
Absorbance (peak height) is measured as a function of mercury concentration and
recorded in the usual manner.
2.2 In addition to inorganic forms of mercury, organic mercurials may also be
present. These organo-mercury compounds will not respond to the flameless
atomic absorption technique unless they are first broken down and converted to
mercuric ions. Potassium permanganate oxidizes many of these compounds but
recent studies have shown that a number of organic mercurials, including phenyl
mercuric acetate and methyl mercuric chloride, are only partially oxidized by this
reagent. Potassium persulfate has been found to give approximately 100%
recovery when used as the oxidarit with these compounds. Therefore, an
automated persulfate oxidation step following the automated addition of the
permanganate has been included to insure that organo-mercury compounds, if
present, will be oxidized to the mercuric ion before measurement.
3. Sample Handling and Preservation
3.1 Until more conclusive data are obtained, samples should be preserved by
acidification with nitric acid to a pH of 2 or lower immediately at the time of
collection/1') If only dissolved mercury is to be determined, the sample should be
filtered before the acid is added. For total mercury the filtration is omitted.
4. Interference (See NOTE 1)
4.1 Some sea waters and wastewaters high in chlorides have shown a positive
interference, probably due to the formation of free chlorine.
127
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4.2 Interference .from certain volatile organic materials which will absorb at this
wavelength is also possible. A preliminary run under oxidizing conditions, without
stannous sulfate, would determine if this typfe of interference is present.
4.3 Formation of a heavy precipitate, in some wastewaters and effluents, has been
reported upon addition of concentrated sulfuric acid. If this is encountered, the
problem sample cannot be analyzed by this method.
4.4 Samples containing solids must be blended and then mixed while being sampled if
total mercury values are to be reported. '
NOTE 1: All the above interferences can be overcome by use of the Manual
Mercury method in this manual.
5. Apparatus
5.1 Technicon Auto Analyzer consisting of:
5.1.1 Sampler II with provision for sample mixing.
5.1.2 Manifold. :
5.1.3 Proportioning Pump II or III.
5.1.4 High temperature heating bath with two distillation coils (Technicon
Part #116-0163) in series.
5.2 Vapor-liquid separator (Figure 1).
5.3 Absorption cell, 100 mm long, 10 mm diameter with quartz windows.
5.4 Atomic Absorption Spectrophotometer (See Note 2): Any atomic absorption unit
having an open sample presentation area in which to mount the absorption cell is
suitable. Instrument settings recommended by the particular manufacturer should
be followed.
NOTE 2: Instruments designed specifically for the measurement of mercury using
the cold vapor technique are commercially available and may be substituted for
the atomic absorption spectrophotometer.
5.5 Mercury Hollow Cathode Lamp: Westinghouse WL-22847, argon filled, or
equivalent.
5.6 Recorder: Any multi-range variable speed recorder that is compatible with the UV
detection system is suitable.
5.7 Source of cooling water for jacketed mixing coil and connector A-7.
5.8 Heat lamp: A small reading lamp with 60W bulb may be used to prevent
condensation of moisture inside the cell. The lamp is positioned to shine on the
absorption cell maintaining the air temperature in the cell about 10°C above
ambient.
128
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Reagents
6.1 Sulfuric Acid, Cone: Reagent grade
6.1.1 Sulfuric acid, 2 N: Dilute 56 ml of cone, sulfuric acid to 1 liter with
distilled water.
6.1.2 Sulfuric acid, 10%: Dilute 100 ml cone, sulfuric acid to 1 liter with
distilled water.
6.2 Nitric acid, Cone: Reagent grade of low mercury content.
6.2.1 Nitric Acid, 0.5% Wash Solution: Dilute 5 ml of cone, nitric acid to 1
liter with distilled water;
6.3 Stanhous Sulfate: Add 50 g stannous sulfate to 500 ml of 2 N sulfuric acid
(6.1.1). This mixture is a suspension and should be stirred continuously during
use.
NOTE 3: Stannous chloride may be used in place of stannous sulfate.
6.4 Sodium Chloride-Hydroxylamine Sulfate Solution: Dissolve 30 g of sodium
chloride and 30 g of hydroxylamine sulfate in distilled water to 1 liter.
NOTE 4: Hydroxylamine hydrochloride may be used in place of hydroxylamine
sulfate.
6.5 Potassium Permanganate: 0.5% solution, w/v. Dissolve 5 g of potassium
permanganate in 1 liter of distilled water.
6.6 Potassium Permanganate, 0.1 N: Dissolve 3.16 g of potassium permanganate in
distilled water and dilute to 1 liter.
6.7 Potassium Persuifate: 0.5% solution, w/v. Dissolve 5 g of potassium persulfate in 1
liter of distilled water.
6.8 Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in 75 ml of
distilled water. Add 10 ml of cone, nitric acid and adjust the volume to 100.0 ml.
1.0 ml = l.OmgHg.
6.9 Working Mercury Solution: Make successive dilutions of the stock mercury
solution (6.8) to obtain a working standard containing 0.1 fig per ml. This
working standard and the dilutions of the stock mercury solution should be
prepared fresh daily. Acidity of the working standard should be maintained at
0.15% nitric acid. This acid should be added to the flask as needed before the
addition of the aliquot. From this solution prepare standards containing 0.2, 0.5,
1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 /ng Hg/1.
6.10 Air Scrubber Solution: Mix equal volumes of 0.1 N .potassium permanganate
(6.6) and 10% sulfuric acid (6.1.2).
129
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7. Procedure
7.1 Set up manifold as shown in Figure 2.
7.2 Feeding all the reagents through the system with acid wash solution (6.2.1)
through the sample line, adjust heating bath to 105°C.
7.3 Turn on atomic absorption spectrophotometer, adjust instrument settings as
recommended by the manufacturer, align absorption cell in light path for
maximum transmittance and place heat lamp directly over absorption cell. .
7.4 Arrange working mercury standards from 0.2 to 20.0 jug Hg/1 in sampler and start
sampling. Complete loading of sample tray with unknown samples.
7.5 Prepare standard curve by plotting peak height of processed standards against
concentration values. Determine concentration of samples by comparing sample
peak height with standard curve.
NOTE 5: Because of the toxic nature of mercury vapor, precaution must be taken
to avoid its inhalation. Venting the mercury vapor into an exhaust hood or
passing the vapor through some absorbing media such as:
a) equal volumes of 0.1 N KMnO4 (6.6) and 10%H2SO4 (6.1.2).
b) 0.25% iodine in a 3% KI solution, is recommended.
A specially treated charcoal that will adsorb mercury vapor is also available from
Barnebey and Cheney, E. 8th Ave. and North Cassidy St., Columbus, Ohio 43219,
Cat. #580-13 or #580-22.
7.6 After the analysis is complete put all lines except the H2 SO4 line in distilled water
to wash out system. After flushing, wash out the H2SO4 line. Also flush the coils
in the high temperature heating bath by pumping stannous sulfate (6.3) through
the sample lines followed by distilled water. This will prevent build-up of oxides
of manganese.
8. Precision and Accuracy
8.1 In a single laboratory (SEWL), using distilled water standards at concentrations of
0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 jug Hg/1, the standard deviations were ±0.04,
±0.07, ±0.09, ±0.20, ±0.40 and ±0.84 jug/1, respectively.
8.2 In a single laboratory (SEWL), using surface water samples spiked with ten
organic mercurials at the 10 jug/1 level, recoveries ranged from 87 to 117%.
Recoveries of the same ten organic mercurials in distilled water at the 10 jug/1
level, ranged from 92 to 125%.
130
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Bibliography
1. Wallace, R. A., Fulkerson, W., Shults, W. D., and Lyon, W. S., "Mercury in the
EnvironmentThe Human Element", Oak Ridge National Laboratory, ORNL-NSF-
EP-l,p 31, (January, 1971).
2. Hatch, W. R. and Ott, W. L., "Determination of Sub-Microgram Quantities of Mercury
by Atomic Absorption Spectrophotometry", Anal. Chem. 40, 2085 (1968).
3. Bran denberger, H. and Bader, H., "The Determination of Nanogram Levels of Mercury
in Solution by a Flameless Atomic Absorption Technique", Atomic Absorption
Newsletter 6, 101 (1967).
4. Brandenberger, H. and Bader, H., "The Determination of Mercury by Flameless
Atomic Absorption II, A Static Vapor Method", Atomic Absorption Newsletter 7, 53
(1968).
5. Goulden, P. D. and Afghan, B. K., "An Automated Method for Determining Mercury
in Water", Technicon, Adv. in Auto. Anal. 2, p 317 (1970).
131
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AIR AND
SOLUTION
IN
l4cm
0.4cm 10
"SOLUTION
OUT
FIGURE 1. VAPOR LIQUID SEPARATOR
132
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SPECTROPHOTOMETER
Dl
OOOflOOQQ
OJ
SAMPLER II
PS 3
0000
HEATING BATH
JD~!i
HI
I PS4
ml/min
90
3.90
3.90
2.00 ) DO
3.90
3.90
3.90
Fi
3.90
3.90
3.90
3.90 GO
1.20
t
GO Air
AIR
SCRUBBER
10%
2;50
3% NaCl
+
3% (NH2OH)2
1.20
.5% K2S208
2.76
2.76
3.90
3.90 IGO
3.95
?
(Con) H2S04 **
Sample
2.00
Air
1.20
.5%
Proportioning Pump III
FIGURE 2 MERCURY MANIFOLD AA-I
* Acid Flex SCRUBBER
-------
MERCURY IN SEDIMENT
(Manual Cold Vapor Technique)
1. Scope and Application
1.1 This procedure*1) measures total mercury (organic + inorganic) in soils,
sediments, bottom deposits and sludge type materials.
1.2 The range of the method is 0.2 to 5 jug/g. The range may be extended above or
below the normal range by increasing or decreasing sample size or through
instrument and recorder control.
2. Summary of Method
2.1 A weighed portion of the sample is digested in aqua regia for 2 minutes at 95°C,
followed by oxidation with potassium permanganate. Mercury in the digested
sample is then measured by the conventional cold vapor technique.
2.2 An alternate digestion*2) involving the use of an autoclave is described in (8.2).
3. Sample Handling and Preservation
3.1 Because of the extreme sensitivity of the analytical procedure and the
omnipresence of mercury, care must be taken to avoid extraneous contamination.
Sampling devices and sample containers should be ascertained to be free of
mercury; the sample should not be exposed to any condition in the laboratory
that may result in contact or air-borne mercury contamination.
3.2 While the sample may be analyzed without drying, it has been found to be more
convenient to analyze a dry sample. Moisture may be driven off in a drying oven
at a temperature of 60°C. No mercury losses have been observed by using this
drying step. The dry sample should be pulverized and thoroughly mixed before
the aliquot is weighed.
4. Interferences
4.1 The same types of interferences that may occur in water samples are also possible
with sediments, ie., sulfides, high copper, high chlorides, etc.
4.2 Volatile materials which absorb at 253.7 nm will cause a positive interference. In
order to remove any interfering volatile materials, the dead air space in the BOD
bottle should be purged before the addition of stannous sulfate.
5. Apparatus
5.1 Atomic Absorption Spectrophotometer (See Note 1): Any atomic absorption unit
having an open sample presentation area in which to mount the absorption cell is
suitable. Instrument settings recommended by the particular manufacturer should
be followed.
134
/
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NOTE !: Instruments designed specifically for the measurement of mercury using
the cold vapor technique are commercially available and may be substituted for
the atomic absorption spectrophotometer.
5.2 Mercury Hollow Cathode Lamp: Westinghouse WL-22847, argon filled, or
equivalent.
5.3 Recorder: Any multi-range variable speed recorder that is compatible with the UV
detection system is suitable..
5.4 Absorption Cell: Standard-spectrophotometer cells 10 cm long, having quartz end
windows may be used. Suitable cells may be constructed from plexiglass tubing,
1" O.D. X 4-1/2". >The ends are ground perpendicular to the longitudinal axis and
quartz windows (1" diameter X 1/16" thickness) are cemented in place. Gas inlet
and outlet ports (also of plexiglass but 1/4" O.D.) are attached approximately
1/2" from each end. The cell is strapped to a burner for support and aligned in the
light beam to give the maximum transmittance.
NOTE 2: Two 2" X 2" cards with one inch diameter holes may be placed over
each end of the cell to assist in positioning the cell for maximum transmittance.
5.5 Air Pump: Any peristaltic pump capable of delivering 1 liter of air per minute
may be used. A Masterflex pump with electronic speed control has been found to
be satisfactory. (Regulated compressed air can be used in an open one-pass
system.)
5.6 Flowmeter: Capable of measuring an air flow of 1 liter per minute.
5.7 Aeration Tubing: Tygon tubing is used for passage of the mercury vapor from the
sample bottle to the absorption cell and return. Straight glass tubing terminating
in a coarse porous frit is used for sparging air into the sample.
5.8 Drying Tube: 6" X 3/4" diameter tube containing 20 g of magnesium perchlorate
(See Note 3). The apparatus is assembled as shown in the accompanying diagram.
NOTE 3: In place of the magnesium perchlorate drying tube, a small reading lamp
with 60W bulb may be used to prevent condensation of moisture inside the cell.
The lamp is positioned to shine on the absorption cell maintaining the air
temperature in the cell about 10°C above ambient.
6. Reagents
6.1 Aqua Regia: Prepare immediately before use by carefully adding three volumes of
cone. HC1 to one volume of cone. HNO3.
6.2 Sulfuric Acid, 0.5 N: Dilute 14.0 ml of cone, sulfuric acid to 1 liter.
6.3 Stannous Sulfate: Add 25 g stannous sulfate to 250 ml of 0.5 N sulfuric acid
(6.2). This mixture is a suspension and should be stirred continuously during use.
135
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6.4 Sodium Chloride-Hydroxylamine Sulfate Solution: Dissolve 12 g of sodium
chloride and 12 g of hydroxylamine sulfate in distilled water and dilute to 100
ml.
NOTE 4: A 10% solution of stannous chloride may be substituted for (6.3) and
hydroxylamine hydrochloride may be used in place of hydroxylamine sulfate in
(6.4).
6.5 Potassium Permanganate: 5% solution, w/v. Dissolve 5 g of potassium perman-
ganate in 100 ml of distilled water.
6.6 Stock Mercury Solution: Dissolve 0.1354 g of mercuric chloride in 75 ml of
distilled water. Add 10 ml of cone, nitric acid and adjust the volume to 100.0 ml.
1.0 ml = l.OmgHg.
6.7 Working Mercury Solution: Make successive dilutions of the stock mercury
solution (6.6) to obtain a working standard containing 0.1 Mg/ml. This working
standard and the dilution of the stock mercury solutions should be prepared fresh
daily. Acidity of the working standard should be maintained at 0.15% nitric acid.
This acid should be added to the flask as needed before the addition of the
aliquot.
7. Calibration
7.1 Transfer 0, 0.5, 1.0, 2.0, 5.0 and 10 ml aliquots of the working mercury solution
(6.7) containing 0 to 1.0 /ug of mercury to a series of 300 ml BOD bottles. Add
enough distilled water to each bottle to make a total volume of 10 ml. Add 5 ml
of aqua regia (6.1) and heat 2 minutes in a water bath at 95°C. Allow the sample
to cool and add 50 ml distilled water and 15 ml of KMnO4 solution (6.5) to each
bottle and return to the water bath for 30 minutes. Cool and add 6 ml of sodium
chloride-hydroxylamine sulfate solution (6.4) to reduce the excess permanganate.
Add 50 ml of distilled water. Treating each bottle individually, add 5 ml of
stannous sulfate solution (6.3) and immediately attach the bottle to the aeration
apparatus. At this point, the sample is allowed to stand quietly without manual
agitation. The circulating pump, which has previously been adjusted to a rate of 1
liter per minute, is allowed to run continuously. The absorbance, as exhibited
either on the spectrophotometer or the recorder, will increase and reach
maximum within 30 seconds. As soon as the recorder pen levels off, approximate-
ly 1 minute, open the bypass valve and continue the aeration until the absorbance
returns to its minimum value (See Note 5). Close the bypass valve, remove the
fritted tubing from the BOD bottle and continue the aeration. Proceed with the
standards and construct a standard curve by plotting peak height versus
micrograms of mercury.
136
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NOTE 5: Because of the toxic nature of mercury vapor precaution must be taken
to avoid its inhalation. Therefore, a bypass has been included in the system to
either vent the mercury vapor into an exhaust hood or pass the vapor through
some absorbing media, such as:
a) equal volumes of 0.1 N KMnO4 and 10% H2SO4
b) 0.25% iodine in a 3% KI solution.
A specially treated charcoal that wall absorb mercury vapor is also available from
Bamebey and Cheney, E. 8th Ave. and North Cassidy St., Columbus, Ohio 43219,
Cat. #580-13 or #580-22.
8. Procedure
8.1 Weigh triplicate 0.2 g portions of dry sample and place in bottom of a BOD
bottle. Add 5 ml of distilled water and 5 ml of aqua regia (6.1). Heat 2 minutes in
a water bath at 95°C. Cool, add 50 ml distilled water and 15 ml potassium
permanganate solution (6.5) to each sample bottle. Mix thoroughly and place in
the water bath for 30 minutes at 95°C. Cool and add 6 ml of sodium
chloride-hydroxylamine sulfate (6.4) to reduce the excess permanganate. Add 55
ml of distilled water. Treating each bottle individually, add 5 ml of stannous
sulfate (6.3) and immediately attach the bottle to the aeration apparatus.
Continue as described under (7.1).
8.2 An alternate digestion procedure employing an autoclave may also be used. In this
method 5 ml of cone. H2SO4 and 2 ml of cone. HNO3 are added to the 0.2 g of
sample. 5 ml of saturated KMnO4 solution is added and the bottle covered with a
piece of aluminum foil. The samples are autoclaved at 121°C and 15 Ibs. for 15
minutes. Cool, make up to a volume of 100 ml with distilled water and add 6 ml
of sodium chloride-hydroxylamine sulfate solution (6.4) to reduce the excess
permanganate. Purge the dead air space and continue as described under (7.1).
9. Calculation
9.1 Measure the peak height of the unknown from the chart and read the mercury
value from the standard curve.
9.2 Calculate the mercury concentration in the sample by the formula:
jug Hg in the aliquot
/ug Hg/g = -
wt of the aliquot in gms.
9.3 Report mercury concentrations as follows: Below 0.1 Mg/gm, <0.1; between 0;1
and 1 Mg/gm, to the nearest 0.01 /ug; between 1 and 10/ug/gm, to nearest 0.1 /ug;
above 10 /ug/gm, to nearest /ug.
137
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10. Precision and Accuracy
10.1 The following standard deviations on replicate sediment samples were recorded at
the indicated levels; 0.29 /ug/g ±0.02 and 0.82 ;ug/g ±0.03. Recovery of mercury at
these levels, added as methyl mercuric chloride, was 97 and 94%, respectively.
Bibliography
1. Bishop, J. N., "Mercury in Sediments", Ontario Water Resources Comm., Toronto,
Ontario, Canada, 1971.
2. Salma, M., private communication, EPA Cal/Nev Basin Office, Almeda, California.
138
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MOLYBDENUM
(Standard Conditions)
STORETNO. Total 01062
Optimum Concentration Range: 0.5-20 mg/1 using a wavelength of 313.3 nm
Sensitivity: 0.3 mg/1
Detection Limit: 0.1 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.840 g of ammonium molybdate (NH4 )6 Mo7O24 '4H2O
(analytical reagent grade) in deionized distilled water and dilute to 1 liter. 1 ml= 1
mg Mo (1000 mg/1).
2. Aluminum nitrate solution: Dissolve 139 g aluminum nitrate, A1(NO3)3 -9H2O, in
150 ml of deionized distilled water; heat to effect solution. Allow to cool and
make up to 200 ml.
3. Prepare dilutions of the stock molybdenum solution to be used as calibration
standards at the time of analysis. To each 100 ml of standard and sample alike,
add 2 ml of the aluminum nitrate solution. The calibration standards should be
prepared using the same type of acid (HC1 or HNO3) and at the same
concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Molybdenum hollow cathode lamp
2. Wavelength: 313.3 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous Oxide
5. Type of flame: Fuel rich
Interferences
1. With the recommended nitrous oxide-acetylene flame, interferences of calcium
and other ions may be controlled by adding 1000 mg/1 of a refractory metal such
as aluminum' [Anal. Chem. Acta 44, 437 (1969)]. This should be done to both
samples and standards alike.
139
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Notes
1. For low levels of molybdenum an oxine extraction procedure may be useful.
(Ref: Chau et.al., Anal. Chem. Acta 48, 205, 1969).
2. Data to be entered into STORET must be reported as pig/1.
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.30, 1.5 and 7.5 mg Mo/1, the standard deviations
were ±0.007, ±0.02 and ±0.07, respectively. Recoveries at these levels were 100%,
96% and 95%, respectively.
140
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NICKEL
(Standard Conditions)
STORETNO. Total 01067
Optimum Concentration Range: 0.3-10 mg/1 using a wavelength of 232.0 nm
Sensitivity: 0.15 mg/1
Detection List: 0.02 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 4.953 g of nickel nitrate, Ni(NO3)2 -6H2O (analytical
reagent grade) in deionized distilled water. Add 10 ml of cone, tiitric acid and
dilute to 1 liter with deionized distilled water. 1 ml = 1 mg Ni (1000 mg/1).
2. Prepare dilutions of the stock nickel solution to be used as calibration standards
at the time of analysis. The calibration standards should be prepared using the
same type of acid (HC1 or HNO3) and at the same concentration as the samples
for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods .section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Nickel hollow cathode lamp
2. Wavelength: 232.0 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of Flame: Oxidizing
Interferences
1. The 352.4 nm wavelength is less susceptible to nonatomic absorbance and may be
used. The calibration curve is more linear at this wavelength; however, there is
some loss of sensitivity.
Notes
1. For levels of nickel below 50 pg/1, the extraction procedure is recommended.
2. Data to be entered into STORET must be reported as j/g/1.
3. The heptoxime method may be used (Standard Methods, 13th Edition, p 443).
141
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Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.20, 1.0 and 5.0 mg Ni/1, the standard deviations
were ±0.011, ±0.02 and ±0.04, respectively. Recoveries at these levels were 100%,
97% and 93%, respectively.
142
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POTASSIUM
(Standard Conditions)
STORE! NO. Total 00937
Optimum Concentration Range: 0.1-2 mg/1 using a wavelength of 766.5 nm
Sensitivity: 0.04 mg/1
Detection Limit: 0.005 mg/1
Preparation of Standard Solution
1. Stock Solution; Dissolve 0.1907 g of KC1 (analytical reagent grade), dried at
110°C, in deionized distilled water and make up to 1 liter. 1 ml = 0.10 mg K (100
mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis.
Sample Preparation
1. For the analysis of total postassium in domestic and industrial effluents, the
procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
2. For ambient waters, a representative aliquot of a well-mixed sample may also be
used directly for analysis. If suspended solids are present in sufficient amounts to
clog the nebulizer, the sample may be allowed to settle and the supernatant liquid
analyzed directly.
3. Samples should be preserved with (1:1) nitric acid to a pH of 2 at the time of
collection.
Instrumental Parameters (General)
1. Potassium hollow cathode lamp
2. Wavelength: 766.5 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Slightly oxidizing
Notes
1. The Osram potassium vapor-discharge lamp may also be used in the Perkin-Elmer
303. In this case the current should be 350 ma or the optimum operating current.
2. Sodium may interfere if present at much higher levels than the potassium. This
effect can be compensated by approximately matching the sodium content of the
potassium standards with that of the sample.
143
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3. Potassium absorption is enhanced in the presence of Na, Li and Cs, especially in a
high-temperature flame. This enhancement effect of sodium can be eliminated by
changing the burner height and the type of flame used. The burner assembly is set
approximately 0.05 cm below the optical light path so that the optical light path
is sliced at the bottom by the burner head. A fuel-rich flame is used. .
4. The 404.4 nm line may also be used. This line has a sensitivity of 5 mg/1 for 1%
absorption.
5. To cover the range of potassium values normally observed in surface waters
(0.1-20 mg/1), it is suggested that the burner be rotated 75°.
6. The flame photometric or colorimetric methods may be used (Standard Methods,
13th Edition, p 283 & 285).
7. Data to be entered into STORET must be reported as mg/1.
Precision and Accuracy
1. In a single laboratory (MDQARL), using distilled water samples at concentrations
of 1.6 and 6.3 mg/1, the standard deviations were ±0.2 and ±0.5, respectively.
Recoveries at these levels were 103% and 102%.
144
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SELENIUM
(Gaseous Hydride Method)
STORETNO. Total 01147
1. Scope and Application
1.1 The gaseous hydride method determines inorganic selenium when present in
concentrations at or above 2 /ig/1. The method is applicable to most fresh and
saline waters, in the absence of high concentrations of chromium, cobalt, copper,
mercury, molybdenum, nickel and silver.
2. Summary of Method
2.1 Selenium in the sample is reduced from the +6 oxidation state to the +4 oxidation
state by the addition of SnCl2. Zinc is added to the acidified sample, producing
hydrogen and converting the selenium to the hydride, SeH2. The gaseous
selenium hydride is swept into an argon-hydrogen flame of an atomic absorption
spectrophotometer. The working range of the method is 2-20 /ig/1 using the 196.0
nm wavelength.
3. Comments
3.1 In analyzing most surface and ground waters, interferences are rarely en-
countered. Industrial waste samples should be spiked with a known amount of
selenium to establish adequate recovery.
3.2 Organic forms of selenium must be converted to an inorganic form and organic
matter must be oxidized before beginning the analysis.
3.3 Data to be entered into STORET must be reported as vg/l.
4. Precision and Accuracy
4.1 Ten replicate solutions of selenium oxide at the 5, 10 and 15 /ig/1 level were
analyzed by a single laboratory (Caldwell, Et.Al.). Standard deviations at these
levels were ±0.6, ±1.1 and ±2.9 with recoveries of 100, 100 and 101%.
Bibliography
1. Caldwell, J. S., Lishka, R. J., and McFarren, E. F., "Evaluation of a Low-Cost Arsenic
and Selenium Determination at Microgram per Liter Levels", JAWWA, vol 65, p. 731,
Nov. 1973.
145
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SILVER
(Standard Conditions)
STORETNO. Total 01077
Optimum Concentration Range: 0.1-4 mg/1 using a wavelength of 328.1 nm
Sensitivity: 0.06 mg/1
Detection Limit: 0.01 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.575 g of AgNO3 (analytical reagent grade) in
deionized distilled water, add 10 ml cone. HNO3 and make up to 1 liter. 1 ml = 1
mgAg( 1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. Maintain an acid strength of 0.15% HNO3 in all calibration
standards.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory; however, the residue must be taken up in dilute nitric acid rather
than hydrochloric to prevent precipitation of AgCl.
Instrumental Parameters (General)
1. Silver hollow cathode lamp
2. Wavelength: 328.1 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. For levels of silver below 20 jug/1, the extraction procedure is recommended.
2. Silver nitrate standards are light sensitive. Dilutions of the stock should be
discarded after use as concentrations below 10 mg/1 are not stable over long
periods of time.
3. The 338.2 nm wavelength may also be used. This has a relative sensitivity of 3.
4. Data to be entered into STORET must be reported as /ug/1.
5. The dithizone colorimetric method may be used (Standard Methods, 13th
Edition, p 310).
146
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SODIUM
(Standard Conditions)
STORET NO. Total 00929
Optimum Concentration Range: 0.03-1.0 mg/1 using a wavelength of 589.6 nm
Sensitivity: 0.015 mg/1
Detection Limit: 0.002 mg/1
Preparation of Standard Solutions
1. Stock Solution: Dissolve 2.542 g of NaCl (analytical reagent grade), dried at
140°C, in deionized distilled water and make up to 1 liter. 1 ml = 1 mg Na (1000
mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis.
Sample Preparation
1. For the analysis of total sodium in domestic and industrial effluents, the
procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
2. For ambient waters, a representative aliquot of a well-mixed sample may be used
directly for analysis. If suspended solids are present in sufficient amounts to clog
the nebulizer, the sample may be allowed to settle and the supernatant liquid
analyzed directly.
3. Samples should be preserved with (1:1) nitric acid to a pH of 2 at the time of
collection.
Instrumental Parameters (General)
1. Sodium hollow cathode lamp
2. Wavelength: 589.6 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. The 330.2 nm resonance line of sodium yields a sensitivity of about 3 mg/1
sodium for 1% absorption and provides a convenient way to avoid the need to
dilute more concentrated solutions of sodium.
147
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2. Low-temperature flames increase sensitivity by reducing the extent of ionization
of this easily ionized metal. Ionization may also be controlled by adding
potassium (1000 mg/1) to both standards and samples.
3. Data to be entered into STORET must be reported as mg/1.
4. The flame photometric method may be used (Standard Methods, 13th Edition, p
317).
Precision and Accuracy
1. In a single laboratory (MDQARL), using distilled water samples at levels of 8.2
and 52 mg/1, the standard deviations were ±0.1 and ±0.8, respectively. Recoveries
at these levels were 102% and 100%.
148
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THALLIUM
(Standard Conditions)
STORETNO. Total 01059
Optimum Concentration Range: 1-20 mg/1 using a wavelength of 276.8 nm
Sensitivity: 0.5 mg/1
Detection Limit: 0.1 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.303 g of thallium nitrate, T1NO3 (analytical reagent
grade) in deionized distilled water. Add 10 ml of cone, nitric acid and dilute to 1
liter with deionized distilled water. 1 ml = 1 mg Tl (1000 mg/1).
2. Prepare dilutions of the stock thallium solution to be used as calibration standards
at .the time of analysis. The calibration standards should be prepared using the
same type of acid (HC1 or HNO3 ) and at the same concentration as the samples
for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Thallium hollow cathode lamp
2. Wavelength: 276.8 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. Data to be entered into STORET must be reported as Mg/1-
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 0.60, 3.0 and 15 mg Tl/1, the standard deviations
were ±0.018, ±0.05 and ±0.2, respectively. Recoveries at these levels were 100%,
98% and 98%, respectively.
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TIN
(Standard Conditions)
STORE! NO. Total 01102
Optimum Concentration Range: 10-200 mg/1 using a wavelength of 286.3 nm
Sensitivity: 4 mg/1
Detection Limit: 0.8 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.000 g of tin metal (analytical reagent grade) in 100 ml
of cone. HC1 and dilute to 1 liter with deionized distilled water. 1 ml = 1 mg Sn
(1000 mg/1).
2. Prepare dilutions of the stock tin solution to be used as calibration standards at
the time of analysis. Maintain an acid concentration of 10% HC1 in all solutions.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Tin hollow cathode lamp
2. Wavelength: 286.3 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Fuel rich
Notes
1. Data to be entered into STORET must be reported as jzg/1.
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 4.0, 20 and 60 mg Sn/1, the standard deviations were
±0.25, ±0.5 and ±0.5, respectively. Recoveries at these levels were 96%, 101%
and 101%, respectively.
150
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TITANIUM
(Standard Conditions)
STORETNO.Total01152
Optimum Concentration Range: 5-100 mg/1 using a wavelength of 365.3 nm
Sensitivity: 2.0 mg/1
Detection Limit: 0.3 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 4.008 g of titanium sulfate, Ti2(SO4)3, in dilute HC1
and make up to 1 liter with deionized distilled water. 1 ml = 1 mg Ti (1000 mg/1).
2. Potassium chloride solution: Dissolve 95 g potassium chloride, KC1, in distilled
water and make up to 1 liter.
3. Prepare dilutions of the stock titanium solution to be used as calibration
standards at the time of analysis. To each 100 ml of standard and sample alike,
add 2 ml of potassium chloride solution.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual must be modified by the
addition of 3 ml of cone, sulfuric acid in addition to the nitric acid. This is
necessary to keep any titanium that may be present in solution.
Instrumental Parameters (General)
1. Titanium hollow cathode lamp
2. Wavelength: 365.3 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous Oxide
5. Type of flame: Fuel rich
Interferences
1. A number of .elements increase the sensitivity of titanium. To control this
problem, potassium (1000 mg/1) must be added to standards and samples alike.
[Atomic Absorption Newsletter 6, p 86 (1967)]
Notes
1. Data to be entered into STORET must be reported as Mg/1.
151
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Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 2.0, 10 and 50 mg Ti/1, the standard deviations were
±0.07, ±0.1 and ±0.4, respectively. Recoveries at these levels were 97%, 91% and
88%, respectively.
152
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VANADIUM
(Standard Conditions)
STORE! NO. Total 01087
Optimum Concentration Range: 1-100 mg/1 using a wavelength of 318.4 nm
Sensitivity: 0.8 mg/1.
Detection Limit: 0.2 mg/1
Preparation of Standard Solution
1. Stock Solution: Dissolve 1.7854 g of vanadium pentoxide, V2Os (analytical
reagent) in 10 ml of cone, nitric acid and dilute to 1 liter with deionized distilled
water. 1 ml = 1 mg V (1000 mg/1).
2. Aluminum nitrate solution: Dissolve 139 g aluminum nitrate, A1(NO3)3 '9H2O, in
150 ml of deionized distilled water; heat to effect solution. Allow to cool and
make up to 200 ml.
3. Prepare dilutions of the stock vanadium solution to be used as calibration
standards at the time of analysis. To each 100 ml of standard and sample alike,
add 2 ml of the aluminum nitrate solution. The calibration standards should be
prepared using the same type of acid (HC1 or HNO3) and at the same
concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4-1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters (General)
1. Vanadium hollow cathode lamp
2. Wavelength: 318.4 nm
3. Fuel: Acetylene
4. Oxidant: Nitrous oxide
5. Type of flame: Fuel rich
Interferences
1. It has been reported that high concentrations of aluminum and titanium increase
the sensitivity of vanadium. This interference can be controlled by adding excess
aluminum (1000 ppm) to both samples and standards. [Talanta 15, 871 (1968)].
153
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Notes
1. Data to be entered into STORET must be reported as //g/1.
2. The gallic acid colorimetric method may be used (Standard Methods, 13th
Edition, p 357).
Precision and Accuracy
1. In a single laboratory (MDQARL), using a mixed industrial-domestic waste
effluent at concentrations of 2.0, 10 and 50 mg V/l, the standard deviations were
±0.10, ±0.1 and ±0.2, respectively. Recoveries at these levels were 100%, 95%
and 97%, respectively.
154
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ZINC
(Standard Conditions)
STORET NO. Total 01092
Optimum Concentration Range: 0.05-2 mg/1 using a wavelength of 213.9 nm
Sensitivity: 0.02 mg/1
Detection Limit: 0.005 mg/1
Preparation of Standard Solution
1. Stock Solution: Carefully weigh 1.00 g of zinc metal (analytical reagent grade)
and dissolve cautiously in 10 ml HNO3. When solution is complete make up to 1
liter with deionized distilled water. 1 ml = 1 mg Zn (1000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration standards at the
time of analysis. The calibration standards should be prepared using the same type
of acid (HC1 or HNO3) and at the same concentration as the samples for analysis.
Sample Preparation
1. The procedure for the determination of total metals as given in part 4.1.3 of the
Atomic Absorption Methods section of this manual has been found to be
satisfactory.
Instrumental Parameters
1. Zinc hollow cathode lamp
2. Wavelength: 213.9 nm
3. Fuel: Acetylene
4. Oxidant: Air
5. Type of flame: Oxidizing
Notes
1. High levels of silicon may interfere.
2. The air-acetylene flame absorbs about 25% of the energy at the 213.9 nm line.
3. The sensitivity may be increased by the use of low-temperature flames.
4. Data to be entered into STORET must be reported as /ug/1.
5. The dithizone colorimetric method may be used (Standard Methods, 13th
Edition, p 444).
Precision and Accuracy
1. An interlaboratory study on trace metal analyses by atomic absorption was
conducted by the Quality Assurance and Laboratory Evaluation Branch of
MDQARL. Six synthetic concentrates containing varying levels of aluminum.
155
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cadmium, chromium, iron, manganese, lead and zinc were added to natural water
samples. The statistical results for zinc were as follows:
Standard
Number True Values Mean Value Deviation Accuracy as
of Labs pig/liter jug/liter jug/liter % Bias
86 281 284 97 1.2
89 310 308 114 -0.7
82 56 62 28 11.3
81 70 75 28 6.6
62 7 22 26 206
61 11 17 18 56.6
156
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METHYLENE BLUE ACTIVE SUBSTANCES (MBAS)
(Methylene Blue Method)
STORET NO. 38260
1. Scope and Application
1.1 This method is applicable to the measurement of methylene blue active
substances (MBAS) in drinking waters, surface waters, domestic and industrial
wastes. It is not applicable to measurement of surfactant-type materials in saline
waters.
1.2 It is not possible to differentiate between linear alkyl sulfonate (LAS) and alkyl
benzene sulfonate (ABS) or other isomers of these types of compounds. However,
LAS has essentially replaced ABS on the surfactant market so that measurable
surfactant materials will probably be LAS type materials.
1.3 The .method is applicable over the range of 0.025 to 100 mg/1 LAS.
2. Summary of Method
2.1 The dye, methylene blue, in aqueous solution reacts with anionic-type surface
active materials to form a blue colored salt. The salt is ex tractable with
chloroform and the intensity of color produced is proportional to the
concentration of MBAS.
3. Comments
3.1 Materials other than man-made surface active agents which react with methylene
blue are organically bound sulfates, sulfonates, carboxylates, phosphates, phenols,
cyanates, thiocyanates and some inorganic ions such as nitrates and chlorides.
However, the occurrence of these materials at interference levels is relatively rare
and with the exception of chlorides may generally be disregarded.
3.2 Chlorides at concentration of about 1000 mg/1 show a positive interference but
the degree of interference has not been quantified. For this reason the method is
not applicable to brine samples.
3.3 Naturally occurring organic materials that react with methylene blue are relatively
insignificant. Except under highly unusual circumstances, measurements of MBAS
in finished waters, surface waters and domestic sewages may be assumed to be
accurate measurements of man-made surface active agents.
4. Precision and Accuracy
4.1 On a sample of filtered river water, spiked with 2.94 mg LAS/liter, 110 analysts
obtained a mean of 2.98 mg/liter with a standard deviation of ±0.272.
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4.2 On a sample of tap water spiked with 0.48 mg LAS/liter, analysts obtained a
mean of 0.49 mg/1 with a standard deviation of ±0.048.
4.3 On a sample of distilled water spiked with 0.27 mg LAS/liter, 110 analysts
obtained a mean of 0.24 mg/1 with a standard deviation of ±0.036.
4.4 Analytical Reference Service, Water Surfactant No. 3, Study No. 32, (1968).
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewaters, 13th Edition, p
339-342, Method No. 159A(1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 492, Method
02330-68(1973).
158
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NITROGEN, AMMONIA
(DistiDation Procedure)
STORETNO. 00610
1. Scope and Application
1.1 This distillation method covers the determination of ammonia-nitrogen exclusive
of total Kjeldahl nitrogen, in drinking, surface, and saline waters, domestic and
industrial wastes. It is the method of choice where economics and sample load do
not warrant the use of automated equipment.
1.2 The method covers the range from about 0.05 to 1.0 mg/1 NH3-N/1 for the
colorimetric procedures, from 1.0 to 25 mg/1 for the titrimetric procedure, and
from 0.05 to 1400.mg/1 for the electrode method.
1.3 This method is described for macro glassware; however, micro distillation
equipment may also be used.
2. Summary of Method
2.1 The sample is buffered at a pH of 9.5 with a borate buffer in order to decrease
hydrolysis of cyanates and organic nitrogen compounds, and is then distilled into
a solution of boric acid. The ammonia in the distillate can be determined
colorimetrically by nesslerization, titrimetrically with standard sulfuric acid with
the use of a mixed indicator, or potentiometrically by the ammonia electrode.
The choice between the first two procedures depends on the concentration of the
ammonia.
3. Sample Handling and Preservation
3.1 Samples may be preserved with 2 ml of cone. H2SO4 or 40 mg HgQ2 per liter
and stored at 4°C.
4. Interferences
4.1 A number of aromatic and aliphatic amines, as well as other compounds, both
organic and inorganic, will cause turbidity upon the addition of Nessler reagent,
so direct nesslerization (i.e., without distillation), has been discarded as an official
method.
4.2 Cyanate, which may be encountered in certain industrial effluents, will hydrolyze
to some extent even at the pH of 9.5 at which distillation is carried out. Volatile
alkaline compounds, such as certain ketones, aldehydes, and alcohols, may cause
an off-color upon nesslerization in the distillation method. Some of these, such as
formaldehyde, may be eliminated by boiling off at a low pH (approximately 2 to
3) prior to distillation and nesslerization.
159
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4.3 Residual chlorine must also be removed by pretreatment of the sample with
sodium thiosulfate before distillation.
4.4 // the sample has been preserved with a mercury salt, the mercury ion must be
complexed with sodium thiosulfate (0.2 g) prior to distillation.
5. Apparatus
5.1 An all-glass distilling apparatus with an 800-1000 ml flask.
5.2 Spectrophotometer or filter photometer for use at 425 nm and providing a light
path of 1 cm or more.
5.3 Nessler tubes: Matched Nessler tubes (APHA Standard) about 300 mm long, 17
mm inside diameter, and marked at 225 mm ±1.5 mm inside measurement from
bottom.
5.4 Erlenmeyer flasks: The distillate is collected in 500 ml glass-stoppered flasks.
These flasks should be marked at the 350 and the 500 ml volumes. With such
marking, it is not necessary to transfer the distillate to volumetric flasks.
6. Reagents
6.1 Distilled water should be free of ammonia. Such water is best prepared by passage
through an ion exchange column containing a strongly acidic cation exchange
resin mixed with a strongly basic anion exchange resin. Regeneration of the
column should be carried out according to the manufacturer's instructions.
NOTE 1: All solutions must be made with ammonia-free water.
6.2 Ammonium chloride, stock solution:
1.0 ml = 1.0 mg NH3-N. Dissolve 3.819 g NH4C1 in distilled water and bring to
volume in a 1 liter volumetric flask.
6.3 Ammonium chloride, standard solution:
1.0 ml = 0.01 mg. Dilute 10.0 ml of stock solution (6.2) to 1 liter in a volumetric
flask.
6.4 Boric acid solution (20 g/1): Dissolve 20 g H3BO3 in distilled water and dilute to
1 liter.
6.5 Mixed indicator: Mix 2 volumes of 0.2% methyl red in 95% ethyl alcohol with 1
volume of 0.2% methylene blue in 95% ethyl alcohol. This solution should be
prepared fresh every 30 days.
NOTE 2: Specially denatured ethyl alcohol conforming to Formula 3A or 30 of
the U.S. Bureau of Internal Revenue may be substituted for 95% ethanol.
6.6 Nessler reagent: Dissolve 100 g of mercuric iodide and 70 g of potassium iodide in
a small amount of water. Add this mixture slowly, with stirring, to a cooled
solution of 160 g of NaOH in 500 ml of water. Dilute the mixture to 1 liter. If
160
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this reagent is stored in a Pyrex bottle out of direct sunlight, it will remain stable
for a period of up to 1 year.
NOTE 3: This reagent should give the characteristic color with ammonia within
10 minutes after addition, and should not produce a precipitate with small
amounts of ammonia (0.04 mg in a 50 ml^volume).
6.7 Borate buffer: Add 88 ml of 0.1 N NaOH solution to 500 ml of 0.025 M sodium
tetraborate solution (5.0 g anhydrous Na2B4O7 or 9.5 g Na2B4O7 '10H2O per
liter) and dilute to 1 liter.
6.8 Sulfuric acid, standard solution: (0.02 N, 1 ml = 0.28 mg NH3-N). Prepare a
\
stock solution of approximately 0.1 N acid by diluting 3 ml of cone. H2 SO4 (sp.
gr. 1.84) to 1 liter with CO2-free distilled water. Dilute 200 ml of this solution to
1 liter with CO2-free distilled water.
NOTE 4: An alternate and perhaps preferable method is to standardize the
approximately 0.1 N H2SO4 solution against a 0.100 N Na2CO3 solution. By
proper dilution the 0.02 N acid can then be prepared.
6.8.1 Standardize the approximately 0.02 N acid against 0.0200 N Na2CO3
solution. This last solution is prepared by dissolving 1.060 g anhydrous
Na2CO3) oven-dried at 140°C, and diluting to 1000 ml with CO2-free
distilled water.
6.9 Sodium hydroxide, 1 N: Dissolve 40 g NaOH in ammonia-free water and dilute to
1 liter.
6.10 Dechlorinatirig reagents: A number of dechlorinating reagents may be used to
remove residual chlorine prior to distillation. These include:
a. Sodium thiosulfate (1/70 N): Dissolve 3.5 g Na2S2O3 in distilled water and
dilute to 1 liter. One ml of this solution will remove 1 mg/1 of residual chlorine in
500 ml of sample.
b. Sodium arsenite (1/70 N): Dissolve 1.0 g NaAsO2 in distilled water and dilute
to 1 liter.
7. Procedure
7.1 Preparation of equipment: Add 500 ml of distilled water to an 800 ml Kjeldahl
flask. The addition of boiling chips which have been previously treated with dilute
NaOH will prevent bumping. Steam out the distillation apparatus until the
distillate shows no trace of ammonia with Nessler reagent.
7.2 Sample preparation: Remove the residual chlorine in the sample by adding
dechlorinating agent equivalent to the chlorine residual. To 400 ml of sample add
1 N NaOH (6.9); until the pH is 9.5, checking the pH during addition with a pH
meter or by use of a short range pH paper.
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7.3 Distillation: Transfer the sample, the pH of which has been adjusted to 9.5, to an
800 ml Kjeldahl flask and add 25 ml of the borate buffer (6.7). Distill 300 ml at
the rate of 6-10 ml/rnin. into 50 ml of 2% boric acid (6.4) contained in a 500 ml
Erlenmeyer flask.
NOTE 5: The condenser tip or an extention of the condenser tip must extend
below the level of the boric acid solution.
Dilute the distillate to 500 ml with distilled water and nesslerize an aliquot to
obtain an approximate value of the ammonia-nitrogen concentration. For
concentrations above 1 mg/1 the ammonia should be determined titrimetrically.
For concentrations below this value it is determined colorimetrically. The
electrode method may also be used.
7.4 Determination of ammonia in distillate: Determine the ammonia content of the
distillate titrimetrically, colorimetrically or potentiometrically as described below.
7.4.1 Titrimetric determination: Add 3 drops of the mixed indicator to the
distillate and titrate the ammonia with the 0.02 N H2 SO4, matching the
end point against a blank containing the same volume of distilled water
and H3BO3 solution.
7.4.2 Colorimetric determination: Prepare a series of Nessler tube standards as
follows:
ml of Standard
1.0 ml = 0.01 mgNH3-N mg NH3 -N/50.0 ml
0.0 0.0
0.5 0.005
1.0 0.01
2.0 0.02
3.0 0.03
4.0 0.04
5.0 0.05
8.0 0.08
10.0 0.10
Dilute each tube to 50 ml with distilled water, add 1.0 ml of Nessler
reagent (6.6) and mix. After 20 minutes read the optical densities at 425
nm against the blank. From the values obtained plot optical density
(absorbance) vs. mg NH3 -N for the standard curve.
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7.4.3 Potentiometric determination: Consult the method entitled Nitrogen,
Ammonia: Selective Ion Electrode Method in this manual.
7.4.4 It is not imperative that all standards be distilled in the same manner as
the samples. It is recommended that at least two standards (a high and
low) be distilled and compared to similar values on the curve to insure
that the distillation technique is reliable. If distilled standards do not
agree with undistilled standards the operator should find the cause of the
apparent error before proceeding.
7.5 Determine the ammonia in the distillate by nesslerizing 50 ml or an aliquot
diluted to 50 ml and reading the optical density at 425 nm as described above for
the standards. Ammonia-nitrogen content is read from the standard curve.
8. Calculations
8.1 Titrimetric
AX 0.28X1000
mg/1 NH3 -N =
where:
A = ml 0.02 N H2 SO4 used.
S = ml sample.
8.2 Spectrophotometric
AX 1000 B
mg/!NH3-N= X
DC
where:
A = mg NH3 N read from standard curve.
B = ml total distillate collected, including boric acid and dilution.
C = ml distillate taken for nesslerization.
D = ml of original sample taken.
8.3 Potentiometric
500
mg/!NH3-N= X A
D
where:
A =
-------
9. Precision and Accuracy
9.1 Twenty-four analysts in sixteen laboratories analyzed natural water samples
containing exact increments of an ammonium salt, with the following results:
Increment as
Nitrogen, Ammonia
mg N/liter
0.21
0.26
1.71
1.92
Precision as
Standard Deviation
mg N/liter
0.122
0.070
0.244
0.279
Accuracy as
Bias,
%
- 5.54
-18.12
+ 0.46
- 2.01
Bias,
mg N/liter
-0.01
-0.05
+0.01
-0.04
(FWPCA Method Study 2, Nutrient Analyses)
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NITROGEN, AMMONIA
(Selective Ion Electrode Method)
STORE! NO. 00610
1. Scope and Application
1.1 This method is applicable to the measurement of ammonia-nitrogen in drinking,
surface, and saline waters, domestic and industrial wastes.
1.2 This method covers the range from 0.03 to 1400 mg NH3-N/1. Color and
turbidity have no effect on the measurements and distillation is not necessary.
2. Summary of Method
2.1 The ammonia is determined potentiometrically using a selective ion ammonia
electrode and a pH meter having an expanded millivolt scale or a specific ion
meter.
2.2 The ammonia electrode uses a hydrophobic gas-permeable membrane to separate
the sample solution from an ammonium chloride internal solution. Ammonia in
the sample diffuses through the membrane and alters the pH of the internal
solution, which is sensed by a pH electrode. The constant level of chloride in the
internal solution is sensed by a chloride selective ion electrode which acts as the
reference electrode.
3. Sample Handling and Preservation
3.1 Preserve by refrigeration at 4°C; analyze within 24 hours. If longer holding times
are desired, preserve with 2 ml cone. H2SO4 per liter (pH<2).
4. Interferences
' 4.1 Volatile amines act as a positive interference.
4.2 Mercury interferes by forming a strong complex with ammonia. Thus the samples
cannot be preserved with mercuric chloride.
5. Apparatus
5.1 Electrometer (pH meter) with expanded mV scale or a specific ion meter.
5.2 Ammonia selective electrode, such as Orion Model 95-10 or EIL Model 8002-2.
5.3 Magnetic stirrer, thermally insulated, and Teflon-coated stirring bar.
6. Reagents
6.1 Distilled water: Special precautions must be taken to insure that the distilled
water is free of ammonia. This is accomplished by passing distilled water through
an ion exchange column containing a strongly acidic cation exchange resin mixed
with a strongly basic anion exchange resin.
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6.2 Sodium hydroxide, ION: Dissolve 400 g of sodium hydroxide in 800 ml of
distilled water. Cool and dilute to 1 liter with distilled water (6.1).
6.3 Ammonium chloride, stock solution: 1.0 ml = 1.0 mg NH3-N. Dissolve 3.819 g
NH4 Q in water and bring to volume in a 1 liter volumetric flask using distilled
water (6.1).
6.4 Ammonium chloride, standard solution: 1.0 ml = 0.01 mg NH3N. Dilute 10.0
ml of the stock solution (6.3) to 1 liter with distilled water (6.1) in a volumetric
flask.
NOTE 1: When analyzing saline waters, standards must be made up in synthetic
ocean water (SOW); found in Nitrogen, Ammonia: Automated Colorimetric
Phenate Method.
7. Procedure
7.1 Preparation of standards: Prepare a series of standard solutions covering the
concentration range of the samples by diluting either the stock or standard
solutions of ammonium chloride.
7.2 Calibration of electrometer: Place 100 ml of each standard solution in clean 150
ml beakers. Immerse electrode into standard of lowest concentration and add 1
ml of ION sodium hydroxide solution while mixing. Keep electrode in the
solution until a stable reading is obtained.
NOTE 2: The pH of the solution after the addition of NaOH must be above 11.
Caution: Sodium hydroxide must not be added prior to electrode immersion, for
ammonia may be lost from a basic solution.
7.3 Repeat this procedure with the remaining standards, going from lowest to highest
concentration. Using semilogarithmic graph paper, plot the concentration of
ammonia in mg NH3-N/1 on the log axis vs. the electrode potential developed in
the standard on the linear axis, starting with the lowest concentration at the
bottom of the scale.
7.4 Calibration of a specific ion meter: Follow the directions of the manufacturer for
the operation of the instrument.
7.5 Sample measurement: Follow the procedure in (7.2) for 100 ml of sample in 150
ml beakers. Record the stabilized potential of each unknown sample and convert
the potential reading to the ammonia concentration using the standard curve. If a
specific ion meter is used, read the ammonia level directly in mg NH3 N/l.
8. Precision and Accuracy
8.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 1.00, 0.77, 0.19, and 0.13 mg NH3-N/1, standard deviations were ±0.038,
±0.017, ±0.007, and ±0.003, respectively.
166
-------
8.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.19 and 0.13 NH3 -N/l, recoveries were 96% and 91%, respectively.
Bibliography
1. Booth, R L., and Thomas, R. F., "Selective Electrode Determination of Ammonia in
Water and Wastes", Envir. Sci. Technology, 7, p 523-526 (1973).
2. Banwart, W. L., Bremner, J. M., and Tabatabai, M. A., "Determination of Ammonium
in Soil Extracts and Water Samples by an Ammonia Electrode", Comm. Soil Sci. Plant
Anal, 3, p 449 (1972).
3. Midgley, D., and Torrance, K., "The Determination of Ammonia in Condensed Steam
and Boiler Feed-Water with a Potentiometric Ammonia Probe", Analyst, 97, p 626-633
(1972).
167
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NITROGEN, AMMONIA
(Automated Colorimetric Phenate Method)
STORE! NO. 00610
1. Scope and Application
1.1 This method covers the determination of ammonia in drinking, surface, and saline
waters, domestic and industrial wastes in the range of 0.01 to 2.0 mg/1 NH3 as N.
This range is for photometric measurements made at 630-660 nm in a 15 mm or
50 mm tubular flow cell. Higher concentrations can be determined by sample
dilution. Approximately 20 to 60 samples per hour can be analyzed.
2. Summary of Method
2.1 Alkaline phenol and hypochlorite react with ammonia to form indophenol blue
that is proportional to the ammonia concentration. The blue color formed is
intensified with sodium nitroprusside.
3. Sample Handling and Preservation
3.1 Preservation by addition of 2 ml cone. H2SO4 or 40 mg HgCl2 per liter and
refrigeration at 4°C. Note HgQ2 interference under (4.2).
4. Interferences
4.1 In sea water, calcium and magnesium ions are present in concentrations sufficient
to cause precipitation problems during the analysis. This problem is eliminated by
using 5% EDTA.
4.2 Mercury chloride, used as a preservative, gives a negative interference by
complexing with the ammonia. This is overcome by adding a comparable amount
of HgCl2 to the ammonia standards used for the preparation of the standard
curve.
4.3 Sample turbidity and color may interfere with this method. Turbidity must be
removed by filtration prior to analysis. Sample color that absorbs in the
photometric range used will also interfere.
5. Apparatus
5.1 Technicon Auto Analyzer Unit (AAI or AAII) consisting of:
5.1.1 Sampler.
5.1.2 Manifold (AAI) or Analytical Cartridge (AAII).
5.1.3 Proportioning pump.
5.1.4 Heating bath with double delay coil (AAI).
5.1.5 Colorimeter equipped with 15 mm tubular flow cell and 630-660 nm
filters. .
168
-------
5.1.6 Recorder.
5.1.7 Digital printer for AAII (optional).
6. Reagents
6.1 Distilled water: Special precaution must be taken to insure that distilled water is
free of ammonia. Such water is prepared by passage of distilled water through an
ion exchange column comprised of a mixture of both strongly acidic cation and
strongly basic anion exchange resins. The regeneration of the ion exchange
column should be carried out according to the instruction of the manufacturer.
NOTE 1: All solutions must be made using ammonia-free water.
6.2 Sulfuric acid 5N: Air scrubber solution. Carefully add 139 ml of cone, sulfuric
acid to approximately 500 ml of ammonia-free distilled water. Cool to room
temperature and dilute to 1 liter with ammonia-free distilled water.
6.3 Sodium phenolate: Using a 1 liter Erlenmeyer flask, dissolve 83 g phenol in 500
ml of distilled water. In small increments, cautiously add with agitation, 32 g of
NaOH. Periodically, cool flask under water faucet. When cool, dilute to 1 liter
with distilled water.
6.4 Sodium hypochlorite solution: Dilute 250 ml of a bleach solution containing
5.25% NaOCl (such as "Clorox") to 500 ml with distilled water. Available
chlorine level should approximate 2 to 3%. Since "Clorox" is a proprietary
product, its formulation is subject to change. The analyst must remain alert to
detecting any variation in this product significant to its use in this procedure. Due
to the instability of this product, storage over an extended period should be
avoided.
6.5 Disodium ethylenediamine-tetraacetate (EDTA) (5%): Dissolve 50 g of EDTA
(disodium salt) and approximately six pellets of NaOH in 1 liter of distilled water.
NOTE 2: On salt water samples where EDTA solution does not prevent
precipitation of cations, sodium potassium tartrate solution may be used to
advantage. It is prepared as follows:
6.5.1 Sodium potassium tartrate solution: 10% NaKC4H4O6-4H2O. To 900
ml of distilled water add 100 g sodium potassium tartrate. Add 2 pellets
of NaOH and a few boiling chips, boil gently for 45 minutes. Cover, cool,
and dilute to 1 liter with ammonia-free distilled water. Adjust pH to
5.2±.05 with H2SO4. After allowing to settle overnight in a cool place,
filter to remove precipitate. Then add 1/2, ml Brij-35 (available from
Technicon Corporation) solution and store in stoppered bottle.
6.6 Sodium nitroprusside (0.05%): Dissolve 0.5 g of sodium nitroprusside in 1 liter of
distilled water.
169
-------
6.7 Stock solution: Dissolve 3.819 g of anhydrous ammonium chloride, NH4C1,
dried at.lQ5°C, in distilled water, and dilute to 1000 ml. 1.0 ml = 1.0 mg
NH3-N.
6.8 Standard solution A: Dilute 10.0 ml of stock solution (6.7) to. 1000 ml with
distilled water. 1.0 ml = 0.01 mg NH3 -N.
6.9 Standard solution B: Dilute 10.0 ml of standard solution A (6.8) to 100.0 ml
with distilled water. 1.0 ml = 0.001 mg NH3-N.
6.10 Using standard solutions A and B, prepare the following standards in 100 ml
volumetric flasks (prepare fresh daily):
NH3 -N, mg/1 ml Standard Solution/100 ml
Solution B
(O-Qli. 1.0
0.02; 2.0
,:0.05 5.0
0.10 10.0
;:... Solution A
;0.20 2.0
0.50 5.0
0,80 8.0
1,00 10.0
1.50 15.0
2.00 20.0
NOTE 3: When saline water samples are analyzed, Substitute Ocean Water
(SOW) should be used for preparing the above standards used for the
calibration curve; otherwise, distilled water is used. If SOW is used, subtract its
blank background response from the standards before preparing the standard
curve.
Substitute Ocean Water (SOW)
g/1
24.53
5.20
4.09
1.16
0.70
NaHCO3
KBr
H3BO3
SrCl2
NaF
NaCl . 24.53 NaHCO3 0.20
MgCl2 5.20 KBr 0.10
Na2SO4 4.09 H3BO3 0.03
CaCl2 1.16 SrCl, 0.03
KC1 0.70 NaF 0.003
170
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7. Procedure
7.1 Any marked variation in acidity or alkalinity among samples should be
eliminated, since the intensity of the color used to quantify the concentration is
pH dependent. Likewise, the pH of the wash water and the standard ammonia
solutions should approximate that of the samples. For example, if the samples
have been preserved with 2 ml cone. H2 SO4 /liter, the wash water and standards
should also contain 2 ml cone. H2 SO4 /liter.
7.2 For a working range of 0.01 to 2.00 mg NH3N/l (AAI), set up the manifold as
shown in Figure 1. -For a working range of .01 to 1.0 mg NH3 -N/l (AAII), set up
the manifold as shown in Figure 2. Higher concentrations may be accommodated
by sample dilution.
7.3 Allow both colorimeter and recorder to warm up for 30 minutes. Obtain a stable
baseline with all reagents, feeding distilled water through sample line.
7.4 For the AAI system, sample at a rate of 20/hr, 1:1. For the AAII use a 60/hr 6:1
cam with a common wash.
7.5 Arrange ammonia standards in sampler in order of decreasing concentration of
nitrogen. Complete loading of sampler tray with unknown samples.
7.6 Switch sample line from distilled water to sampler and begin analysis.
8. Calculations
8.1 Prepare appropriate standard curve derived from processing ammonia standards
through manifold. Compute concentration of samples by comparing sample peak-
heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory, (MDQARL), using surface water samples at concentrations
of 1.41, 0.77, 0:59, and 0.43 mg NH3-N/1, the standard deviation was ±0.005.
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.16 and 1.44 mg NH3-N/1, recoveries were 107% and 99%, respectively.
Bibliography
1. Hiller, A., and Van Slyke, D., "Determination of Ammonia in Blood", J. Biol. Chem.
702, p 499 (1933).
2. O'Connor, B., Doobs, R., Villiers, B., and Dean, R., "Laboratory Distillation of
Municipal Waste Effluents", JWPCF 39, R 25 (1967).
3. Fiore, J., and O'Brien, J. E., "Ammonia Determination by Automatic Analysis",
Wastes Engineering 33, p 352 (1962).
4. A wetting agent recommended and supplied by the Technicpn- Corporation for use in
AutoAnalyzers.
171
-------
5. ASTM "Manual on Industrial Water and Industrial Waste Water", 2nd Ed., 1966
printing, p 418.
6. Booth, R. L., and Lobring, L. B., "Evaluation of the AutoAnalyzer II: A Progress
Report" on Advances in Automated Analysis: 1972 Technicon International Congress,
Vol. 8, p 7-10, Mediad Incorporated, Tarrytown, N.Y., (1973).
172
-------
PROPORTIONING
OJ.
_L MIXING CO
!GE MIXING C
1
HEATING (
BATH 37°C V
1
WASH WATER
TO SAMPLER
IL SM
OIL 0000
LM
OOOOOOOO
f
LM
OOOOOOOO
SM OOOO
) f
i
MB»
1
1
WASTE
r~
<
PUMP
P B
G G
R R
G G
W W
W W
R R|
ml/mln
2.9 WASH
2.0 SAMPLE °
SAMPLER
0.8 EDTA 20/hr.
2.0 AIR*
0.6 PHENOLATE
0.6 HYPOCHLORITE
0.6 NITROPRUSSIDE
P PI2.5
« | WASTE
RECORDER
*r
i " i
-- ''
COLORIMETER
15mm FLOW CELL
650-660 nm FILTER
5N H ^
2 4
FIGURE 1 AMMONIA MANIFOLD AA I
-------
HEATING
BATH
50° C
WASH WATER
TO SAMPLER
OQQQ
WASTE
PROPORTIONING
PUMP
ml/min.
2.0 WASH
SAMPLER
GO/ hr.
6 = 1
W
BLACK
BLUE
0.23 AIR'
0.42 SAMPLE
0.8 EDTA
0.42 PHENOLATE
0.32 HYPOCHLORITE
0.42 NITROPRUSSIDE
1.6
WASTE
DIGITAL
PRINTER
COLORIMETER
50 mm FLOW CELL
650-660 nm FILTER
'SCRUBBED THROUGH
5N H2S04
FIGURE 2. AMMONIA MANIFOLD AA II
-------
NITROGEN, KJELDAHL, Total
STORET NO. 00625
1. Scope and Application
1.1 This method covers the determination of total Kjeldahl nitrogen in drinking,
surface, and saline waters, domestic and industrial wastes. The procedure converts
nitrogen components of biological origin such as amino acids, proteins and
peptides to ammonia, but may not convert the nitrogenous compounds of some
industrial wastes such as amines, nitro compounds, hydrazones, oximes, semi-
carbazones and some refractory tertiary amines.
1.2 Three alternatives are listed for the determination of ammonia after distillation:
the titrimetric method which is applicable to concentrations above 1 mg N/liter;
the Nesslerization method which is applicable to concentrations below 1 mg
N/liter; and the potentiometric method applicable to the range 0.05 to 1400 mg/1.
1.3 This method is described for macro and micro glassware systems.
2. Definitions
2.1 Total Kjeldahl nitrogen is defined as the sum of free-ammonia and organic
nitrogen compounds which are converted to ammonium sulfate (NH4)2SO4,
under the conditions of digestion described below.
2.2 Organic Kjeldahl nitrogen is defined as the difference obtained by subtracting the
free-ammonia value (cf Nitrogen, Ammonia, this manual) from the total Kjeldahl
nitrogen value. This may be determined directly by removal of ammonia before
digestion.
3. Summary of Method
3.1 The sample is heated in the presence of cone, sulfuric acid, K2SO4 and HgSO4
and evaporated until SO3 fumes are obtained and the solution becomes colorless
or pale yellow. The residue is cooled, diluted, and is treated and made alkaline
with a hydroxide-thiosulfate solution. The ammonia is distilled and determined
after distillation by Nesslerization, titrimetry, or potentiometrically.
4. Sample Handling and Preservation
4.1 Samples may be preserved by addition of 2 ml of cone. H2SO4 or 40 mg HgCl2
per liter and stored at 4°C. Even when preserved in this manner, conversion of
organic nitrogen to ammonia may occur. Preserved samples should be analyzed as
soon as possible.
175
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5. Apparatus
5.1 Digestion apparatus: A Kjeldahl digestion apparatus with 800 or 100 ml flasks
and suction takeoff to remove SO3 fumes and water.
5.2 Distillation apparatus: The macro Kjeldahl flask is connected to a condenser and
an adaptor so that the distillate can be collected. Micro Kjeldahl steam distillation
apparatus is commercially available.
5.3 Spectrophotometer for use at 400 to 425 nm with a light path of 1 cm or longer.
6. Reagents
6.1 Distilled water should be free of ammonia. Such water is best prepared by the
passage of distilled water through an ion exchange column containing a strongly
acidic cation exchange resin mixed with a strongly basic anion exchange resin.
Regeneration of the column should be carried out according to the manufac-
turer's instructions.
NOTE 1: All solutions must be made with ammonia-free water.
6.2 Mercuric sulfate solution: Dissolve 8 g red, mercuric oxide (HgO) in 50 ml of 1:5
sulfuric acid (10.0 ml cone. H2SO4: 40 ml distilled water) and dilute to 100ml
with distilled water.
6.3 Sulfuric acid-mercuric sulfate-potassium sulfate solution: Dissolve 267 g K2SO4
in 1300 ml distilled water and 400 ml cone. H2SO4. Add 50 ml mercuric sulfate
solution (6.2) and dilute to 2 liters with distilled water.
6.4 Sodium hydroxide-sodium thiosulfate solution: Dissolve 500 g NaOH and 25 g
Na2S2O3 -5H2O in distilled water and dilute to 1 liter.
6.5 Phenolphthalein indicator solution: Dissolve 5 g phenolphthalein in 500 ml 95%
ethyl alcohol or isopropanol and add 500 ml distilled water. Add 0.02 N NaOH
dropwise until a faint, pink color appears.
6.6 Mixed indicator: Mix 2 volumes of 0.2% methyl red in 95% ethanol with 1
volume of 0.2% methylene blue in ethanol. Prepare fresh every 30 days.
6.7 Boric acid solution: Dissolve 20 g boric acid, H3BO3, in water and dilute to 1 liter
with distilled water.
6.8 Sulfuric acid, standard solution: (0.02 N) 1 ml = 0.28 mg NH3-N. Prepare a
stock solution of approximately 0.1 N acid by diluting 3 ml of cone. H2SO4 (sp.
gr. 1.84) to 1 liter with CO2-free distilled water. Dilute 200 ml of this solution to
1 liter with CO2-free distilled water. Standardize the approximately 0.02 N acid
so prepared against 0.0200 N Na2CO3 solution. This last solution is prepared by
dissolving 1.060 g anhydrous Na2CO3, oven-dried at 140°C, and diluting to 1 liter
with C02-free distilled water.
176
-------
NOTE 2: An alternate and perhaps preferable method is to standardize the
approximately 0.1 N H2SO4 solution against a 0.100 N NaCO3 solution. By
proper dilution the .02 N acid can then be prepared.
6.9 Ammonium chloride, stock solution: 1.0 ml = 1.0 mg NH3N. Dissolve 3.819 g
NH4 Cl in water and make up to 1 liter in a volumetric flask with distilled water.
6.10 Ammonium chloride, standard solution: 1.0 ml = 0.01 mg NH3-N. Dilute 10.0
ml of the stock solution (6.9) with distilled water to 1 liter in a volumetric flask.
6.11 Nessler reagent: Dissolve 100 g of mercuric iodide in a small volume of distilled
water. Add this mixture slowly, with stirring, to a cooled solution of 160 g of
NaOH in 500 ml of distilled water. Dilute the mixture to 1 liter. The solution is
stable for at least one year if stored in a pyrex bottle out of direct sunlight.
NOTE 3: Reagents 6.8, 6.9, 6.10, and 6.11 are identical to reagents 6.8, 6.2, 6.3,
and 6.6 described under Nitrogen, Ammonia (Distillation Procedure).
7. Procedure
7.1 The distillation apparatus should be pre-steamed before use by distilling a 1:1
mixture of distilled water and sodium hydroxide-sodium thiosulfate solution (6.4)
until the distillate is ammonia-free. This operation should be repeated each time
the apparatus is out of service long enough to accumulate ammonia (usually 4
hours or more).
7.2 Macro Kjeldahl system
7.2.1 Place a measured sample or the residue from the distillation in the
ammonia determination (for Organic Kjeldahl only) into an 800 ml
Kjeldahl flask. The sample size can be determined from the following
table:
Kjeldahl Nitrogen Sample Size
in Sample, ing/1 ml
0-5 500
5- 10 250
10- 20 100
20-50 50.0
50 - 500 25.0
Diluic the sample, if required, to 500 ml with distilled water, and add
100 ml sulfuric acid-mercuric sulfate-potassium sulfate solution (6.3)
177
-------
(Note 4), and evaporate the mixture in the Kjeldahl apparatus until SO3
fumes are given off and the solution turns colorless or pale yellow.
Continue heating for 30 additional minutes. Cool the residue and add
300 ml distilled water.
NOTE 4: Digesting the sample with 1 Kel-Pac (Olin-Matheson) and 20 ml
cone. H2SO4 is acceptable. Cut the end from the package and empty the
contents into the digestion flask; discard the container.
7.2.2 Make the digestate alkaline by careful addition of 100 ml of sodium
hydroxide-thiosulfate solution (6.4) without mixing.
NOTE 5: Slow addition of the heavy caustic solution down the tilted
neck of the digestion flask will cause heavier solution to underlay the
aqueous sulfuric acid solution without loss of free-ammonia. Do not mix
until the digestion flask has been connected to the distillation apparatus.
7.2.3 Connect the Kjeldahl flask to the condenser with the tip of condenser
(or an extension of the condenser tip) below the level of the boric acid
solution (6.7) in the receiving flask.
7.2.4 Distill 300 ml at the rate of 6-10 ml/min., into 50 ml of 2% boric acid
(6.7) contained in a 500 ml Erlenmeyer flask.
7.2.5 Dilute the distillate to 500 ml in the flask. These flasks should be marked
at the 350 and the 500 ml volumes. With such marking, it is not
necessary to transfer the distillate to volumetric flasks. For concentra-
tions above 1 mg/1, the ammonia can be determined titrimetrically. For
concentrations below this value, it is determined colorimetrically. The
potcntiometric method is applicable to the range 0.05 to 1400 mg/1.
7.3 Micro Kjeldahl system
7.3.1 Place 50.0 ml of sample or an aliquot diluted to 50 ml in a 100 ml
Kjeldahl flask and add 10 ml sulfuric acid-mercuric sulfate-potassium
sulfatc solution (6.3). Evaporate the mixture in the Kjeldahl apparatus
until SO3 fumes are given off and the solution turns colorless or pale
yellow. Then digest for an additional 30 minutes. Cool the residue and
add 30 ml distilled water.
. 7.3.2 Make the digestate alkaline by careful addition of 10 ml of sodium
hydroxide-thiosulfate solution (6.4) without mixing. Do not mix until
the digestion flask has been connected to the distillation apparatus.
7.3.3 Connect the Kjeldahl flask to the condenser with the tip of condenser or
an extension of the condenser tip below the level of the boric acid
solution (6.7) in the receiving flask or 50 ml short-form Nessler tube.
178
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7.3.4, Steam distill 30 ml at the rate of 6-10 ml/min., into 5 ml of 2% boric
/ acid (6.7).
7.3.5 Dilute the distillate to 50 ml. For concentrations above 1 mg/1 the
ammonia can be determined titrimetrically. For concentrations below
this value, it is determined colorimetrically. The potentiometric method
is applicable to the range 0.05 to 1400 mg/1.
7.4 Determination of ammonia in distillate: Determine the ammonia content of the
distillate titrimetrically, colorimetrically, or potentiometrically, as described
below.
7.4.1 Titrimetric determination: Add 3 drops of the mixed indicator (6.6) to
the distillate and titrate the ammonia with the 0.02 N H2SO4, (6.8),
matching the endpoint against a blank containing the same volume of
distilled water and H3BO3 (6.7) solution.
7.4.2 Colorimetric determination: Prepare a series of Nessler tube standards as
follows:
ml as Standard
1.0 ml =0.01 mgNH3-N mg NH3-N/50.0 ml
0.0 0.0
0.5 0.005
i.O 0.010
2.0 0.020
4.0 ' 0.040
5.0 0.050
8.0 0.080
10.0 0.10
To the standards and distilled samples, add 1 ml of Nessler reagent (6.11)
and mix. After 20 minutes read the optical densities at 425 nm against
the blank. From the values obtained for the standards plot optical
density (absorbance) vs. mg NH3N for the standard curve. Read the
ammonia-nitrogen in mg for the samples from the standard curve.
7.4.3 Potentiometric determination: Consult the method entitled Nitrogen,
Ammonia: Selective Ion Electrode Method, in this manual.
7.4.4 It is not imperative that all standards be treated in the same manner as
179
-------
the samples. It is recommended that at least 2 standards (a high and low)
be digested, distilled, and compared to similar values on the curve to insure
that the digestion-distillation technique is reliable. If treated standards
do not agree with untreated standards the operator should find the cause
of the apparent error before proceeding.
8. Calculation
8.1 If the titrimetric procedure is used calculate Total Kjeldahl Nitrogen, in mg/1, in
the original sample as follows:
(A-B)N X F X 1000
Total Kjeldahl nitrogen, mg/1 =
where:
A = milliliters of standard 0.020 N H2 SO4 solution used in titrating sample.
B = milliliters of standard 0.020 N H2SO4 solution used in titrating blank.
N = normality of sulfuric acid solution.
F = milliequivalent weight of nitrogen (14 mg).
S = milliliters of sample digested.
If the sulfuric acid is exactly 0.02 N the formula is shortened to:
(A-B) X 280
TKN, mg/1 =
S
8.2 If the Nessler procedure is used, calculate the Total Kjeldahl Nitrogen, in mg/1, in
the original sample as follows:
AX 1000 B
TKN, mg/1 = X
ml sample C
where:
A = mg NH3 N read from curve.
B = ml total distillate collected including the H3 BO3.
C= ml distillate taken for Nesslerization.
8.3 Calculate Organic Kjeldahl Nitrogen in mg/1, as follows:
Organic Kjeldahl Nitrogen = TKN -(NH3 -N.)
8.4 Potentiometric determination: Calculate Total Kjeldahl Nitrogen, in mg/1, in the
original sample as follows:
500
TKN, mg/l= XA
180
-------
where:
A = mg NH3 -N/l from electrode method standard curve.
D = ml of original sample taken.
9. Precision
9.1 Thirty-one analysts in twenty laboratories analyzed natural water samples
containing exact increments of organic nitrogen, with the following results: .
Increment as
Nitrogen, Kjeldahl
mg N/liter
0.20
0.31
4.10
4.61
Precision as
Standard Deviation
mg N/liter
0.197
0.247
1.056
1.191
Accuracy as
Bias,
%
+15.54
+ 5.45
+ 1.03
- 1.67
Bias,
mg N/liter
+0.03
+0.02
+0.04
-0.08
(FWPCA Method Study 2, Nutrient Analyses)
181
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NITROGEN, KJELDAHL, TOTAL
(Automated Phenate Method)
STORET NO. 00625
1. Scope and Application
1.1 This automated method may be used to determine Kjeldahl nitrogen in surface
and saline waters. The applicable range is 0.05 to 2.0 mg N/l. Approximately 20
samples per hour can be analyzed.
2. Summary of Method
2.1 The sample is automatically digested with a sulfuric acid solution containing
potassium sulfate and mercuric sulfate as a catalyst to convert organic nitrogen to
ammonium sulfate. The solution is then automatically neutralized with sodium
hydroxide solution and treated with alkaline phenol reagent and sodium
hypochlorite reagent. This treatment forms a blue color designated as indophenol.
Sodium nitroprusside, which increases the intensity of the color, is added to
obtain necessary sensitivity for measurement of low level nitrogen.
3. Definitions
3.1 Total Kjeldahl nitrogen is defined as the sum of free-ammonia and of organic
nitrogen compounds which are converted to (NH4 )2 SO4 under the conditions of
digestion which are specified below.
3.2 Organic Kjeldahl nitrogen is defined as the difference obtained by subtracting the
free-ammonia value from the total Kjeldahl nitrogen value. Also, organic Kjeldahl
nitrogen may be determined directly by removal of ammonia before digestion.
4. Sample Handling and Preservation
4.1 Samples may be preserved by addition of 2 ml of cone. H2 SO4 or 40 mg HgCl2
per liter and refrigeration at 4°C.
5. Interferences
5.1 Iron and chromium ions tend to catalyze while copper ions tend to inhibit the
indophenol color reaction.
6. Apparatus
6.1 Technicon AutoAnalyzer consisting of:
, 6.1.1 Sampler II, equipped with continuous mixer.
6.1.2 Two proportioning pumps.
182
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6.1.3 Manifold I.
6.1.4 Manifold II.
6.1.5 Continuous digester.
6.1.6 Planetary pump.
6.1.7 Five-gallon Carboy fume-trap.
6.1.8 80°C Heating bath.
6.1.9 Colorimeter equipped with 50 mm tubular flow cell and 630 nm filters.
6.1.10 Recorder equipped with range expander.
6.1.11 Vacuum pump.
7. Reagents
7.1 Distilled water: Special precaution must be taken to insure that distilled water is
free of ammonia. Such water is prepared by passage of distilled water through an
ion exchange column comprised of a mixture of both strongly acidic cation and
strongly basic anion exchange resins. Furthermore, since organic contamination
may interfere with this analysis, use of the resin Dowex XE-75 or equivalent
which also tends to remove organic impurities is advised. The regeneration of the
ion exchange column should be carried out according to the instruction of the
manufacturer.
NOTE 1: All solutions must be made using ammonia-free water.
7,2 Sulfuric acid: As it. readily absorbs ammonia, special precaution must also be
taken with respect to its use. Do not store bottles reserved for this determination
in areas of potential ammonia contamination.
7.3 EDTA (2% solution): Dissolve 20 g disodium ethylenediamine tetraacetate in 1
liter of distilled water. Adjust pH to 10.5-11 with NaOH (7.4).
7.4 Sodium hydroxide (30% solution): Dissolve 300 g NaOH in 1 liter of distilled
water.
NOTE 2: The 30% sodium hydroxide should be sufficient to neutralize the
digestate. In rare cases it may be necessary to increase the concentration of
sodium hydroxide in this solution to insure neutralization of the digested sample
in the manifold at the water jacketed mixing coil.
7.5 Sodium nitroprusside, (0.05% solution): Dissolve 0.5 g Na2Fe(CN)5NO-2H2O in
1 liter distilled water.
7.6 Alkaline phenol reagent: Pour 550 ml liquid phenol (88-90%) slowly with mixing
into 1 liter of 40% (400 g per liter) NaOH. Cool and dilute to 2 liters with
distilled water.
183
-------
7.7 Sodium hypochlorite (1% solution): Dilute commercial "Clorox"-200 ml to 1
liter with distilled water. Available chlorine level should be approximately 1%.
Due to the instability of this product, storage over an extended period should be
avoided.
7.8 Digestant mixture: Place 2 g red HgO in a 2 liter container. Slowly add, with
stirring, 300 ml of acid water (100 ml H2SO4 + 200 ml H2O) and stir until cool.
Add 100 ml 10% (10 g per 100 ml) K2SO4. Dilute to 2 liters with cone, sulfuric
acid (approximately 500 ml at a time, allowing time for cooling). Allow 4 hours
for the precipitate to settle or filter through glass fiber filter.
7.9 Stock solutions: Dissolve 4.7619 g of pre-dried (1 hour at 105°C) ammonium
sulfate in distilled water and dilute to 1.0 liter in a volumetric flask. 1.0 ml = 1.0
mg N.
7.10 Standard solution: Dilute 10.0 ml of stock solution (7.9) to 1000 ml. 1.0 ml =
0.01 mgN.
7.11 Using the standard solution (7 .10), prepare the following standards in 100 ml
volumetric flasks:
Cone., mg N/l ml Standard Solution/100 ml
0.00 0.0
0.05 0.5
0.10 1.0
0.20 2.0
0.40 4.0
0.60 '' 6.0
0.80 8.0
1.00 10.0
1.50 15.0
2.00 20.0
8. Procedure
8.1 Set up manifolds as shown in Figures 1, 2, and 3.
8.1.1 In the operation of manifold No. 1, the control of four key factors is
required to enable manifold No. 2 to receive the mandatory representa-
tive feed. First, the digestant flowing into the pulse chamber (PC-1)
must be bubble free; otherwise, air will accumulate in A-7, thus altering
184
-------
the ratio of sample to digestant in digester. Second, in maintaining even
flow from the digestor helix, the peristaltic pump must be adjusted to
cope with differences in density of the digestate and the wash water.
Third, the sample pick-up rate from the helix must be precisely adjusted
to insure that the entire sample is aspirated into the mixing chamber.
And finally, the contents of the "Mixing Chamber" must.be kept
homogeneous by the proper adjustment of the air bubbling rate.
8.1.2 In the operation of manifold No. 2, it is important in the neutralization
of the digested sample to adjust the concentration of the NaOH so that
the waste from the C-3 debubbler is slightly acid to Hydrion B paper.
8.1.3 The digestor temperature is 390°C for the first stage and 360°C for the
second and third stages.
8.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain stable baseline.
8.3 Set sampling rate of Sampler II at 20 samples per hour, using a sample to wash
ratio of 1 to 2 (1 minute sample, 2 minute wash).
8.4 Arrange various standards in sampler cups in order of increasing concentration.
Complete loading of sampler tray with unknown samples.
8.5 Switch sample line from distilled water to sampler and begin analysis.
9. Calculation
9.1 Prepare standard curve by plotting peak heights of processed standards against
concentration values. Compute concentration of samples by comparing sample
peak heights with standard curve.
9.2 Any sample that has a computed concentration that is less than 10% of the
sample run immediately prior to it must be rerun.
10. Precision and Accuracy
10.1 Six laboratories analyzed four natural water samples containing exact increments
of organic nitrogen compounds, with the following results:
185
-------
Increment as
Kjeldahl-Nitrogen
mg N/liter
1.89
2.18
5.09
5.81
Precision as
Standard Deviation
Kjeldahl-N mg N/liter
0.54
0.61
1.25
1.85
Accuracy as
Bias,
%
-24.6
-28.3
-23.8
-21.9
Bias,
mg N/liter
-0.46
-0.62
-1.21
-1.27
(FWQA Method Study 4, Automated Methods In preparation).
Bibliography
1. Kammerer, P. A., Rodel, M. G., Hughes, R. A., and Lee, G. F., "Low Level Kjeldahl
Nitrogen Determination on the Technicon AutoAnalyzer." Environmental Science and
Technology 7,340(1967).
2. McDaniel, W. H., Hemphill, R. N., Donaldson, W. T., "Automatic Determination of
Total Kjeldahl Nitrogen in Estuarine Waters." Presented at Technicon Symposium on
Automation in Analytical Chemistry, New York, October 3, 1967.
3. B. O'Connor, Dobbs, Villiers, and Dean, "Laboratory Distillation of Municipal Waste
Effluents". JWPCF 39, R 25, 1967.
186
-------
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FIGURE 1. KJELDAHL NITROGEN - MANIFOLD 1 AA-I
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CONTINUOUS DIGESTER & MIXING CHAMBER ASSEMBLY
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FIGURE 2. KJELDAHL NITROGEN AA-I
-------
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FIGURE 3. KJELDAHL NITROGEN MANIFOLD 2. A A-I
-------
NITROGEN, KJELDAHL, TOTAL
(Automated Selenium Method)
STORET NO. 00625
1. Scope and Application
1.1 This automated method may be used to determine total Kjeldahl nitrogen in
drinking and surface waters, domestic and industrial wastes. This method cannot
be used on saline waters. The applicable range is 0.1 to 10.0 mg/1. Approximately
15 samples per hour can be analyzed.
2. Summary .of Method
2.1 The sample is automatically digested with a 'sulfuric acid solution containing
selenium dioxide and perchloric acid to convert organic nitrogen to ammonium
sulfate. The solution is then treated with sodium hydroxide, alkaline phenol and
sodium hypochlorite to form a blue color designated as indophenol. Sodium
nitroprusside, which increases the intensity of the color, is added to obtain
necessary sensitivity and eliminate interference of iron and manganese.
3. Definitions
3.1 Total Kjeldahl nitrogen is defined as the sum of free ammonia and of organic
compounds which are converted to (NH4 )2 SO4 under the conditions of digestion
which are specified below.
3.2 Organic Kjeldahl nitrogen is defined as the difference obtained by subtracting the
free ammonia from the total Kjeldahl nitrogen value.
4. Sample Handling and Preservation
4.1 Samples may be preserved by addition of 2 ml of cone. H2SO4 or 40 mg HgCl2
per liter and refrigeration at 4°C.
5. Apparatus
5.1 Technicon AutoAnalyzer consisting of:
5.1.1 Sampler.
5.1.2 Two manifolds (See Figures 1 and 2 or 3).
5.1.3 Two proportioning pumps.
5.1.4 Continuous digestor (speed 6.7 rpm).
5.1.5 Vacuum pump.
5.1.6 Two five gallon glass carboys.
5.1.7 Colorimeter equipped with a 15 or 50 mm flow cell and a 630 or 650 nm
filter.
5.1.8 Recorder.
190
-------
6. Reagents for AAI
6.1 Ammonia-free water: Ammonia-free water is prepared by passage of distilled
water through an ion exchange column comprised of a mixture of both strongly
acidic cation and strongly basic anion exchange resins. Dowex XE-75 or
equivalent is advised.
NOTE 1: All solutions must be made using ammonia-free water.
6.2 Sulfuric acid: As it readily absorbs ammonia, special precaution must also be
taken with respect to its use. Do not store bottles reserved for this determination
in areas of potential ammonia contamination.
6.3 Digestion mixture: Dissolve 3 g selenium dioxide in 50 ml of distilled water. Add
20 ml of perchloric acid (67-70%). Dilute slowly to 1 liter .with cone, sulfuric acid
(6.2).
6.4 Sodium hydroxide-tartrate: Dissolve 350 g NaOH and 50 g of KNaC4H4O6
4H2O in 700 ml of distilled water. Allow to cool and dilute to 1 liter.
6.5 Alkaline phenol: Dissolve 120 g of phenol in 500 ml of distilled water. Add 31 g
NaOH. Dilute to 1 liter with distilled water.
6.6 Sodium hypochlorite: Dilute 200 ml of fresh "Clorox" to 1 liter with distilled
water. Caution: Do not store "Clorox" for extended periods; it is not stable.
6.7 Sodium nitroprusside: Dissolve 0.5 g of sodium nitroprusside in 1 liter of distilled
water.
6.8 Stock solution: Dissolve 3.819 g of predried (1 hour at 105°C) ammonium
chloride in distilled water and dilute to 1 liter in a volumetric flask. 1.0 ml = 1.0
mgN.
6.9 Standard solution A: Dilute 100.0 ml of stock solution' (6.8) to 1 liter in a
volumetric flask. 1.0 ml = 0.10 mg N.
6.10 Standard solution B: Dilute 10.0 ml of standard solution A (6.9) to 100.0 ml. 1.0
ml = 0.01mgN. (See dilution table on p. 192.)
7. Reagents for AAII
All reagents listed for AAI, Section 6, except the digestion mixture and the sodium
hydroxide-tartrate solution.
7.1 Digestion mixture: Dissolve 3 g selenium dioxide in 100 ml of distilled water. Add
3 ml of perchloric acid (67-70%). Dilute slowly to 1 liter with cone, sulfuric acid
(6.2).
7.2 Sodium hydroxide-tartrate: Dissolve 270 g NaOH and 50 g KNaC4H4O6 4H2O
in 700 ml of distilled water. Allow to cool and dilute to 1 liter.
191
-------
ml of Standard Solution/100 ml Cone, mg N/l
Solution B
1.0 0.1
2.0 0.2
5.0 0.5
10.0 1.0
Solution A
2.0 2.0
5.0 5.0
8.0 8.0
10.0 10.0
8. Procedure ,
8.1 Set up manifolds as shown in Figures 1 and 2 or 3.
8.1.1 In the operation of manifold No. 1, the acidflex tubing should be in
good condition at all times. The life of the tubing can be extended by air
drying after each run.
8.1.2 The digestor temperature is 390-400°C (4.2 ampere) for the first stage
and 370-380°C (7.0 ampere) for the second and third stages.
8.2 Allow digestor, colorimeter and recorder to warm up for 30 minutes. Run a
baseline with all reagents, feeding distilled water through the sample line.
8.3 Sampling rate:
8.3.1 Large sampler use 1 minute, 15 second sample and 2 minute, 30
second wash.
8.3.2 Sampler II or IV set sampling rate at 20 samples per hour using a
sample to wash ratio of 1:2 (1 minute sample, 2 minute wash).
8.4 Arrange series of standards in sampler cups or test tubes in order of decreasing
concentration.
8.5 Switch sample line from distilled water sample and begin analysis.
NOTE 2: During sampling, sample must be agitated;
9. Calculations
9.1 Prepare standard curve by plotting peak heights of processed standard against
concentration values. Compute concentration of samples by comparing sample
peak heights with standard curve.
192
-------
10. Precision and Accuracy
10.1 In a single laboratory (MDQARL), using surface water samples of concentra-
tions of 0.32, 1.05, 1.26, and 4.30 mg N/l, the precision was ±0.09, ±0.05,
±0.09, and ±0.14 mg N/l, respectively.
10.2 In a single laboratory (MDQARL), using a variety of domestic and industrial
wastes ranging from 23 to 68 mg N/l, recoveries were 91 to 102%.
Bibliography
1. Technicon Auto Analyzer Methodology Industrial Method, 30-69A, (1969).
193
-------
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FIGURE 2. TKN, AUTOMATED SELENIUM METHOD, MANIFOLD 2 AA-I
-------
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METHOD, MANIFOLD 2 AA II
-------
NITROGEN, NITRATE
(Brucine)
STORET NO. 00620
1. Scope and Application
1.1 This method is applicable to the analysis of drinking, surface, and saline waters,
domestic and industrial wastes. Modification can be made to remove or correct
for turbidity, color, salinity, or dissolved organic compounds in the sample.
1.2 The applicable range of concentrations is 0.1 to 2 mg NO3 N/liter.
2. Summary of Method
2.1 This method is based upon the reaction of the nitrate ion with brucine sulfate
in a 13 N H2SO4 solution at a temperature of 100°C. The color of the resulting
complex is measured at 410 nm. Temperature control of the color reaction is
. extremely critical.
3. Sample Handling and Preservation
3.1 Samples may be preserved by addition of 2 ml cone. H2SO4/liter or by
addition of 40 mg HgQ2 per liter and storage at 4°C.
4. Interferences
4.1 Dissolved organic matter will cause an off color in 13 N H2SO4 and must be
compensated for by additions of all reagents except the brucine-sulfanilic acid
reagent. This also applies to natural color present not due to dissolved organics.
4.2 The effect of salinity is eliminated by addition of sodium chloride to the
blanks, standards and samples.
4.3 All strong oxidizing or reducing agents interfere. The presence of oxidizing
agents may be determined by the addition of orthotolidine reagent.
4.4 Residual chlorine interference is eliminated by the addition of sodium arsenite.
4.5 Ferrous and ferric iron and quadrivalent manganese give slight positive
interferences, but in concentrations less than 1 mg/1 these are negligible.
4.6 Uneven heating of the samples and standards during the reaction time will
result in erratic values. The necessity for absolute control of temperature
during the critical color development period cannot be too strongly em-
phasized.
5. Apparatus
5.1 Spectrophotometer or filter photometer suitable for measuring optical densities
at410nm.
197
-------
5.2 Sufficient number of 40-50 nil glass sample tubes for reagent blanks, standards,
and samples.
5.3 Neoprene coated wire racks to hold sample tubes.
5.4 Water bath suitable for use at 100°C. This bath should contain a stirring
mechanism so that all tubes are at the same temperature and should be of
sufficient capacity to accept the required number of tubes without significant
drop in temperature when the tubes are immersed.
5.5 Water bath suitable for use at 10-15°C.
6. Reagents
6.1 Distilled water free of nitrite and nitrate is to be used in preparation of all
reagents arid standards.
6.2 Sodium chloride solution'(300 g/1): Dissolve 300 g NaCl in distilled water and
dilute to 1 liter.
6.3 Sulfuric acid solution: Carefully add 500 ml cone. H2SO4 to 125 ml distilled
water. Cool and keep tightly stoppered to prevent absorption of atmospheric
moisture.
6.4 Brucine-suifanilic acid reagent: Dissolve 1 g brucine sulfate [(C23H26N2
O4)2-H2SO4-7H2O] and 0.1 gsulfanilic acid (NH2C6H4SO3H-H2O) in 70 ml
hot distilled water. Add 3 ml cone. HC1, cool, mix and dilute to 100 ml with
distilled water. Store in a dark bottle at 5°C. This solution is stable for several
months; the pink color that develops slowly does not effect its usefulness. Mark
bottle with warning; CA UTION: Brucine Sulfate is toxic; take care to avoid
ingestion.
6.5 Potassium nitrate stock solution: 1.0 ml = 0.1 mg NO3-N. Dissolve 0.7218 g
anhydrous potassium nitrate (KNO3 ) in distilled water and dilute to 1 liter in a
volumetric flask. Preserve with 2 ml chloroform per liter. This solution is stable
for at least 6 months.
6.6 Potassium nitrate standard solution: 1.0 ml = 0.001 mg NO3-N. Dilute 10.0
ml of the stock solution (6.5) to 1 liter in a volumetric flask. This standard
solution should be prepared fresh weekly.
6.7 Acetic acid (1 + 3): Dilute 1 volume glacial acetic acid (CH3COOH) with 3
volumes of distilled water.
6.8 Sodium hydroxide: Dissolve 40 g of NaOH in distilled water. Cool and dilute to
1 liter.
7. Procedure
7.1 Adjust the pH of the samples to approximately 7 with acetic acid (6.7) or
sodium hydroxide (6.8). If necessary, filter to remove turbidity.
198
-------
7.2 Set up the required number of sample tubes in the rack to handle reagent
blank, standards and samples. Space tubes evenly throughout the rack to allow
for even flow of bath water between the tubes. This should assist in achieving
uniform heating of all tubes.
7.3 If it is necessary to correct for color or dissolved organic matter which will
cause color on heating, a set of duplicate samples must be run to which all
reagents except the brucine-sulfanilic acid have been added.
7.4 Pipette 10.0 ml of standards and samples or an aliquot of the samples diluted
to 10.0 ml into the sample tubes.
7.5 If the samples are saline, add 2 ml of the 30% sodium chloride solution (6.2) to
the reagent blank, standards and samples. For fresh water samples, sodium
chloride solution may be omitted. Mix contents of tubes by swirling and place
rack in cold water bath (0-10°C).
7.6 Pipette 10.0 ml of sulfuric acid solution (6.3) into each tube and mix by
swirling. Allow tubes to come to thermal equilibrium in the cold bath. Be sure
that temperatures have equilibrated in all tubes before continuing.
7.7 Add 0.5 ml brucine-sulfanilic acid reagent (6.4) to each tube (except the
interference control tubes, 7.3) and carefully mix by swirling, then place the
rack of tubes in the 100°C water bath for exactly 25 minutes.
Caution: Immersion of the tube rack into the bath should not decrease the
temperature of the bath more than 1 to 2°C. In order to keep this temperature
decrease to an absolute minimum, flow of bath water between the tubes should
not be restricted by crowding too many tubes into the rack. If color
development in the standards reveals discrepancies in the procedure, the
operator should repeat the procedure after reviewing the temperature control
steps.
7.8 Remove rack of tubes from the hot water bath and immerse in the cold water
bath and allow to reach thermal equilibrium (20-25°C).
7.9 Read absorbance against the reagent blank at 410 nm using a 1 cm or longer
cell.
8. Calculation
8.1 Obtain a standard curve by plotting the absorbance of standards run by the
above procedure against mg NO3N/l. (The color reaction does not always
follow Beer's law).
8.2 Subtract the absorbance of the sample without the brucine-sulfanilic reagent from
the absorbance of the sample containing brucine-sulfanilic acid and determine mg
199
-------
NO3 N/l. Multiply by an appropriate dilution factor if less than 10 ml of sample
is taken.
9. Precision and Accuracy
9.1 Twenty-seven analysts in fifteen laboratories analyzed natural water samples
containing exact increments of inorganic nitrate, with the following results:
Increment as
Nitrogen, Nitrate
mg N/liter
0.16
0.19
1.08
1.24
Precision as
Standard Deviation
mg N/liter
0.092
0:083
0.245
0.214
Accuracy as
Bias,
%
-6.79
+8.30
+4.12
+2.82
Bias,
mg N/liter
-0.01
+0.02
+0.04
+0.04
(FWPCA Method Study 2, Nutrient Analyses).
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 461,
Method 213-C, (1971).
2. ASTM Standards, Part 23, Water; Atmospheric Analysis, D 992-71, p 363, (1973).
3. Jenkins, D., and Medsken, L., "A Brucine Method for the Determination of Nitrate in
Ocean, Estuarine, and Fresh Waters", Anal Chem., 36, p 610, (1964).
200
-------
NITROGEN, NITRATE-NITRITE
(Cadmium Reduction Method)
STORET NO. 00630
1. Scope and Application
1.1 This method pertains to the determination of nitrite singly, or nitrite and nitrate
combined in drinking, surface, and saline waters, domestic and industrial wastes.
The applicable range of this method is 0.01 to 1.0 mg/1 nitrate-nitrite nitrogen.
The range may be extended with sample dilution.
2. Summary of Method
2.1 A filtered sample is passed through a column containing granulated copper-
cadmium to reduce nitrate to nitrite. The nitrite (that originally present plus
reduced nitrate) is determined by diazotizing with sulfanilamide and coupling
with N(1-naphthyl)ethylenediamine dihydrochloride to form a highly colored
azo dye which is measured spectrophotometrically. Separate, rather than
combined nitrate-nitrite values, are readily obtained by carrying out the
procedure first with, and then without, the initial Cu-Cd reduction step.
3. Sample Handling and Preservation
3.1 Analysis should be made as soon as possible. If analysis can be made within 24
hours, the sample should be preserved by refrigeration at 4°C. When samples must
be stored for more than 24 hours, they should be preserved with sulfuric acid (2
ml H2SO4 per liter) and refrigeration.
Caution: Samples for reduction column must not be preserved with mercuric
chloride.
4. Interferences
4.1 Build up of suspended matter in the reduction column will restrict sample flow.
Since nitrate-nitrogen is found in a soluble state, the sample may be pre-filtered
through a glass fiber filter or a 0.45^ membrane filter. Highly turbid samples may
be pretreated with zinc sulfate before filtration to remove the bulk of particulate
matter present in the sample.
4.2 Low results might be obtained for samples that contain high concentrations of
iron, copper or other metals. EDTA is added to the samples to eliminate this
interference.
4.3 Samples that contain large concentrations of oil and grease will coat the surface of
the cadmium. This interference is eliminated by pre-extracting the sample with an
organic solvent.
201
-------
4.4 This procedure determines both nitrate and nitrite. If only nitrate is desired, a
separate determination must be made for nitrite and subsequent corrections
made. The nitrite may be determined by the procedure below without the
reduction step.
5. Apparatus
5.1 Reduction column: The column in Figure I was constructed from a 100 ml pipet
by removing the top portion. This column may also be constructed from two
pieces of tubing joined end to end. A 10 cm length of 3 cm I.D. tubing is joined to
a 25 cm length of 3.5 mm I.D. tubing.
5.2 Spectrophotometer for use at 540 nm, providing a light path of 1 cm or longer.
6. Reagents
6.1 Granulated cadmium: 40-60 mesh (E M Laboratories, Inc., 500 Exec. Blvd.,
Elmsford, NY 10523, Cat. 2001 Gadmium, Coarse Powder).
6.2 Copper-Cadmium: The cadmium granules (new or used) are cleaned with dilute
HC1 and copperized with 2% solution of copper sulfate in the following manner:
6.2.1 Wash the cadmium with dilute HC1 (6.10) and rinse with distilled water.
6.2.2 Swirl 25 g cadmium in 100 ml portions of a 2% solution of copper
sulfate (6.11) for 5 minutes or until blue color partially fades, decant
and repeat with fresh copper sulfate until a brown colloidal precipitate
forms.
6.2.3 Wash the copper-cadmium with distilled water (at least 10 times) to
remove all the precipitated copper.
202
-------
10cm
80-85 ml
3cm I.D.
3.5 mm I.D.
GLASS WOOL PLUG
FIGURE 1. REDUCTION COLUMN
203
-------
6.3 Preparation of reaction column: Insert a glass wool plug into the bottom of the
reduction column and fill with distilled water. Add sufficient copper-cadmium
granules to produce a column 18.5 cm in length. Maintain a level of distilled water
above the copper-cadmium granules to eliminate entrapment of air. Wash the
column with 200 ml of dilute ammonium chloride solution (6.5). The column is
then activated by passing through the column 100 ml of a solution composed of
25 ml of a 1.0 mg/1 NO3N standard and 75 ml of ammonium chloride EDTA
solution (6.4). Use a flow rate between 7 and 10 ml per minute. -
6.4 Ammonium chloride - EDTA solution: Dissolve 13 g ammonium chloride and
1.7 g disodium ethylenediamine tetracetate in 900 ml of distilled water. Adjust
the pH to 8.5 with cone, ammonium hydroxide (6.9) and dilute to 1 liter:
6.5 Dilute ammonium chloride-EDTA solution: Dilute 300 ml of ammonium
chloride-EDTA solution (6.4) to 500 ml with distilled water.
6.6 Color reagent: Dissolve 10 g sulfanilamide and 1 g N(l-naphthyl)ethylene-
diamine dihydrochloride in a mixture of 100 ml cone, phosphoric acid and 800
ml of distilled water and dilute to 1 liter with distilled water.
6.7 Zinc sulfate solution: Dissolve 100 g ZnSO4 -7H2O in distilled water and dilute to
1 liter.
6.8 Sodium hydroxide solution, 6N: Dissolve 240 g NaOH in 500 ml distilled water,
cool and dilute to 1 liter.
6.9 Ammonium hydroxide, cone.
6.10 Dilute hydrochloric acid, 6N: Dilute 50 ml of cone. HC1 to 100 ml with distilled
water.
6.11 Copper sulfate solution, 2%: Dissolve 20 g of CuSO4 -5H2O in 500 ml of distilled
water and dilute to 1 liter.
6.12 Stock nitrate solution: Dissolve 7.218 g KNO3 in distilled water and dilute to
1000 ml. Preserve with 2 ml of chloroform per liter. This solution is stable for at
least 6 months. 1.0 ml = 1.00 mg NO3 -N.
6.13 Standard nitrate solution: Dilute 10.0 ml of nitrate stock solution (6.12) to 1000
ml with distilled water. 1.0 ml = 0.01 mg NO3 -N.
6.14 Stock nitrite solution: Dissolve 6.072 g KNO2 in 500 ml of distilled water and
dilute to 1000 ml. Preserve with 2 ml of chloroform and keep under refrigeration.
Stable for approximately 3 months. 1.0 ml = 1.00 mg NO2 N.
6.15 Standard nitrite solution: Dilute 10.0 ml of stock nitrite solution (6.14) to 1000
ml with distilled water. 1.0 ml = 0.01 mg NO2-N.
6.16 Using standard nitrate solution (6.13) prepare the following standards in 100ml
volumetric flasks:
204
-------
Conc.,mg-NO3-N/l ml of Standard Solution/100.0 ml
0.00 0.0
0.05 0.5
0.10 1.0
0.20 2.0
0.50 - 5.0
1.00 10.0
7. Procedure
7.1 Turbidity removal: One of the following methods may be used to remove
suspended matter.
7.1.1 Filter sample through a glass fiber filter or a 0.45/1 membrane filter.
7.1.2 Add 1 ml zinc sulfate solution (6.7) to 100 ml of sample and mix
thoroughly. Add 0.4-0.5 ml sodium hydroxide solution (6.8) to obtain a
pH of 10.5 as determined with a pH meter. Let the treated sample stand
a few minutes to allow the heavy flocculent precipitate to settle. Clarify
by filtering through a glass fiber filter or a 0.4 5/z membrane filter.
7.2 Oil and grease removal: Adjust the pH of 100 ml of filtered sample to 2 by
addition of cone. HC1. Extract the oil and grease from the aqueous solution with
two 25 ml portions of a non-polar solvent (Freon, chloroform or equivalent).
7.3 If the pH of the sample is below 5 or above 9, adjust to between 5 and 9 with
either cone. HC1 or cone. NH4OH. This is done to insure a sample pH of 8.5 after
step (7.4).
7.4 To 25.0 ml of sample or an aliquot diluted to 25.0 ml, add 75 ml of ammonium
chloride-EDTA solution (6.4) and mix.
7.5 Pour sample into column and collect sample at a rate of 7-10 ml per minute.
7.6 Discard the first 25 ml, collect the rest of the sample (approximately 70 ml) in
the original sample flask.
7.7 Add 2.0 ml of color reagent (6.6) to 50.0 ml of sample. Allow 10 minutes for
color development. Within 2 hours measure the absorbance at 540 nm against a
reagent blank.
NOTE: If the concentration of sample exceeds 1.0 mg NO3N/l, the remainder
of the reduced sample may be used to make an appropriate dilution before
proceeding with step (7.7).
7.8 Standards: Carry out the reduction of standards exactly as described for the
samples. At least one nitrite standard should be compared to a reduced nitrate
205
-------
standard at the same concentration to verify the efficiency of the reduction
column.
8. Calculation
8.1 Obtain a standard curve by plotting the absorbance of standards run by the above
procedure against N03 N mg/1. Compute concentration of samples by comparing
sample absorbance with standard curve.
8.2 If less than 25 ml of sample is used for the analysis the following equation should
be used:
AX 25
mgNO2 +NO3-N/1 = -
ml sample used
where:
A = Concentration of nitrate from standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using sewage samples at concentrations of 0.04,
0.24, 0.55 and 1.04 mg NO3 + NQ2-N/1, the standard deviations were ±0.005,
±0.004, ±0.005 and ±0.01, respectively.
9.2 In a single laboratory (MDQARL), using sewage samples at concentrations of
0.24, 0.55, and 1.05 mg NO3 + NO2-N/1, the recoveries were 100%, 102% and
100%, respectively.
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 458,
(1.971).
2. Henrikson, A., and Selmer-Olsen, "Automatic Methods for Determining Nitrate and
Nitrite in Water and Soil Extracts". Analyst, May 1970, Vol. 95, p 514-518.
3. Grasshoff, K., "A Simultaneous Multiple Channel System for Nutrient Analysis in Sea
Water with Analog and Digital Data Record", "Advances in Automated Analysis",
Technicon International Congress, 1969, Vol. 11, p 133-145.
4. Brewer, P. G., Riley, J. P., "The Automatic Determination of Nitrate in Sea Water",
Deep Sea Research, 1965, Vol. 12, p 765-772.
206
-------
NITROGEN, NITRATE-NITRITE
(Automated Cadmium Reduction Method)
STORET NO. 00630
1. Scope and Application
1.1 This method pertains to the determination of nitrite singly, or nitrite and nitrate
combined in surface and saline waters, and domestic and industrial wastes. The
applicable range of this method is 0.05 to 10.0 mg/1 nitrate-nitrite nitrogen. The
range may be extended with sample dilution.
2. Summary of Method
2.1 A filtered sample is passed through a column containing granulated copper-
cadmium to reduce nitrate to nitrite. The nitrite (that originally present plus
reduced nitrate) is determined by diazotizing with sulfanilamide and coupling
with N(1-napthyl)ethylenediamine dihydrochloride to form a highly colored
azo dye which is measured colorimetrically. Separate, rather than combined
nitrate-nitrite values, are readily obtained by carrying out the procedure first
with, and then without, the initial Cu-Cd reduction step.
3. Sample Handling and Preservation
3.1 Analysis should be made as soon as possible. If analysis can be made within 24
hours, the sample should be preserved by refrigeration at 4°C. When samples must
be stored for more than 24 hours^ they should be preserved with sulfuric acid (2
ml coric. H2 SO4 per liter) and refrigeration.
Caution: Samples for reduction column must not be preserved with mercuric
chloride.
4. Interferences
4.1 Sample turbidity and color may interfere with this method. Turbidity must be
removed by filtration prior to analysis. Sample color that absorbs in the
photometric range used for analysis will also interfere.
5. Apparatus
5.1 Technicon Auto Analyzer (AAI or AAII) consisting of the following components:
5.1.1 Sampler.
5.1.2 Manifold (AAI) or analytical cartridge (AAII).
5.1.3 Colorimeter equipped with a 15 mm or 50 mm tubular flow cell and 540
nm filters.
5.1.4 Recorder.
207
-------
5.1.5 Digital printer for AAII (Optional).
6. Reagents
6.1 Granulated cadmium: 40-60 mesh (E M Laboratories, Inc., 500 Exec. Blvd.,
Elmsford, NY 10523, Cat. 2001 Cadmium, Coarse Powder).
6.2 Copper-cadmium: The cadmium granules (new or used) are cleaned with dilute
HC1 (6.7) and copperized with 2% solution of copper sulfate (6.8) in the
following manner:
6.2.1 Wash the cadmium with HC1 (6.7) and rinse with distilled water.
6.2.2 Swirl 10 g cadmium in 100 ml portions of 2% solution of copper sulfate
(6.8) for five minutes or until blue color partially fades, decant and
repeat with fresh copper sulfate until a brown colloidal precipitate
forms.
6.2.3 Wash the cadmium-copper with distilled water (at least 10 times) to
remove all the precipitated copper.
6.3 Preparation of reduction column AAI: The reduction column is an 8 by 50 mm
glass tube with the ends reduced in diameter to permit insertion into the system.
Copper-cadmium granules (6.2) are placed in the column between glass wool
plugs. The packed reduction column is placed in an up-flow 20° incline to
minimize channeling. See Figure 1.
6.4 Preparation of reduction column AAII: The reduction column is a U-shaped, 35
cm length, of 2 mm I.D. glass tubing (Note 1). Fill the reduction column with
distilled water to prevent entrapment of air bubbles during the filling operations.
Transfer the copper-cadmium granules (6.2) to the reduction column and place a
glass wool plug in each end. To prevent entrapment of air bubbles in the
reduction column be sure that all pump tubes are filled with reagents before
putting the column into the analytical system.
NOTE 1: A 0.081 I.D. pump tube (purple) can be used in place of the 2 mm glass
tube.
6.5 Distilled water: Because of possible contamination, this should be prepared by
passage through an ion exchange column comprised of a mixture of both strongly
acidic-cation and strongly basic-anion exchange resins. The regeneration of the ion
exchange column should be carried out according to the manufacturer's
instructions.
6.6 Color reagent: To approximately 800 ml of distilled water, add, while stirring,
100 ml cone, phosphoric acid, 40 g sulfanilamide, and 2 g N-l naphthyjethylene-
diamine dihydrochloride. Stir until dissolved and dilute to 1 liter. Store in brown
208
-------
INDENTATIONS FOR
SUPPORTING CATALYST
GLASS WOOL
Cd-TURNINGS
TILT COLUMN TO 20° POSTION
FIGURE 1. COPPER-CADMIUM REDUCTION COLUMN
(1 1/2 ACTUAL SIZE)
209
-------
bottle and keep in the dark when not in use. This solution is stable for several
months.
6.7 Dilute hydrochloric acid, 6N: Dilute 50 ml of cone. HC1 to 100 ml with distilled
water.
6.8 Copper sulfate solution, 2%: Dissolve 20 g of CuSO4 -5H2O in 500 ml of distilled
water and dilute to 1 liter.
6.9 Wash solution: Use distilled water for unpreserved samples; samples preserved
. with H2SO4, use 2 ml H2SO4 per liter of wash water.
6.10 Ammonium chloride solution (8.5% NH4C1): Dissolve 85 g of reagent grade
ammonium chloride in distilled water and dilute to 1 liter with distilled water.
Add 1/2 ml Brij-35 (Available from Technicon Corporation).
6.11 Stock nitrate solution: Dissolve 7.218 g KNO3 and dilute to 1 liter in a
volumetric flask with distilled water. Preserve with 2 ml of chloroform per liter.
Solution is stable for 6 months. 1 ml = 1.0 mg NO3 -N.
6.12 Stock nitrite solution: Dissolve 6.072 g KNO2 in 500 ml of distilled water and
dilute to 1 liter in a volumetric flask. Preserve with 2 ml of chloroform and keep
under refrigeration. 1.0 ml = 1.0 mg NO2 N.
6.13 Standard nitrate solution: Dilute 10.0 ml of stock nitrate solution (6.11) to 1000
ml. 1.0 ml = 0.01 mg NO3N. Preserve with 2 ml of chloroform per liter.
Solution is stable for 6 months.
6.14 Standard nitrite solution: Dilute 10.0 ml of stock nitrite (6.12) solution to 1000
ml. 1.0 ml = 0.01 mg NO2N. Solution is unstable; prepare as required.
6.15 Using standard nitrate solution (6.13), prepare the following standards in 100.0
ml volumetric flasks. At least one nitrite standard should be compared to a nitrate
standard at the same concentration to verify the efficiency of the reduction
column.
Cone., mg NO2 -N or NO3 -N/l ml Standard Solution/100 ml
0.0 0
0.05 0.5
0.10 1.0
0.20 2.0
0.50 5.0
1.00 10.0
2.00 20.0
4.00 40.0
6.00 60.0
210
-------
NOTE 2: When the samples to be analyzed are saline waters. Substitute Ocean
Water (SOW) should be used for preparing the standards; otherwise, distilled
water is used. A tabulation of SOW composition follows:
NaCl - 24.53 g/1 MgCl2 - 5.20 g/1 Na2 SO4 - 4.09 g/1
CaCl2 - 1.16 g/1 KCL - 0.70 g/1 NaHCO3 - 0.20 g/1
KBr - 0.10 g/1 H3BO3 - 0.03 g/1 SrCl2 - 0.03 g/1
NaF - 0.003 g/1
7. Procedure
7.1 If the pH of the sample is below 5 or above 9, adjust to between 5 and 9 with
either cone. HC1 or cone. NH4OH.
7.2 Set up the manifold as shown in Figure 1 (AAI) or Figure 2 (AAII). Note that
reductant column should be in 20° incline position (AAI). Care should be taken
not to introduce air into reduction column on the AAII.
7.3 Allow both colorimeter and recorder to warm up for 30 minutes. Obtain a stable
baseline with all reagents, feeding distilled water through the sample line.
7.4 Place appropriate nitrate and/or nitrite standards in sampler in order of decreasing
concentration of nitrogen. Complete loading of sampler tray with unknown
samples.
7.5 For the AAI system, sample at a rate of 30/hr, 1:1. For the AAII, use a 40/hr, 4:1
cam and a common wash.
7.6 Switch sample line to sampler and start analysis.
8. Calculations
8.1 Prepare appropriate standard curve or curves derived from processing NO2 and/or
NO3 standards through manifold. Compute concentration of samples by
comparing sample peak heights with standard curve.
9. Precision and Accuracy
9.1 Three laboratories analyzed four natural water samples containing exact in-
crements of inorganic nitrate, with the following results:
211
-------
Increment as
Nitrate Nitrogen
mg N/liter
0.29
0.35
2.31
2.48
Precision as
Standard Deviation
mg N/liter
0.012
0.092
0.318
0.176
Accuracy as
Bias,
%
+ 5.75
+18.10
+ 4.47
- 2.69
Bias,
mg N/liter
+0.017
+0.063
+0.103
-0.067
J
(FWQA Method Study 4, Automated Methods In preparation)
Bibliography
1. Fiore, J., and O'Brien, J. E., "Automation in Sanitary Chemistry parts 1 & 2
Determination of Nitrates and Nitrites", Wastes Engineering 33, 128 & 238 (1962).
2. Armstrong, F. A., Stearns, C. R., and Strickland, J. D., "The Measurement of
Upwelling and Subsequent Biological Processes by Means of the Technicon Auto-
Analyzer and Associated Equipment", Deep Sea Research 14, p 381-389 (1967).
3. ASTM Manual on Industrial Water and Industrial Waste Water, Method D 1254, p 465
(1966).
4. Chemical Analyses for Water Quality Manual, Department of the Interior, FWPCA,
R. A. Taft Sanitary Engineering Center Training Program, Cincinnati, Ohio 45226
(January, 1966).
5. "ASTM Manual on Industrial Water and Industrial Waste Water", Substitute Ocean
Water, Table 1, p 418, 1966 Edition.
212
-------
TO SAMPLE WASH
SAMPLER 2
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J
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FIGURE 2. NITRATE NITRITE MANIFOLD AA-I
-------
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FIGURE 3 NITRATE-NITRITE MANIFOLD AA II
-------
NITROGEN, NITRITE
STORET NO. 00615
1. Scope and Application
1.1 This method is applicable to the determination of nitrite in drinking, surface, and
saline waters, domestic and industrial wastes.
1.2 The method is applicable in the range from 0.01 to 1.0 mg NO2 N/l.
2. Summary of Method
2.1 The diazonium compound formed by diazotation of sulfanilamide by nitrite in
water under acid conditions is coupled with N(1-naphthyl)ethylenediamine to
produce a red dish-purple color which is read in a spectrophotometer at 540 nm.
3. Sample Handling and Preservation
3.1 Samples should be analyzed as soon as possible. They may be stored for 24 to 48
hours at 4°C.
4. Interferences
4.1 There are very few known interferences at concentrations less than 1,000 times
that of the nitrite; however, the presence of strong oxidants or reductants to the
samples will readily affect the nitrite concentrations. High alkalinity (>600 mg/1)
will give low results due to a shift in pH.
5. Apparatus
5.1 Spectrophotometer equipped with 1 cfn or larger cells for use at 540 nm.
5.2 Nessler tubes, 50 ml or volumetric flasks, 50 ml.
6. Reagents
6.1 Distilled water free of nitrite and nitrate is to be used in preparation of all
reagents and standards.
6.2 Buffer-color reagent: To 250 ml of distilled water, add 105 ml cone, hydrochloric
acid, 5.0 g sulfanilamide and 0.5 g N(1-naphthyl) ethylenediamine dihydro-
chloride. Stir until dissolved. Add 136 g of sodium acetate (CH3COONa-3H2O)
and again stir until dissolved. Dilute to 500 ml with distilled water. This solution
is stable for several weeks if stored in the dark.
6.3 Nitrite stock solution: 1.0 ml = 0.10 mg NO2-N. Dissolve 0.4926 g of dried
anhydrous sodium nitrite (24 hours in desiccator) in distilled water and dilute to
1000 ml. Preserve with 2 ml chloroform per liter.
6.4 Nitrite standard solution: 1.0 ml = 0.001 mg NO2 -N. Dilute 10.0 ml of the stock
solution (6.3) to 1000ml.
215
-------
7. Procedure
7.1 If the sample has a pH greater than 10 or a total alkalinity in excess of 600 mg/1,
adjust to approximately pH 6 with 1:3 HC1.
7.2 If necessary, filter the sample through a 0.45 n pore size filter using the first
portion of filtrate to rinse the filter flask.
7.3 Place 50 ml of sample, or an aliquot diluted to 50 ml, in 50 ml Nessler tube; hold
until preparation of standards is completed.
7.4 At the same time prepare a series of standards in 50 ml Nessler tubes as follows:
ml of Standard Solution Cone., When Diluted to
1.0 ml = 0.001 mgNO2-N 50 ml, mg/1 of NO2-N
0.0 (Blank) 0.0
0.5 0.01
1.0 0.02
1.5 0.03
2.0 0.04
3.0 0.06
4.0 0.08
5.0 0.10
10.0 0.20
7.5 Add 2 ml of buffer-color reagent (6.2) to each standard and sample, mix and
allow color to develop for at least 15 minutes. The color reaction medium should
be between pH 1.5 and 2.0.
7.6 Read the color in the spectrophotometer at 540 nm against the blank and plot
concentration of NO2 N against absorbance.
8. Calculation
8.1 Read the concentration of NO2N directly from the curve.
8.2 If less than 50.0 ml of sample is taken, calculate mg/1 as follows:
mg/1 from standard curve X 50
NO2-N, mg/1 =
ml sample used
9. Precision and Accuracy
9.1 Precision and accuracy data are not available at this time.
216
-------
NTA
(Zinc-Zincon Method)
STORET NO. 00695
1. Scope and Application
1.1 In this method, NTA refers to the tri-sodium salt of nitrilotriacetic acid,
N(CH2COONa)3.
1.2 This method is applicable to surface waters in the range of 0.510.0 mg/1 NTA.
2. Summary of Method*' >
2.1 Zinc forms a blue-colored complex with 2 carboxy-2'-hydroxy-5'-sulfoformazyl-
benzene (Zincon) in a solution buffered to pH 9.2. When NTA is added, the
Zn-Zincon complex is broken which reduces the optical density in proportion to
the amount of NTA present.
3. Sample Handling and Preservation
3.1 Samples should be analyzed as soon as possible, as NTA has been shown to be
biodegradable*2).
4. Interferences
4.1 Cations, such as calcium, magnesium, zinc, copper, iron, and manganese, complex
with NTA and give a negative interference. These ions are removed by batch
treating samples with ion-exchange resin. At concentrations higher than expected
in typical river waters*3), only zinc, copper, and iron were not completely
removed with ion-exchange treatment. Results are summarized in Table 1.
TABLE 1
Interference of Common Metals
mg/1
Metal added
Blank 0.0
. Zinc 2.0
1.0 mg/1
NTA
5.0 mg/1
NTA
Recoveries
1.1
<0.5
5.5
0.6
217
-------
TABLE l-(Cont'd)
Metal
Boron
Iron
Molybdenum
Manganese
Aluminum
Copper
Strontium
mg/1
added
5.0
5.0
2.0
4.0
3.0
0.5
5.0
1.0 mg/1
NTA
5.0 mg/1
NTA
Recoveries
1.1
0.95
1.0
1.1
0.85
<0.5
1.0
5.5
4.6
5.5
5.6
5.2
3.4
5.4
4.2 This method has not been found applicable to salt waters.
5. Apparatus
5.1 Shaking machine, tray type, for stirring sample-resin mixtures in 125 ml
Erlenmeyer flasks.
5.2 Photometer, suitable for measurements at 620 nm.
6. Reagents
6.1 Sodium hydroxide, 6N: Dissolve 120 g NaOH in distilled water and dilute to 500
ml.
6.2 Buffer: Dissolve 31 g boric acid and 37 g potassium chloride in 800 ml distilled
water. Adjust pH to 9.2 with 6N NaOH (6.1). Dilute to 1 liter.
6.3 Hydrochloric acid, 2N: Dilute 83 ml cone. HC1 to 500 ml with distilled water.
6.4 Zinc: Dissolve 0.44 g ZnSO4 -7H2O in 100 ml 2N HC1 (6.3) and dilute to 1 liter
with distilled water.
6.5 Sodium hydroxide, IN: Dissolve 4 g NaOH in distilled water and dilute to 100
ml.
6.6 Zinc-Zincon: Dissolve 0.13 g Zincon (2-carboxy-2'-hydroxy-5'-sulfoformazyl
benzene) in 2 ml IN NaOH (6.5). Add 300 ml buffer"(6.2). While stirring, add 15
ml Zinc solution (6.4) and dilute to 1 liter with distilled water.
6.7 Ion-exchange resin: Dowex 50W-X8, 50-100 mesh, Na+ form (or equivalent).
218
-------
6.8 Stock NTA solution: Dissolve 1.0700 g N(CH2COONa)3 -H2O in distilled water
and dilute to 1000 ml. 1.0 ml = 0.01 mg NTA.
7. Procedure
7.1 Filter approximately 50 ml of well-mixed sample through a 0.45/u membrane
filter.
7.2 Prepare a series of standards from 0.5 to 10 mg/1 NTA, including a blank of
distilled water. Treat standards and blank in same manner as filtered samples.
7.3 To a 25 ml sample in a 125 ml Erlenmeyer flask add approximately 2.5 g
ion-exchange resin. Agitate sample for at least 15 minutes.
7.4 Filter through coarse filter paper to remove resin. Pipette 15.0 ml of filtrate into a
50 ml beaker. Add 25.0 ml Zinc-Zincon (6.6) by pipette.
7.5 Read absorbance against distilled water at 620 nm in a 1 cm or 2 cm cell.
8. Calculation
8.1 Prepare standard curve by plotting absorbance of standards vs. NTA concentra-
tions. Calculate concentrations of NTA, mg/1, directly from this curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using spiked surface water samples at
concentrations of 0.5, 2, 6, and 10 mg/1 NTA, standard deviations were ±0.17,
±0.14, ±0.1, and ±0.16, respectively.
9.2 In a single laboratory (MDQARL), using spiked surface water samples at
concentrations of 1.0 and 7.5 mg/1 NTA, recoveries were 120% and 103%,
respectively.
Bibliography
1. Thompson, J. E., and Duthie, J. R., "The Biodegradability and Treatment of NTA".
Jour. WPCF, 40, No. 2, 306 (1968).
2. Shumate, K. S. et al, "NTA Removal by Activated Sludge - Field Study", ibid., 42,
No. 4, 631 (1970).
3. Kopp, J. F., and Kroner, R. C., "Trace Metals in Waters of the United States", USDI,
FWPCA, DPS, 1014 Broadway, Cincinnati, Ohio 45202.
219
-------
NTA
(Automated Zinc-Zincon Method)
STORET NO. 00695
1. Scope and Application
1.1 In this method, NTA refers to the tri-sodium salt of nitrilotriacetic acid,
N(CH2COONa)3.
1.2 This method is applicable to surface waters in the range of 0.04 to 1.0 mg/1 and
0.5 to 10.0 mg/1 NTA, depending on which manifold system is used. It does not
apply to saline waters; a positive interference of 0.5 to 1.0 mg/1 is present in
sewage-type samples.
1.3 Approximately 13 samples per hour can be analyzed.
2. Summary of Method*1 >
2.1 Zinc forms a blue-colored complex with 2-carboxy-2'-hydroxy-5'-sulfoformazyl-
benzene (Zincon) in a solution buffered to pH 9.2. When NTA is added, the
Zn-Zincon complex is broken which reduces the optical density in proportion to
the amount of NTA present.
3. Sample Handling and Preservation
3.1 Samples should be analyzed as soon as possible, as NTA has been shown to be
biodegradable.*2*
4. Interferences
4.1 Cations, such as calcium, magnesium, zinc, copper, iron, and manganese, complex
with NTA and give a negative interference. These ions are removed automatically
by passing the sample through an ion-exchange column. At concentrations higher
than expected in typical river waters,*3* only iron was not completely removed
by this column treatment. Results, summarized in Tables 1 and 2, show that iron
gives a negative interference in concentrations above 3.0 mg/1 NTA.
220
-------
TABLE 1
Interference of Common Metals
Metal
Blank
Zinc
Iron
Manganese
Copper
mg/1
added
0.0
2.0
5.0
4.0
0.5
1.0 mg/1
NTA
1.0
0.9
0.8
1.0
1.2
5.0 mg/1
NTA
Recoveries
5.0.
4.9
3.8
4.9
4.9
TABLE 2
Effect of Iron on NTA Recovery in River Water
Iron Added NTA Recovered, mg/1
mg/1 (0.5 mg/1 added)
0.0 0.52
0.5 0.52
1.0 0.52
2.0 . 0.52
3.0 0.48
4.0 0.45
5.0 0.39
4.2 At concentration levels below 0.05 mg/1 NTA, negative peaking may occur during
analyses.
5. Apparatus
5.1 Technicon AutoAnalyzer consisting of:
5.1.1 Sampler I or II.
5.1.2 Manifold.
5.1.3 Proportioning pump.
221
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5.1.4 Colorimeter equipped with 15 mm tubular flow cell and 600 or 625 nm
filter.
5.1.5 Recorder.
6. Reagents
6.1 Sodium hydroxide, 6N: Dissolve 120 g NaOH in distilled water and dilute to 500
ml.
6.2 Buffer: Dissolve 31 g boric acid and 37 g potassium chloride in 800 ml distilled
water. Adjust pH of solution to 9.2 with 6N NaOH (6.1). Dilute to 1 liter.
6.3 Hydrochloric acid, 2N: Dilute 83 ml cone. HC1 to 500 ml with distilled water.
6.4 Zinc: Dissolve 0.44 g ZnSO4 -7H2O in 100 ml 2N HC1 (6.3). Dilute to 1 liter with
distilled water.
6.5 Sodium hydroxide, IN: Dissolve 4 g NaOH in distilled water and dilute to 100
ml.
6.6 Zinc-Zincon reagent A (0.04-1.0 mg/1 NTA): Dissolve 0.065 g Zincon powder
(2-carboxy-2'-hydroxy-5'-sulfoformazyl benzene) in 2 ml of 1 N NaOH (6.5). Add
300 ml buffer (6.2). Stir on a magnetic stirrer and add 7.5 ml zinc solution (6.4).
Dilute to 1 liter with distilled water. This solution is stable for 12 hours.
6.7 Zinc-Zincon reagent B (0.5-10 mg/1 NTA): Dissolve 0.13 g Zincon in 2 ml IN
NaOH (6.5). Stir on magnetic stirrer and add 300 ml buffer (6.2) and 15 ml zinc
solution (6.4). Dilute to 1 liter with distilled water. Stable for 1 week.
6.8 Ion-exchange resin, H+ form: 20-50 mesh or 30-80 mesh, Dowex 50W-XB or
equivalent.
NOTE: Column is prepared by sucking a water slurry of the resin into 12 inches
of 3/16-inch OD sleeving. This may be conveniently done by using a pipette and a
loose-fitting glass wool plug in the sleeve.
6.9 Stock NTA solution: Dissolve 1.0700 g of N(CH2COONa)3-H2O in 500 ml of
distilled water and dilute to 1000 ml. 1.0 ml = 1.0 mg NTA.
6.10 Working solution A: Dilute 10.0 ml of stock NTA solution to 100.0 ml with
distilled water. 1.0 ml = 0.1 mg NTA. Prepare daily.
6.11 Working solution B: Dilute 10.0 ml of Solution A to 100.0 ml with distilled
water. 1.0 ml = 0.01 mg NTA. Prepare daily.
6.12 Working solution C: Dilute 10.0 ml of Solution B to 100.0 ml with distilled
water. 1.0 ml = 0.001 mg NTA. Prepare daily.
6.13 Prepare a series of standards by diluting suitable volumes of working solutions to
100.0 ml with distilled water. The following dilutions are suggested:
222
-------
ml of Solution C/100 ml Cone., mg NT A/1
2 0.02
4 0.04
6 0.06
8 0.08
10 0.10
ml of Solution B/100 ml
2 0.20
4 0.40
6 0.60
8 0.80
10 1.00
ml of Solution A/100 ml
2 2.0
4 4.0
6 6.0
8 8.0
10 10.0
7. Procedure
7.1 Set up manifold as shown in Figure 1.
7.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain suitable baseline.
7.3 Place wash water tubes in sampler in sets of two, leaving every third position
vacant. Set sampling time at 1.5 minutes.
7.4 Place NTA standards in sampler in order of increasing or decreasing concentration.
Complete filling of sample tray with unknown samples.
7.5 Switch sample line from distilled water to sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed NTA standards
against known concentrations. Compute concentration of samples by comparing
sample peak "heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.1, 0.18, 0.27, and 0.44 mg/1, the standard deviations were ±0.01, ±0.004,
223
-------
±0.004, and ±0.005, respectively. At concentrations of 1.3, 4.0, 5.8, and 7.4 mg/1,
the standard deviations were ±0.05, ±0.05, ±0.07, and ±0.1, respectively.
9.2 In a single laboratory (MDQARL), using surface water samples at concentrations
of 0.18 and 0.27 mg/1, recoveries were 101% and 106%, respectively. At
concentrations of 4.0 and 5.8 mg/1, the recoveries were 98% and 96%,
respectively.
Bibliography
1. Thompson, J. E., and Duthie, J. R., "The Biodegradability and Treatment of NTA."
Jour. WPCF, 40, No. 2, 306 (1968).
2. Shumate, K. S. et al, "NTA Removal by Activated Sludge - Field Study." ibid, 42,
No. 4, 631 (1970).
3. Kopp, J. F. and Kroner, R. C., "Trace Metals in Waters of the United States." USDI,
FWPCA, DPS, 1014 Broadway, Cincinnati, Ohio 45202.
224
-------
ION EXCHANGE
WASTE*"
OOQOQOOO
K).
N>
LARGE
MIXING
COILS
WASTE
ml/ min
2.90 C-l SAMPLE
0.
2.00
0.42
0_8
0.8
2.00
PROPORTIONING PUMP
SAMPLING TIME-1.5min
WASH TUBES (2)-3.0min
IOX
*]
COLORIMETER
RECORDER
15 mm Tubular l/c
6OO-625 mp Filters
SAMPLE
AO
WASTE
BUFFER
ZINC-ZINCON (SEE 6.6)
AIR
FOR CONCENTRATION RANGE
OF 0.5 to 10.0 mq/l NTA (RECORDER ot 2X>
ml /min
1
R
0
G
R
R
W
G
R
|0.8 I
^0.23
,2.0
^0.8
SAMPLE
BUFFER
ZINC-ZINCON (s«6.T)
AIR
FIGURE ]. NTA MANIFOLD (0.04-1.0 mg/l NTA) AA-I
-------
OIL AND GREASE, Total, Recoverable
(Soxhlet Extraction)
STORETNO. 00550
1. Scope and Application
1.1 This method includes the measurement of Freon extractable matter from surface
and saline waters, industrial and domestic wastes. It is applicable to the
determination of relatively non-volatile hydrocarbons, vegetable oils, animal fats,
waxes, soaps, greases and related matters.
1.2 The method is not applicable to measurement of light hydrocarbons that
volatilize at temperatures below 70°C. Petroleum fuels from gasoline through #2
fuel oil are completely or substantially lost in the solvent removal operation.
1.3 The method covers the range from 5 to 1000 mg/1 of extractable material.
2. Summary of Method
2.1 The sample is acidified to a low pH (<2) to remove the oils and greases from
solution. After they are isolated by filtration, they are extracted with Freon using
a Soxhlet extraction. The solvent is evaporated from the extract and the residue
weighed.
3. Definitions
3.1 The definition of grease and oil is based on the procedure used. The source of the
oil and/or grease, and the presence of extractable non-oily matter will influence
the material measured and interpretation of results.
4. Sampling and Storage
4.1 A representative 1 liter sample should be collected in a wide-mouth glass bottle. If
analysis is to be delayed for more than a few hours, the sample is preserved by the
addition of 5 ml H2 SO4 or HC1 (6.1) at the time of collection.
4.2 Because losses of grease will occur on sampling equipment, the collection of a
composite sample is impractical. Individual portions collected at prescribed time
intervals must be analyzed separately to obtain the average concentration over an
extended period.
5. Apparatus
5.1 Extraction apparatus consisting of:
5.1.1 Soxhlet extractor, medium size (Corning No. 3740 or equivalent).
^ 5.1.2 Soxhlet thimbles, to fit in Soxhlet extractor, (5.1.1).
5.1.3 Flask, 125 ml (Corning No. 4100 or equivalent).
226
-------
5.1.4 Condenser, Allihn (bulb) type, to fit extractor.
5.1.5 Electric heating mantle.
5.2 Vacuum pump, or other source of vacuum.
5.3 .Buchner funnel, 12 cm.
5.4 Filter paper, Whatman No. 40, 11 cm.
5.5 Muslin cloth discs, 11 cm (muslin cloth available at sewing centers). The muslin
discs are cut to the size of the filter paper and pre-extracted with Freon before
use.
6. Reagents
6.1 Sulfuric acid, 1:1. Mix equal volumes of cone. H2SO4 and distilled water. (Cone.
hydrochloric acid may be substituted directly for cone, sulfuric for this reagent.)
6.2 Freon 113, b.p. 48°C, l,l,2-trichloro-l,2,2-trifluoroethane. At this time, reagent
grade Freon is not commercially available. Freon 113 is available from E. I.
DuPont de Nemours, Inc., and its distributors in 5-gallon cans. It is best handled
by filtering one gallon quantities through paper into glass containers, and
maintaining a regular program of solvent blank monitoring.
6.3 Diatomaceous - silica filter aid suspension, 10 g/1 in distilled water.
NOTE: Hyflo Super-Cel (Johns-Manville Corp.) or equivalent is used in the
preparation of the filter aid suspension.
7. Procedure
7.1 In the following procedure, all steps must be rigidly adhered to if consistent
results are to be obtained.
7.2 Mark the sample bottle at the water meniscus for later determination of sample
volume. If the sample was not acidified at the time of collection, add 5 ml sulfuric
acid or hydrochloric acid (6.1) to the sample bottle. After mixing the sample,
check the pH by touching pH-sensitive paper to the cap to insure that the pH is 2
or lower. Add more acid if necessary.
7.3 Prepare a filter consisting of a muslin cloth disc overlaid with filter paper. Place
trie assembled filter in the Buchner funnel and wet the filter, pressing down the
edges to secure a seal. With vacuum on, put 100 ml of the filter aid suspension
through the filter and then wash with three 100 ml volumes of distilled water.
Continue the vacuum until no more water passes through the filter.
7.4 Filter the acidified sample through the prepared filter pad under vacuum and
continue the vacuum until no more water passes through the filter.
7.5 Using forceps, transfer the filter paper and all solid material on the muslin to a
watch glass. Wipe the inside and cap of the sample bottle and the inside of the
227
-------
Buchner funnel with pieces of filter paper soaked in Freon to remove all oil film.
Fold the pieces of filter paper and fit them into an extraction thimble. Wipe the
watch glass in a similar manner and add the filter paper and all solid matter to the
thimble.
7.6 Fill the thimble with small glass beads or glass wool, and dry in an oven at 103°C
for exactly 30 minutes.
7.7 Weigh the distilling flask (pre-dried in oven at 103°C and stored in desiccator),
add the Freon, and connect to the Soxhlet apparatus in which the extraction
thimble has been placed. Extract at the rate of 20 cycles per hour for four hours.
The four hours is timed from the first cycle.
7.8 Evaporate the solvent from the extraction flask in a water bath at 70°C. Dry by
placing the flask on a covered ;80°C water bath for 15 minutes. Draw air through
the flask by means of an applied vacuum for 1 minute.
7.9 Cool the flask in desiccator for 30 minutes and weigh.
8. Calculation
R-B
8.1 mg/1 total grease =
where:
R = residue, gross weight of extraction flask minus the tare weight, in milligrams.
B = blank determination, residue of equivalent volume of extraction solvent, in
milligrams.
V = volume of sample, determined by refilling sample bottle to calibration line
and correcting for acid addition if necessary, in liters.
9. Precision and Accuracy
9.1 The three oil and grease methods in this manual were tested by a single laboratory
(MDQARL) on a sewage. This method determined the oil and grease level in the
sewage to be 14.8 mg/1. When 1 liter portions of the sewage were dosed with 14.0
mg of a mixture of #2 fuel oil and Wesson oil, the recovery was 88% with a
standard deviation of 1.1 mg.
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 409,
Method 209A(1971).
2. Hatfield, W. D., and Symons, G. E., 'The Determination of Grease in Sewage", Sewage
Works!., 17, 16(1945).
3. Blum, K. A., and Taras, M. J., "Determination of Emulsifying Oil in Industrial
Wastewater", JWPCF Research Suppl. 40, R404 (1968).
228
-------
OIL AND GREASE, Total, Recoverable
(Separatory Funnel Extraction)
STORET NO. 00556
1. Scope and Application
1.1 This method includes the measurement of Freon extractable matter from surface
and saline waters, industrial and domestic wastes. It is applicable to the
determination of relatively non-volatile hydrocarbons, vegetable oils, animal fats,
waxes, soaps, greases and related matter.
1.2 The method is not applicable to measurement of light hydrocarbons that
volatilize at temperatures below 70°C. Petroleum fuels from gasoline through #2
fuel oils are completely or substantially lost in the solvent removal operation.
1.3 Some crude oils and heavy fuel oils contain a significant percentage of
residue-type materials that are not soluble in Freon. Accordingly, recoveries of
these materials will be low.
1.4 The method covers the range from 5 to 1000 mg/1 of extractable material.
2. Summary of Method
2.1 The sample is acidified to a low pH (<2) and serially extracted with Freon in a
separatory funnel. The solvent is evaporated from the extract and the residue
weighed.
3. Definitions
3.1 The definition of grease and oil is based on the procedure used. The source of the
oil and/or grease, and the presence of extractable non-oily matter will influence
the material measured and interpretation of results.
4. Sampling and Storage
4.1 A representative sample of 1 liter volume should be collected in a glass bottle. If
analysis is to be delayed for more than a few hours, the sample is preserved by the
addition of 5 ml H2 SO4 or HC1 (6.1) at the time of collection.
4.2 Because losses of grease will occur on sampling equipment, the collection of a
composite sample is impractical. Individual portions collected at prescribed time
-w
intervals must be analyzed separately to obtain the average concentration over an
extended period.
5. Apparatus
*£>.! Separatory funnel, 2000 ml, with Teflon stopcock.
5.2 Vacuum pump, or other source of vacuum.
229
-------
5.3 Flask, distilling, 125 ml (Coming No. 4100 or equivalent).
5.4 Filter paper, Whatman No. 40, 11 cm.
6. Reagents
6.1 Sulfuric acid, 1:1. Mix equal volumes of cone. H2SO4 and distilled water. (Cone.
hydrochloric acid may be substituted directly for cone, sulfuric for this reagent).
6.2 Freon 113, b.p. 48°C, l,l,2-trichloro-l,2,2-trifluoroethane. At this time, reagent
grade Freon is not commercially available. Freon 113 is available from E. I.
'x<
DuPont de Nemours, Inc. and its distributors, in 5-gallon cans. It is best handled
by filtering one gallon quantities through paper into glass containers, and
maintaining a regular program of solvent blank monitoring.
6.3 Sodium sulfate, anhydrous crystal.
7. Procedure
7.1 Mark the sample bottle at the water meniscus for later determination of sample
volume. If the sample was not acidified at time of collection, add 5 ml sulfuric
acid or hydrochloric acid (6.1) to the sample bottle. After mixing the sample,
check the pH by touching pH-sensitive paper to the cap to insure that the pH is 2
or lower. Add more acid if necessary.
7.2 Pour the sample into a separatory funnel.
7.3 Add 30 ml Freon (6.2) to the sample bottle and rotate the bottle to rinse the
sides. Transfer the solvent into the separatory funnel. Extract by shaking
vigorously for 2 minutes. Allow the layers to separate.
7.4 Tare a distilling flask (pre-dried in an oven at 103°C and stored in a desiccator),
and filter the solvent layer into the flask through a funnel containing solvent
moistened filter paper.
NOTE: An emulsion that fails to dissipate can be broken by pouring about 1 g
sodium sulfate (6.3) into the filter paper cone and draining the emulsion through
the salt. Additional 1 g portions can be added to the cone as required.
7.5 Repeat (7.3 and 7.4) twice more, with additional portions of fresh solvent,
combining all solvent in the distilling flask.
7.6 Rinse the tip of the separatory funnel, the filter paper, and then the funnel with a
total of 10-20 ml Freon and collect the rinsings in the flask.
7.7 Evaporate the solvent from the extraction flask in a water bath at 70°C. Dry by
placing the flask on a covered 80°C water bath for 15 minutes. Draw air through
the flask by means of an applied vacuum for 1 minute.
7.8 Cool in desiccator for 30 minutes and weigh.
230
-------
8, Calculation
R-B
8.1 mg/1 total oil and grease =
where:
R = residue, gross weight of extraction flask minus the tare weight, in milligrams.
B = blank determination, residue of equivalent volume of extraction solvent, in
milligrams.
V = volume of sample, determined by refilling sample bottle to calibration line
and correcting for acid addition if necessary, in liters.
9. Precision and Accuracy
9.1 The three oil and grease methods in this manual were tested by a single laboratory
(MDQARL) on a sewage. This method determined the oil and grease level in the
sewage to be 12.6 mg/1. When 1 liter portions of the sewage were dosed with 14.0
mg of a mixture of #2 fuel oil and Wesson oil, the recovery was 93% with a
standard deviation of 0.9 mg.
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 254,
Method 137(1971).
2. Blum, K. A., and Taras, M. J., "Determination of Emulsifying Oil in Industrial
Wastewater", JWPCF Research Suppl. 40, R404 (1968).
231
-------
OIL AND GREASE, Total, Recoverable
(Infrared)
STORE! NO. 00560
1. Scope and Application
1.1 This method includes the measurement of Freon extractable matter from surface
and saline waters, industrial and domestic wastes. It is applicable to the
determination of hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related matter.
1.2 The method is applicable to measurement of most light petroleum fuels, although
loss of about half of any gasoline present during the extraction manipulations can
be expected.
1.3 The method covers the range from 0.2 to 1000 mg/1 of extractable material.
2. Summary of Method
2.1 The sample is acidified to a low pH (<2) and extracted with Freon. The oil and
grease is determined by comparison of the infrared absorbance of the sample
extract with standards.
3. Definitions
.3.1 The definition of grease and oil is based on the procedure used. The source of the
oil and/or grease, and the presence of extractable non-oily matter will influence
the material measured and interpretation of results.
3.2 An "Unknown Oil" is defined as one for which a representative sample of the oil
or grease is not available for preparation of a standard. Examples of unknown oils
are the oil and grease in a mixed sewage or an unidentified oil slick on a surface
water.
3.3 A "Known Oil" is defined as a sample of oil and/or grease that represents the only
material of that type used or manufactured in the processes represented by a
waste water.
4. Sampling and Storage
4.1 A representative sample of 1 liter volume should be collected in a glass bottle. If
analysis is to be delayed for more than a few hours, the sample is preserved by the
addition of 5 ml H2 SO4 or HC1 (6.1) at the time of collection.
4.2 Because losses of grease will occur on sampling equipment, the collection of a
composite sample is impractical. Individual portions collected at prescribed time
intervals must be analyzed separately to obtain the average concentration over an
extended period.
232
-------
5. Apparatus
5.1 Separately funnel, 2000 ml, with Teflon stopcock.
5.2 Infrared spectrophotometer, double beam, recording.
5.3 Cells, quartz, 10 mm, 50 mm, and 100 mm path length.
5.4 Syringes, 10, 25, 50, 100 microliter capacity.
5.5 Filter paper, Whatman No. 40, 11 cm.
6. Reagents
6.1 Sulfuric acid, 1:1. Mix equal volumes of cone. H2SO4 and distilled water. (Cone.
hydrochloric acid may be substituted directly for cone, sulfuric for this reagent.)
6.2 Freon 113, b.p. 48°C, l,l,2-trichloro-l,2,2-trifluoroethane. At this time, reagent
grade Freon is not commercially available. Freon 113 is available from E. I.
DuPont de Nemours, Inc., and its distributors, in 5-gallon cans. It is best handled
by filtering one gallon quantities through paper into glass containers, and
maintaining a regular program of solvent blank monitoring.
6.3 Sodium sulfate, anhydrous crystal.
6.4 Known oil reference standard: Accurately weigh about 0.05 g of known oil
directly into a 100 ml volumetric flask. Add 80 ml Freon and dissolve the oil. If,
as in the case of a heavy fuel oil, all the oil does not go into solution, let stand
overnight. The next day filter through paper into another 100 ml volumetric and
dilute to mark. Treat calculations as if all oil had gone into solution.
6.5 Unknown oil reference standard (lOjul = 7.69 mg oil): Pipette 15.0 ml
n-hexadecane, 15.0 ml isooctane, and 10.0 ml benzene into a 50 ml glass stoppered
bottle. Assume the specific gravity of this mixture to be 0.769 and maintain the
integrity of the mixture by keeping stoppered except when withdrawing aliquots.
7. Procedure
7.1 Mark the sample bottle at the water meniscus for later determination of sample
volume. If the sample was not acidified at time of collection, add 5 ml sulfuric or
hydrochloric acid (6.1) to the sample bottle. After mixing the sample, check the
pH by touching pH-sensitive paper to the cap to insure that the pH is 2 or lower.
Add more acid if necessary.
7.2 Pour the sample into a separatory funnel.
7.3 Add 30 ml Freon (6.2) to the sample bottle and rotate the bottle to rinse the
sides. Transfer the solvent into the separatory funnel. Extract by shaking
vigorously for 2 minutes. Allow the layers to separate. ,
7.4 Filter the solvent layer into a 100 ml volumetric flask through a funnel containing
solvent-moistened filter paper.
233
-------
NOTE 1: An emulsion that fails to dissipate can be broken by pouring about 1 g
sodium sulfate (6.3) into the filter paper cone and draining the emulsion through
the salt. Additional 1 g portions can be added to the cone as required.
7.5 Repeat (7.3 and 7.4) twice more with 30 ml portions of fresh solvent, combining
all solvent in the volumetric flask.
7.6 Rinse the tip of the separatory funnel, filter paper, and the funnel with a total of
10-20 ml Freon and collect the rinsings in the flask. Dilute the extract to 100 ml,
and stopper the flask.
7.7 Select appropriate calibration standards and cell pathlength according to the
following table of approximate working ranges:
Pathlength Range
1 cm 4-40 mg
5 cm 0.5-8 mg
10cm 0.1-4mg
Prepare calibration standards by pipetting appropriate amounts of the known oil
reference standard (6.4) into 100 ml volumetric flasks and diluting to mark with
Freon. Alternately, transfer appropriate amounts of the unknown oil reference
standard (6.5), using microliter syringes, to 100 ml volumetric flasks and diluting
to mark with Freon.
NOTE 2: Ten microliters of the unknown oil is equivalent to 7.69 mg per 100 ml
Freon, and 7.69 mg per sample volume.
7.8 Scan standards and samples from 3200 cm-1 to 2700 cm-1 with Freorijn the
reference beam and record the results on absorbance paper. The absorbances of
samples and standards are measured by constructing a straight baseline over the
range of the scan and measuring the absorbance of the peak maximum at 2930
cm-1 and subtracting the baseline absorbance at that point. If the absorbance
exceeds 0.8 for a sample, select a shorter pathlength or dilute as required.
NOTE 3: Caution must be exercised in the selection of the 2930 cm-1 peak, as it
may not always be the largest peak in the range of the scan. For an example of a
typical oil spectrum and baseline construction, see Gruenfeld*3).
7.9 Use a calibration plot of absorbance vs. mg oil prepared from the standards to
determine the mg oil in the sample solution.
8. Calculation
RXD
8.1 mg/1 total oil and grease =
where:
R = oil in solution, determined from calibration plot, in milligrams.
234
-------
D = extract dilution factor, if used.
V = volume of sample, determined by refilling sample bottle to calibration line
and correcting for acid addition if necessary, in liters.
9. Precision and Accuracy
9.1 The three oil and grease methods in this manual were tested by a jingle laboratory
(MDQARL) on a sewage. This method determined the oil and grease level in the
sewage to be 17.5 mg/1. When 1 liter portions of the sewage were dosed with 14.0
mg of a mixture of #2 fuel oil and Wesson oil, the recovery was 99% with a
standard deviation of 1.4 mg.
Bibliography
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 254,
Method 137(1971).
2. American Petroleum Institute, "Manual on Disposal of Refinery Wastes", Vol. IV,
Method 733-58(1958).
3. Gruenfeld, M., "Extraction of Dispersed Oils from Water for Quantitative Analysis by
Infrared Spectroscopy", Environ. Sci. Technol. 7, 636 (1973).
235
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ORGANIC CARBON
(Total and Dissolved)
STORET NO. Total 00680
Dissolved 00681
1. Scope and Application
1.1 This method includes the measurement of organic carbon in drinking, surface, and
saline waters, domestic and industrial wastes. Exclusions are noted under
Definitions and Interferences.
1.2 The method is most applicable to measurement of organic carbon above 1 mg/1.
2. Summary of Method
2.1 Organic carbon in a sample is converted to carbon dioxide (CO2) by catalytic
combustion or wet chemical oxidation. The CO2 formed can be measured directly
by an infrared detector or converted to methane (CH4) and measured by a flame
ionization detector. The amount of CO2 or CH4 is directly proportional to the
concentration of carbonaceous material in the sample.
3. Definitions
3.1 The carbonaceous analyzer measures all of the carbon in a sample. Because of
various properties of carbon-containing compounds in liquid samples, preliminary
treatment of the sample prior to analysis dictates the definition of the carbon as it
is measured. Forms of carbon that are measured by the method are:
A) soluble, nonvolatile organic carbon; for instance, natural sugars.
B) soluble, volatile organic carbon; for instance, mercaptans.
C) insoluble, partially volatile carbon; for instance, oils.
D) insoluble, particulate carbonaceous materials, for instance, cellulose
fibers. *,.
E) soluble or insoluble carbonaceous materials adsorbed or entrapped on
insoluble inorganic suspended matter; for instance, oily matter
adsorbed on silt particles.
3.2 The final usefulness of the carbon measurement is in assessing the potential
oxygen-demanding load of organic material on a receiving stream. This statement
applies whether the carbon measurement is made on a sewage plant effluent,
industrial waste, or on water taken directly from the stream. In this light,
carbonate and bicarbonate carbon are not a part of the oxygen demand in the
stream and therefore should be discounted in the final calculation or removed
prior to analysis. The manner of preliminary treatment of the sample and
236
-------
instrument settings defines the types of carbon which are measured. Instrument
manufacturer's instructions should be followed.
4. Sample Handling and Preservation
4.1 Sampling and storage of samples in glass bottles is preferable. Sampling and
storage in plastic bottles such as conventional polyethylene and cubitainers is
permissible if it is established that the containers do not contribute contaminating
organics to the samples.
NOTE 1: A brief study performed in the EPA Laboratory indicated that distilled
water stored in new, one quart cubitainers did not show any increase in organic
carbon after two weeks exposure.
4.2 Because of the possibility of oxidation or bacterial decomposition of some
components of aqueous samples, the lapse of time between collection of samples
and start of analysis should be kept to a minimum. Also, samples should be kept
cool (4°C) and protected from sunlight and atmospheric oxygen.
4.3 In instances where analysis cannot be performed within two hours (2 hours) from
time of sampling, it is recommended that the sample is acidified (pH^2) with HC1
orH2SO4.
5. Interferences
5.1 Carbonate and bicarbonate carbon represent an interference under the terms of
this test and must be removed or accounted for in the final calculation.
5.2 This procedure is applicable only to homogeneous samples which can be injected
into the apparatus reproducibiy by means of a microliter type syringe or pipette.
The openings of the syringe or pipette limit the maximum size of particles which
may be included in the sample.
6. Apparatus
6.1 Apparatus for blending or homogenizing samples: Generally, a Waring-type
blender is satisfactory.
6.2 Apparatus for total and dissolved organic carbon:
6.2.1 A number of companies manufacture systems for measuring carbona-
ceous material in liquid samples. Considerations should be made as to the
types of samples to be analyzed, the expected concentration range, and
forms of carbon to be measured.
6.2.2 No specific analyzer is recommended as superior. However, analyzers
which have been found to be reliable are the Dow-Beckman Carbona-
ceous Analyzer Model No. 915, the Dohrmann Envirotech DC-50 Carbon
Analyzer and the Oceanography International Total Carbon Analyzer.
237
-------
7. Reagents
7.1 Distilled water used in preparation of standards and for dilution of samples should
be ultra pure to reduce the size of the blank. Carbon dioxide-free, double distilled
water is recommended. Ion exchanged waters are not recommended because of
the possibilities of contamination with organic materials from the resins.
7.2 Potassium hydrogen phthalate, stock solution, 1000 mg carbon/liter: Dissolve
0.2128 g of potassium hydrogen phthalate (Primary Standard Grade) in distilled
water and dilute to 100.0 ml.
NOTE 2: Sodium oxalate and acetic acid are not recommended as stock solutions.
7.3 Potassium hydrogen phthalate, standard solutions: Prepare standard solutions
from the stock solution by dilution with distilled water.
7.4 Carbonate-bicarbonate, stock solution, 1000 mg carbon/liter: Weigh 0.3500 g of
sodium bicarbonate and 0.4418 g of sodium carbonate and transfer both to the
same 100 ml volumetric flask. Dissolve with distilled water.
7.5 Carbonate-bicarbonate, standard solution: Prepare a series of standards similar to
step 7.3.
NOTE 3: This standard is not required by some instruments.
7.6 Blank solution: Use the same distilled water (or similar quality water) used for the
preparation of the standard solutions.
8. Procedure
8.1 Follow instrument manufacturer's instructions for calibration, procedure, and
calculations.
8.2 For calibration of the instrument, it is recommended that a series of standards
encompassing the expected concentration range of the samples be used.
9. Precision and Accuracy
9.1 Twenty-eight analysts in twenty-one laboratories analyzed distilled water solu-
tions containing exact increments of oxidizable organic compounds, with the
following results:
Increment as
TOC
mg/liter
4.9
107
Precision as
Standard Deviation
TOC, mg/liter
3.93
8.32
Accuracy as
Bias,
%
+15.27
+ 1.01
Bias,
mg/liter
+0.75
+1.08
(FWPCA Method Study 3, Demand Analyses)
238
-------
pH
STORET NO. 00400
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
2. Summary of Method
2.1 The pH of a sample is an electrometric measurement, using either a glass electrode
in combination with a reference potential (saturated calomel electrode) or a
combination electrode (glass and reference).
3. Comments
3.1 The sample must be analyzed as soon as practical; preferably within a few hours.
Do not open sample bottle before analyses.
3.2 Oil and greases, by coating the pH electrode, may interfere by causing sluggish
response.
3.3 At least three buffer solutions must be used to initially standardize the
instrument. They should cover the pH range of the samples to be measured.
3.4 Field pH measurements using comparable instruments are reliable.
4. Precision and Accuracy
4.1 Forty-four analysts in twenty laboratories analyzed six synthetic water samples
containing exact increments of hydrogen-hydroxyl ions, with the following
results: .
Increment as
pH Units
3.5
3.5
7.1
7.2
8.0
8.0
Precision as
Standard Deviation
pH Units
0.10
0.11
0.20
0.18
0.13
0.12
Accuracy as
Bias,
%
-0.29
-0.00
+1.01
-0.03
-0.12
+0.16
Bias,
pH Units
-0.01
+0.07
-0.002
-0.01
+0.01
(FWPCA Method Study 1, Mineral and Physical Analyses)
239
-------
4.2 In a single laboratory (MDQARL), using surface water samples at an average pH
of 7.7, the standard deviation was ±0.1.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
276, Method 144 A (1971).
ASTM Standards, Part 23, Water: Atmospheric Analysis, p 186, Method
01293-65(1973).
240
-------
PHENOLICS
(4-AAP Method With Distillation)
STORET NO. 32730
1. Scope and Application
1.1 This method is applicable to the analysis of drinking, surface, and saline waters,
domestic and industrial wastes.
1.2 The method is capable of measuring phenolic materials from about 5 jug/1 to
about 1000 /Lcg/1 when the colored end product is extracted and concentrated in a
solvent phase using phenol as a standard.
1.3 The method is capable of measuring phenolic materials that contain more than 50
Hg/\ in the aqueous phase (without solvent extraction) using phenol as a standard.
1.4 It is not possible to use this method to differentiate between different kinds of
phenols.
2. Summary of Method
2.1 Phenolic materials react with 4-aminoantipyrine in the presence of potassium
ferricyanide at a pH of 10 to form a stable reddish-brown colored antipyrine dye.
The amount of color produced is a function of the concentration of phenolic
material.
3. Comments
3.1 For most samples a preliminary distillation is required to remove interfering
materials.
3.2 Color response of phenolic materials with 4-amino-antipyrine is not the same for
all compounds. Because phenolic type wastes usually contain a variety of phenols,
it is not possible to duplicate a mixture of phenols to be used as a standard. For
this reason phenol itself has been selected as a standard and any color produced
by the reaction of other phenolic compounds is reported as phenol. This value
will represent the minimum concentration of phenolic-compounds present in the
sample.
3.3 Control of the pH of the reaction may be accomplished using the procedure
detailed in Standard Methods (p 506, 13th Edition), or ASTM, Part 23, p 535
(Nov. 1973), or by use of the ammonium hydroxide-ammonium chloride buffer
used in the water hardness test Standard Methods, 13th Edition, p 181, (1971).
4. Precision and Accuracy
4.1 Using the extraction procedure for concentration of color, six laboratories
241
-------
analyzed samples at concentrations of 9.6, 48.3, and 93.5 Mg/1- Standard
deviations were, respectively, ±0.99, ±3.1 and ±4.2 /ng/1.
4.2 Using the direct photometric procedure, six laboratories analyzed samples at
concentrations of 4.7, 48.2 and 97.0 mg/1. Standard deviations were ±0.18, ±0.48
and ±1.58 mg/1, respectively.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
501-510, Method No. 222 through 222E (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 535, Method
EM 783-70 (1973).
242
-------
PHENOLICS
(Automated 4-AAP Method With Distillation)
STORET NO. 32730
1. Scope and Application
1.1 This method is applicable to the analysis of drinking, surface, and saline waters,
domestic and industrial wastes.
1.2 The method is capable of measuring phenolic materials from 2 to 500 /-tg/1 in the
aqueous phase using phenol as a standard. The working ranges are, 2 to 200 >tg/l
and lOtoSOO^g/l.
2. Summary of Method
2.1 This automated method is based on the distillation of phenol and subsequent
reaction of the distillate with alkaline ferricyanide and 4-aminoantipyrine to form
a red complex which is measured at 505 or 520 nm. The same manifold is used
with the AA1 or A All.
3. Sample Handling and Preservation
3.1 Biological degradation is inhibited by the addition of 1 g/1 of copper sulfate to the
sample and acidification to a pH of less than 4 with phosphoric acid. The sample
should be kept at 5-10°C and analyzed within 24 hours after collection.
4. Interference
4.1 Interferences from sulfur compounds are eliminated by acidifying the sample to a
pH of less than 4.0 with H3PO4 and aerating briefly by stirring and adding
CuSO4.
4.2 Oxidizing agents such as chlorine, detected by the liberation of iodine upon
acidification in the presence of potassium iodide, are removed immediately after
sampling by the addition of an excess of ferrous ammonium sulfate (6.5). If
chlorine is not removed, the phenolic compounds may be partially oxidized and
the results may be low.
4.3 Background contamination from plastic tubing and sample containers is elimi-
nated by filling the wash receptacle by siphon (using Kel-F tubing) and using glass
tubes for the samples and standards.
5. Apparatus
5.1 Technicon AutoAnalyzer (I or II)
5.1.1 Sampler.
5.1.2 Manifold.
243
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5.1.3 Proportioning pump II or III.
5.1.4 Heating bath with distillation coil.
5.1.5 Distillation head.
5.1.6 Colorimeter equipped with a 50 mm flow cell and 505 or 520 nm filter.
5.1.7 Recorder.
6. Reagents
6.1 Distillation reagent: Add 100 ml of cone, phosphoric acid (85% H3PO4) to 800
ml of distilled water, cool and dilute to 1 liter.
6.2 Buffered potassium ferricyanide: Dissolve 2.0 g potassium ferricyanide, 3.1 g
boric acid and 3.75 g potassium chloride in 800 ml of distilled water. Adjust to
pH of 10.3 with 1 N sodium hydroxide (6.3) and dilute to 1 liter. Add 0.5 ml of
Brij-35. Prepare fresh weekly.
6.3 Sodium hydroxide (IN): Dissolve 40 g NaOH in 500 ml of distilled water, cool
and dilute to 1 liter.
6.4 4-Aminoantipyrine: Dissolve 0.65 g of 4-aminoantipyrine in 800 ml of distilled
water and dilute to 1 liter. Prepare fresh each day.
6.5 Ferrous ammonium sulfate: Dissolve 1.1 g ferrous ammonium sulfate in 500 ml
distilled water containing 1 ml H2 SO4 and dilute to 1 liter with freshly boiled and
cooled distilled water.
6.6 Stock phenol: Dissolve 1.00 g phenol in 500 ml of distilled water and dilute to
1000 ml. Add 1 g CuSO4 and 0.5 ml cone. H3PO4 as preservative. 1.0 ml = 1.0
mg phenol.
6.7 Standard phenol solution A: Dilute 10.0 ml of stock phenol solution (6.6) to
1000 ml. 1.0 ml = 0.01 mg phenol.
6.8 Standard phenol solution B: Dilute 100.0 ml of standard phenol solution A (6.7)
to 1000 ml with distilled water. 1.0 ml = 0.001 mg phenol.
6.9 Standard solution C: Dilute 100.0 ml of standard phenol solution B (6.8) to 1000
ml with distilled water. 1.0 ml = 0.0001 mg phenol.
6.10 Using standard solution A, B or C prepare the following standards in 100 ml
volumetric flasks. Each standard should be preserved by adding 0.1 g CuSO4 and
2 drops of cone. H3PO4 to 100.0 ml.
244
-------
ml of Standard Solution Cone, jug/1
Solution C
1.0 1.0
2.0 2.0
3.0 3.0
5.0 5.0
Solution B
1.0 10.0
2.0 20.0
5,0 50.0
10.0 100.0
Solution A
2 200
3 300
5 500
7. Procedure
7.1 Set up the manifold as shown in Figures 1 or 2.
7.2 Fill the wash receptacle by siphon. Use Kel-F tubing with a fast flow (1 liter/hr).
7.3 Allow colorimeter and recorder to warm up for 30 minutes. Run a baseline with
all reagents, feeding distilled water through the sample line. Use polyethylene
tubing for sample line. When new tubing is used, about 2 hours may be required
to obtain a stable baseline. This two hour time period may be necessary to remove
the residual phenol from the tubing.
7.4 Place appropriate phenol standards in sampler in order of decreasing concentra-
tion. Complete loading of sampler tray with unknown samples, using glass tubes.
NOTE 1: If samples have not been preserved as instructed in (3.1), add 0.1 g
CuSO4 and 2 drops of cone. H3PO4 to 100 ml of sample.
7.5 Switch sample line from distilled water to sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of standards against concentra-
tion values. Compute concentration of samples by comparing sample peak heights
with standards.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using sewage samples at concentrations of 3.8,
15, 43 and 89 j/g/1, the standard deviations were ±0.5, ±0.6, ±0.6 and ±1.0jug/l,
245
-------
respectively. At concentrations of 73, 146, 299 and 447 jug/1, the standard
deviations were ±1.0, ±1.8, ±4.2 and ±5.3 jug/1, respectively.
9.2 In a single laboratory (MDQARL), using sewaje samples at concentrations of 5.3
and 82 jug/1, the recoveries were 78% and 98%. At concentrations of 168 and 489
//g/1, the recoveries were 97% and 98%, respectively.
: Bibliography
1. Technicon AutoAnalyzer II Methodology, Industrial Method No. 127-71W, AAII.
2. Standard Methods for the Examination of Water and Wastewater, 13th Edition, p 501,
American Public Health Association, Inc., New York (1971).
246
-------
To Waste
ft ft ft
Ml /min
SAMPLER
to
X"
RESAMPLE
\
/
BATH W
TION C
>
1!
^ASTE TO PUMP
OPOQ - * i
S.M.
ITH
OIL
'JOOd
s.
M.
/
T
^
*
1 t
r
505pm filters
2 mm Tubular f/c
p
P
1)
tT
10 X
BLACK BLACK
^-»
G G
0^0
0^0
GRAY ^ GRAY
BLACK ^ BLACK
Y IT Y
0 ^ W
0 ^. W
GRAY ^ GRAY
0.32 AIR
2.00 SAMPLE
0.42 DISTILLING SOL.
0.42 WASTE FROM
STILL
1.0 RESAMPLE WASTE
0.32 AIR
1.2 RESAMPLE
0.23 4AAP
k i
/***
A-2
0.23 BUFFERED POTASSIUM
FERRI CYANIDE
1.0 WASTE FROM F/C
PROPORTIONING
PUMP
SAMPLE RATE 20/hr. 1:2
* Kel-f
** 100 ACIDFLEX
«»* POLYETHYLENE
COLORIMETER RECORDER
FIGURE 1. PHENOL AUTO ANALYZER I
-------
To Woste
K)
*.
00
Ml /min
SAMPLER
X"
.RESAMPLE
X
/ S.M.
BATH WITH
TION COIL
'
\
\
f
I57-B089
nnnn
\
/
{
505pm filters
50 mm Tubular f/c
^^^
t
s
*"
r1
^j
i'
*
10 X
BLACK ^ BLACK
G ^ G
0^0
0^0
GRAY ^ GRAY
BLACK ^. BLACK
Y < Y
0 ^ W
0 W
GRAY ^. GRAY
0.32 AIR
2.00 SAMPLE
0.42 DISTILLING SOL.
0.42 WASTE FROM
STILL
1.0 RESAMPLE WASTE
0.32 AIR
1.2 RESAMPLE
0.23 4 AAP ..
K
1 '
/***
A- 2
0.23 BUFFERED POTASSIUM
FERR 1 CYANIDE
J .0 WASTE FROM F/ C
PROPORTIONING
PUMP
SAMPLE RATE 20/hr. 1:2
* Kel-f
** 100 ACIDFLEX
**» POLYETHYLENE
COLORIMETER RECORDER
FIGURE 2. PHENOL AUTO ANALYZER II
-------
PHOSPHORUS, ALL FORMS
(Single Reagent Method)
STORET NO. See Section 4
1. Scope and Application
1.1 These methods cover the determination of specified forms of phosphorus in
drinking, surface, and saline waters, domestic and industrial wastes. They may be
applicable to sediment-type samples, sludges, algal blooms, etc., but sufficient
data -is not available at this time to warrant such usage when measurements for
phosphorus content are required.
1.2 The methods are based on reactions that are specific for the orthophosphate ion.
Thus, depending on the prescribed pre-treatment of the sample, the various forms
of phosphorus given in Figure 1 may be determined. These forms are, in turn,
defined in Section 4.
1.2.1 Except for in-depth and detailed studies, the most commonly measured
forms are phosphorus and dissolved phosphorus, and orthophosphate
and dissolved orthophosphate.. Hydrolyzable phosphorus is normally
found only in sewage-type samples and insoluble forms of phosphorus, as
noted, are determined by calculation.
1.3 The methods are usable in the 0.01 to 0.5 mg P/l range.
2. Summary of Method
2.1 Ammonium molybdate and antimony potassium tartrate react in an acid medium
with dilute solutions of phosphorus to form an antimony-phospho-molybdate
complex. This complex is reduced to an intensely blue-colored complex by
ascorbic acid. The color is proportional to the phosphorus concentration.
2.2 Only orthophosphate forms a blue color in this test. Polyphosphates (and some
organic phosphorus compounds) may be converted to the orthophosphate form
by sulfuric-acid-hydrolysis. Organic phosphorus compounds may be converted to
the orthophosphate form by persulfate digestion(2 ).
3. Sample Handling and Preservation
3.1 If benthic deposits are present in the area being sampled, great care should be
taken not to include these deposits.
3.2 Sample containers may be of plastic material, such as cubitainers, or of Pyrex
glass.
3.3 If the analysis cannot be performed the same day of collection, the sample should
249
-------
to
u»
O
Residue
SAMPLE
Total Sample (No Filtration)
\/
V
Direct
Colorimetry
V
H2S°4
Hydrolysis
fn1 n-ri
Orthophosphate
Hydrolyzable §
Orthophosphate
Filter (through 0.45 u membrane filter)
\/
Filtrate
\
Direct
\
Colorimetry
/
Dissolved
Orthophosphate
A
H2S04
Hydrolysis §
. Colorimetry
Diss . Hydrolyzable
§ Orthophosphate
Persulfate
Digestion §
.Colorimetry
Dissolved
Phosphorus
Persulfate
Digestion
dolorimptry
Phosphorus
FIGURE 1. ANALYTICAL SCHEME FOR DIFFERENTIATION
OF PHOSPHORUS FORMS
-------
be preserved by the addition of 2 ml cone. H2 SO4 or 40 rng HgCl2 per liter and
refrigeration at 4°C. Note HgQ2 interference under (5.4).
4. Definitions and Storet Numbers
4.1 Total Phosphorus (P) all of the phosphorus present in the sample, regardless of
form, as measured by the persulfate digestion procedure. (00665)
4.1.1 Total Orthophosphate (P, ortho) inorganic phosphorus [(PO4)""3] in
the sample as measured by the direct colorimetric analysis procedure.
(70507)
4.1.2 Total Hydrolyzable Phosphorus (P, hydro) phosphorus in the sample
as measured by the sulfuric acid hydrolysis procedure, and minus
pre-determined orthophosphates. This hydrolyzable phosphorus includes
polyphosphorus. [(P2O7)-4, (PaOio)"5, etc.] + some organic phos-
phorus. (00669)
4.1.3 Total Organic Phosphorus (P, org) phosphorus (inorganic + oxidizable
organic) in the sample measured by the persulfate digestion procedure,
and minus hydrolyzable phosphorus and orthophosphate. (00670)
4.2 Dissolved Phosphorus (P D) all of the phosphorus present in the filtrate of a
sample filtered through a phosphorus-free filter of 0.45 micron pore size and
measured by the persulfate digestion procedure. (00666)
4.2.1 Dissolved Orthophosphate (PD, ortho) as measured by the direct
colorimetric analysis procedure. (00671)
4.2.2 Dissolved Hydrolyzable Phosphorus (PD, hydro) as measured by the
sulfuric acid hydrolysis procedure and minus pre-determined dissolved
orthophosphates. (00672)
4.2.3 Dissolved Organic Phosphorus (PD, org) as measured by the
persulfate digestion procedure, and minus dissolved hydrolyzable phos-
phorus and orthophosphate. (00673)
4.3 The following forms, when sufficient amounts of phosphorus are present in the
sample to warrant such consideration, may be calculated:
4.3.1 Insoluble Phosphorus (P-I) = (P) - (P-D). (00667) -
4.3.1.1 'Insoluble orthophosphate (P-I, ortho) = (P, ortho) - (P-D,
ortho). (00674)
4.3.1.2 Insoluble Hydrolyzable Phosphorus (P-I, hydro) = (P,.
hydro) - (P-D, hydro). (00675)
4.3.1.3 Insoluble Organic Phosphorus (P-I, org) = (P, org) - (P-D,
org). (00676)
251
-------
4.4 All phosphorus forms shall be reported as P, mg/1, to the third place.
5. Interferences
5.1 It is reported that no interference is caused by copper, iron, or silicate at
concentrations many times greater than their reported concentration in sea water.
However, high iron concentrations can cause precipitation of and subsequent loss
of phosphorus.
5.2 The salt error for samples ranging from 5 to 20% salt content was found to be less
than 1%. . .
5.3 Arsenate is determined similarily to phosphorus and should be considered when
present in concentrations higher than phosphorus. However, at concentrations
found in sea water, it does not interfere.
5.4 Mercury chloride, used as a preservative, interferes when the chloride level of the
sample is low (<50 mg Cl/1). This interference is overcome by spiking samples
with a minimum of 50 mg/1 of sodium chloride.
6. Apparatus
6.1 Photometer A spectrophotometer or filter photometer suitable for measure-
ments at 650 or 880 nm with a light path of 1 cm or longer.
6.2 Acid-washed glassware: All glassware used should be washed with hot 1:1 HC1 and
rinsed with distilled water. The acid-washed glassware should be filled with
distilled water and treated with all the reagents to remove the last traces of
phosphorus that might be adsorbed on the glassware. Preferably, this glassware
should be used only for the determination of phosphorus and after use it should
be rinsed with distilled water and kept covered until needed again. If this is done,
the treatment with 1:1 HC1 and reagents is only required occasionally.
Commercial detergents should never be used.
7. Reagents
7.1 Sulfuric acid solution, 5N: Dilute 70 ml of cone. H2SO4 with distilled water to
500 ml.
7.2 Antimony potassium tartrate solution: Weigh 1.3715 g K(SbO)C4H4O6 1 /2
H2O, dissolve in 400 ml distilled water in 500 ml volumetric flask, dilute to
volume. Store at 4°C in a dark, glass-stoppered bottle.
7.3 Ammonium molybdate solution: Dissolve 20 g(NH4)6Mo7O24 '4H2O in 500 ml
of distilled water. Store in a plastic bottle at 4°C.
7.4 Ascorbic acid, 0.1 M: Dissolve 1.76 g of ascorbic acid in 100 ml of distilled water.
The solution is stable for about a week if stored at 4°C.
7.5 Combined reagent: Mix the above reagents in the following proportions for 100
252
-------
ml of the mixed reagent: 50 m! of 5N H2SO4, (7.!), 5 rnl of antimony potassium
tartrate solution, (7.2), 15 ml of ammonium molybdate solution, (7.3), and 30 ml
of ascorbic acid solution (7.4). Mix after addition of each reagent. All reagents
must reach room temperature before they are mixed and must be mixed in the
order given. If turbidity forms in the combined reagent, shake and let it stand for
a few minutes until the turbidity disappears before proceeding. Since the stability
of this solution is limited, it must be freshly prepared for each run.
. 7.6 Sulfuric acid solution, 11 N: Slowly add 310 ml cone. H2SO4 to 600 ml distilled '
water. When cool, dilute to 1 liter.
7.7 Ammonium persuifate.
7.8 Stock phosphorus solution: Dissolve in distilled water 0.2197 g of potassium
dihydrogen phosphate, KH2PO4, which has been dried in an oven at 105°C.
Dilute the solution to 1000 ml; 1.0 ml = 0.05 mg P.
7.9 Standard phosphorus solution: Dilute 10.0 ml of stock phosphorus solution (7.8)
to 1000 ml with distilled water; 1.0 ml = 0.5 jug P.
7.9.1 Using standard solution, prepare the following standards in 50.0 ml
volumetric flasks:
ml of Standard
Phosphorus Solution (7.9) Cone., mg/1
0 0.00
1.0 0.01
3.0 . 0.03
5.0 0.05
10.0 0.10
20.0 0.20
30.0 0.30
40.0 0.40
50.0 0.50
7.10 Sodium hydroxide, 1 N: Dissolve 40 g NaOH in 600 ml distilled water. Cool and
dilute to 1 liter.
7.11 Phenolphthalein: Dissolve 0.5 g of phenolphthalein in a solution of 50 ml of ethyl
or isopropyl alcohol and 50 ml of distilled water.
8. Procedure
8.1 Phosphorus
8.1.1 Add 1 ml of H2SO4 solution (7.6) to a 50 ml sample in a 125 ml
Erlenmeyer flask.
253
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8.1.2 Add 0.4 g of ammonium persulfate.
8.1.3 Boil gently on a pre-heated hot plate for approximately 30-40 minutes or
until a final volume of about 10 ml is reached. Do not allow sample to go
to dryness. Alternatively, heat for 30 minutes in an autoclave at 121°C
(15-20psi).
8.1.4 Adjust the pH of the sample to 7 ±0.2 with 1 N NaOH (7.10) using a pH
meter. Cool and dilute the sample to 50 ml. If sample is not clear at this
point, filter.
8.1.5 Determine phosphorus as outlined in (8.3.2 Orthophosphate).
8.2 Hydrolyzable Phosphorus
8.2.1 Add 1 ml of H2SO4 solution (7.6) to a 50 ml sample in a 125 ml
Erlenmeyer flask.
8.2.2 Boil gently on a pre-heated hot plate for 30-40 minutes or until a final
volume of about 10 ml is reached. Do not allow sample to go to dryness.
Alternatively, heat for 30 minutes in an autoclave at 121°C (15-20 psi).
8.2.3 Adjust the pH of the sample to 7 ±0.2 with NaOH (7.10) using a pH
meter. Cool and dilute the sample to 50 ml.
8.2.4 The sample is now ready for determination of phosphorus as outlined in
(8.3.2 Orthophosphate).
8.3 Orthophosphate
8.3.1 Add 1 drop of phenolphthalein indicator (7.11) to the 50.0 ml sample. If
a red color develops, add strong-acid solution drop-wise to just discharge
the color.
NOTE: If just Orthophosphate is being measured, i.e., and there has been
no pretreatment of the sample and no subsequent neutralization as
outlined above, the pH of the sample must be adjusted to 7±0.2 using a
pH meter.
8.3.2 Add 8.0 ml of combined reagent (7.5) to sample and mix thoroughly.
After a minimum of ten minutes, but no longer than thirty minutes,
measure the color absorbance of each sample at 650 or 880 nm with a
spectrophotometer, using the reagent blank as the reference solution.
9. Calculation
9.1 Prepare a standard curve by plotting the absorbance values of standards versus the
corresponding phosphorus concentrations.
9.1.1 Process standards and blank exactly as the samples. Run at least a blank
254
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and two standards with each series of samples. If the standards do not
agree within ±2% of the true value, prepare a new calibration curve.
9.2 Obtain concentration value of sample directly from prepared standard curve.
Report results as P, mg/1..
10. Precision and Accuracy
10.1 Thirty-three analysts in nineteen laboratories analyzed natural water samples
containing exact increments of organic phosphate, with the following results:
Increment as Precision as Accuracy as
Total Phosphorus Standard Deviation Bias, Bias
mgP/liter mgP/liter % mg P/liter
0.110 0.033 + 3.09 +0.003
0.132 0.051 +11.99 +0.016
0.772 0.130 + 2.96 +0.023
0.882 0.128 - 0.92 -0.008
(FWPCA Method Study 2, Nutrient Analyses)
10.2 Twenty-six analysts in sixteen laboratories analyzed natural water samples
containing exact increments of orthophosphate, with the following results:
Increment as
Orthophosphate
mg P/liter
0.029
0.038
0.335
0.383
Precision as
Standard Deviation '
mg P/liter
0.010
0.008
0.018
0.023
Accuracy as
Bias,
%
-4.95
-6.00
-2.75
-1.76
Bias,
mg P/liter
-0.001
-0,002
-0.009
-0.007
(FWPCA Method Study 2, Nutrient Analyses)
Bibliography
1. Murphy, J., and Riley, J., "A Modified Single Solution for the Determination of
Phosphate in Natural Waters." Anal. Chim. Acta., 27, 31 (1962).
2. Gales, M., Jr., Julian, E., and Kroner, R., "Method for Quantitative Determination of
Total Phosphorus in Water." Jour. AWWA, 58, No. 10, 1363 (1966).
3. ASTM Standards, Part 23, Water; Atmospheric Analysis, D515-72, p 388 (1973).
255
-------
PHOSPHORUS, ALL FORMS
(Automated Colorimetric Ascorbic Acid Reduction Method)
STORET NO. See Section 4
1. Scope and Application
1.1 These methods cover the determination of specified forms of phosphorus in
drinking, surface, and saline waters, domestic and industrial wastes. They may be
applicable to sediment-type samples, sludges, algal blooms, etc., but sufficient
data is not available at this time to warrant such usage when measurements for
phosphorus content are required.
1.2 The methods are based on reactions that are specific for the orthophosphate ion.
Thus, depending on the prescribed pre-treatment of the sample, the various forms
of phosphorus given in Figure 1 may be determined. These forms are, in turn,
defined in Section 4.
1.2.1 Except for in-depth and detailed studies, the most commonly measured
forms are phosphorus and dissolved phosphorus, and orthophosphate
and dissolved orthophosphate. Hydrolyzable phosphorus is normally
found only in sewage-type samples and insoluble forms of phosphorus, as
noted, are determined by calculation.
1.3 The methods are usable in the 0.001 to 1.0 mg P/l range. Approximately 20-30
samples per hour can be analyzed.
2. Summary of Method
2.1 Ammonium molybdate and antimony potassium tartrate react in an acid medium
with dilute solutions of phosphorus to form an antimony-phospho-molybdate
complex. This complex is reduced to an intensely blue-colored complex by
ascorbic acid. The color is proportional to the phosphorus concentration.
2.2 Only orthophosphate forms a blue color in this test. Poly phosphates (and some
organic phosphorus compounds) may be converted to the orthophosphate form
by manual sulfuric-acid-hydrolysis. Organic phosphorus compounds may be
converted to the orthophosphate form by manual persulfate digestion(2). The
developed color is measured automatically on the Auto Analyzer.
3. Sample Handling and Preservation
3.1 If benthic deposits are present in the area being sampled, great care should be
taken not to include these deposits.
256
-------
Total Sample (No Filtration)
\ f
Direct
Colorimetry
Hydrolysis fi
\ f i'olorimetrv
Orthophosphate
Hydrolyzable 6
Orthophosphate
Filter (through 0.45 u membrane filter)
X,
Direct
Colorimetry
/ \
Dissolved
Orthophosphate
"2S°4
Hydrolysis £
^ Colorimetry ^
Diss. Hydrolyzable
6 Orthophosphate
Persulfate
Digestion 6
/ Colorimetry
Dissolved
Phosphorus
\ /
Tersulfate
Digestion
Phosphorus
FIGURE 1. ANALYTICAL SCHEME FOR DIFFERENTIATION
OF PHOSPHORUS FORMS
257
-------
3.2 Sample containers may be of plastic material, such as cubitainers, or of Pyrex
glass.
3.3 If the analysis cannot be performed the same day of collection, the sample should
be preserved by the addition of 2 ml cone. H2SO4 or 40 mgHgC!2 per liter and
refrigeration at 4°C. Note HgCl2 interference under (5.4).
4. Definitions and Storet Numbers
4.1 Total Phosphorus (P) all of the phosphorus present in the sample regardless of
form, as measured by the persulfate digestion procedure. (00665)
4.1.1 Total Orthophosphate (P-ortho)-inorganic phosphorus [(PO4 )~3 ] in the
sample as measured by the direct colorimetric analysis procedure.
(70507)
4.1.2 Total Hydrolyzable Phosphorus (P-hydro)-phosphorus in the sample as
measured by the sulfuric acid hydrolysis procedure, and minus predeter-
mined orthophosphates. This hydrolyzable phosphorus includes poly-
phosphates [(P2O7)~4, (P3Oj0)~s, etc.] + some organic phosphorus.
(00669)
4.1.3 Total Organic Phosphorus (P-org)phosphorus (inorganic + oxidizable
organic) in the sample as measured by the persulfate digestion procedure,
and minus hydrolyzable phosphorus and orthophosphate. (00670)
4.2 Dissolved Phosphorus (P-D) - all of the phosphorus present in the filtrate of a
sample filtered through a phosphorus-free filter of 0.45 micron pore size and
measured by the persulfate digestion procedure. (00666)
4.2.1 Dissolved Orthophosphate (P-D, ortho) - as measured by^ the direct
colorimetric analysis procedure. (00671)
4.2.2 Dissolved Hydrolyzable Phosphorus (PD, hydro) as measured by the
sulfuric acid hydrolysis procedure and minus predetermined dissolved
orthophosphates. (00672)
4.2.3 Dissolved Organic Phosphorus (P-D, org) - as measured by the
persulfate digestion procedure, and minus dissolved hydrolyzable phos-
phorus and orthophosphate. (00673)
4.3 The following forms, when sufficient amounts of phosphorus are present in the
sample to warrant such consideration, may be calculated:
4.3.1 Insoluble Phosphorus (P-I) = (P) - (P-D). (00667)
4.3.1.1 Insoluble orthophosphate (P-I, ortho) = (P, ortho) - (P-D,
ortho). (00674)
258
-------
4,3.1.2 Insoluble Hydrolyzable Phosphorus (P-I, hydro) = (P,
hydro) - (P-D, hydro). (00675)
4.3.1.3 Insoluble Organic Phosphorus (P-I, org) = (P, org) - (P-D,
org). (00676)
4.4 All phosphorus forms shall be reported as P, mg/1, to the third place.
5. Interferences
5.1 It .is reported that no interference is caused by copper, iron, or silicate at
concentrations many times greater than their reported concentration in sea water.
However, high iron concentrations can cause precipitation of and subsequent loss
of phosphorus.
5.2 The salt error for samples ranging from 5 to 20% salt content was found to be less
than 1%.
5.3 Arsenate is determined similarly to phosphorus and should be considered when
present in concentrations higher than phosphorus. However, at concentrations
found in sea water, it does not interfere.
5.4 Mercury chloride, used as a preservative, interferes. This interference is overcome
in the AAI method by substituting a solution of sodium chloride (2.5 g/1) in place
of the distilled water.
5.5 Sample turbidity must be removed by filtration prior to analysis for ortho-
phosphate. Samples for total or total hydrolyzable phosphorus should be filtered
only after digestion. Sample color that absorbs in the photometric range used for
analysis will also interfere.
6. Apparatus
6.1 Technicon Auto Analyzer consisting of:
6.1.1 Sampler.
6.1.2 Manifold (AAI) or Analytical Cartridge (AAII).
6.1.3 Proportioning pump.
6.1.4 Heating bath, 50°C.
6.1.5 Colorimeter equipped with 15 or 50 mm tubular flow cell.
6.1.6 650-660 or 880 nm filter.
6.1.7 Recorder.
6.1.8 Digital printer for AAII (optional).
6.2 Hot plate or autoclave.
6.3 Acid-washed glassware: All glassware used in the determination should be washed
with hot 1:1 HC1 and rinsed with distilled water. The acid-washed glassware
should be filled with distilled water and treated with all the reagents to remove
259
-------
the last traces of phosphorus that might be adsorbed on the glassware. Preferably,
this glassware should be used only for the determination of phosphorus and after
use it should be rinsed with distilled water and kept covered until needed again. If
this is done, the treatment with 1:1 HC1 and reagents is only required
occasionally. Commercial detergents should never be used.
1. Reagents
7.1 Sulfuric acid solution, 5N: Slowly add 70 ml of cone. H2SO4 to approximately
400 ml of distilled water. Cool to room temperature and dilute to 500 ml with
distilled water.
7.2 Antimony potassium tartrate solution: Weigh 0.3 g K(SbO)C4H4O6-1/2H2O,
dissolve in 50 ml distilled water in 100 ml volumetric flask, dilute to volume.
Store at 4° C in a dark, glass-stoppered bottle.
7.3 Ammonium molybdate solution: Dissolve 4 g (NH4)6Mo7O24 '4H2O in 100 ml
distilled water. Store in a plastic bottle at 4°C.
7.4 Ascorbic acid, 0.1M: Dissolve 1.8 g of ascorbic acid in 100 ml of distilled water.
The solution is stable for about a week if prepared with water containing no more
than trace amounts of heavy metals and stored at 4°C.
7.5 Combined reagent (AAI): Mix the above reagents in the following proportions for
100 ml of the mixed reagent: 50 ml of 5N H2SO4 (7.1), 5 ml of antimony
potassium tartrate solution (7.2), 15 ml of ammonium molybdate solution (7.3),
and 30 ml of ascorbic acid solution (7.4). Mix after addition of each reagent. All
reagents must reach room temperature before they are mixed and must be mixed
in the order given. If turbidity forms in the combined reagent, shake and let it
stand for a few minutes until the turbidity disappears before processing. This
volume is sufficient for 4 hours operation. Since the stability of this solution is
limited, it must be freshly prepared for each run.
NOTE 1: A stable solution can be prepared by not including the ascorbic acid in
the combined reagent. If this is done, the mixed reagent (molybdate, tartrate, arid
acid) is pumped through the distilled water line and the ascorbic acid solution (30
ml of 7.4 diluted to 100 ml with distilled water) through the original mixed
reagent line.
7.6 Sulfuric acid solution, UN: Slowly add 310 ml cone. H2SO4 to 600 ml distilled
water. When cool, dilute to 1 liter.
7.7 Ammonium persulfate.
7.8 Acid wash water: Add 40 ml of sulfuric acid solution (7.6) to 1 liter of distilled
260
-------
water and dilute to 2 liters. (Not to be used when only orthophosphate is being
determined).
7.9 Phenolphthalein indicator solution (5 g/1): Dissolve 0.5 g of phenolphthalein in a
solution of 50 ml of ethyl or isopropyl alcohol and 50 ml of distilled water.
7.10 Stock phosphorus solution: Dissolve 0.4393 g of pre-dried (105°C for 1 hour)
KH2PO4 in distilled water and diluted 1000 ml. 1.0 ml = 0.1 mg P.
7.11 Standard phosphorus solution: Dilute 100.0 ml of stock solution (7.10) to 1000
ml with distilled water. 1.0 ml = 0.01 mg P.
7.12 Standard phosphorus solution: Dilute 100.0 ml of standard solution (7.11) to
1000 ml with distilled water. 1.0 ml = 0.001 mg P.
7.13 Prepare a series of standards by diluting suitable volumes of standard solutions
(7.11) and (7.12) to 100.0 ml with distilled water. The following dilutions are
suggested:
ml of Standard Cone.,
Phosphorus Solution (7.12) mg P/l
0.0 0.00
2.0 ! 0.02
5.0 0.05
10.0 0.10
ml of Standard
Phosphorus Solution (7.11)
2.0 0.20
5.0 0.50
8.0 0.80
10.0 . 1.00
8. Procedure
8.1 Phosphorus
8.1.1 Add 1 ml of sulfuric acid solution (7.6) to a 50 ml sample and standards
in a 125 ml Erlenmeyer flask.
8.1.2 Add 0.4 g of ammonium persulfate.
8.1.3 Boil gently on a pre-heated hot plate for approximately 30-40 minutes or
261
-------
until a final volume of about 10 ml is reached. Do not allow sample to go
to dryness. Alternately, heat for 30 minutes in an autoclave at 121°C
(15-20psi).
8.1.4 Cool and dilute the sample to 50 ml. If sample is not clear at this point,
filter.
8.1.5 Determine phosphorus as outlined in (8.3.2) with acid wash water (7.8)
in wash tubes.
8.2 Hydrolyzable Phosphorus
8.2.1 Add 1 ml of sulfuric acid solution (7.6) to a 50 ml sample and standards
in a 125 ml Erlenmeyer flask.
8.2.2 Boil gently on a pre-heated hot plate for 30-40 minutes or until a final
volume of about 10 ml is reached. Do not allow sample to go to dryness.
Alternatively, heat for 30 minutes in an autoclave at 121°C (15-20 psi).
8.2.3 Cool and dilute the sample to 50 ml. If sample is not clear at this point,
filter.
8.2.4 Determine phosphorus as outlined in (8.3.2) with acid wash water (7.8)
in wash tubes.
8.3 Orthophosphate
8.3.1 Add 1 drop of phenolphthalein indicator solution (7.9) to approximately
50 ml of sample. If a red color develops, add sulfuric acid solution (7.6)
drop-wise to just discharge the color. Acid samples must be neutralized
with 1 N sodium hydroxide (40 g NaOH/1).
8.3.2 Set up manifold as shown in Figure 2, AAI or Figure 3, AAII.
8.3.3 Allow both colorimeter and recorder to warm up for 30 minutes. Obtain
a stable baseline with all reagents, feeding distilled water through the
sample line.
8.3.4 For the AAI system, sample at a rate of 20/hr, 1 minute sample, 2
minute wash. For the AAII system, use a 30/hr, 2:1 cam, and a common
wash.
8.3.5 Place standards in Sampler in order of decreasing concentration.
Complete filling of sampler tray with unknown samples.
8.3.6 Switch sample line from distilled water to Sampler and begin analysis.
9. Calculation
9.1 Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Compute concentrations of samples by comparing sample
peak heights with standard curve. Any sample whose computed value is less than
i
262
-------
5% of its immediate predecessor must be rerun.
10. Precision and Accuracy (AAI system)
10.1 Six laboratories analyzed four natural water samples containing exact increments
of orthophosphate, with the following results:
Increment as
Orthophosphate
mg P/liter
0.04
0.04
0.29
0.30
Precision as
Standard Deviation
mg P/liter
0.019
0.014
0.087
0.066
Accuracy as
Bias,
%
+16.7
- 8.3
-15.5
-12.8
Bias,
mg P/liter
+0.007
-0.003
-0.05
-0.04
(FWPCA Method Study 4, Automated Methods - In preparation).
10.2 In a single laboratory, (MDQARL), using surface water samples at concentrations
of 0.04, 0.19, 0.35, and 0.84 mg P/l, standard deviations were ±0.005, ±0.000,
±0.003, and ±0.000, respectively.
10.3 In a single laboratory, (MDQARL), using surface water samples at concentrations
of 0.07 and 0.76 mg P/l, recoveries were 99% and 100%, respectively.
Bibliography
1. Murphy, J. and Riley, J., "A Modified Single Solution for the Determination of
Phosphate in Natural Waters." Anal. Chim. Acta., 27, 31 (1962).
2. Gales, M., Jr., Julian, E., and Kroner, R., "Method for Quantitative Determination of
Total Phosphorus in Water." Jour AWWA, 55, No. 10, 1363 (1966).
3. Lobring, L. B. and Booth, R. L., "Evaluation of the Auto Analyzer II; A Progress
Report." Technicon International Symposium, June, 1972. New York, N.Y.
4. ASTM Standards, Part 23, Water; Atmospheric Analysis, 515-72, p 388 (1973).
263
-------
0\
SM= SMALL MIXING
LM= LARGE MIXING
COIL
COIL
WASH WATER
TO SAMPLER
SM
oono
50°C(
HEATINGV
BATH
1
cc
LM
nnnnnnnn
)
i
f
SM
nnnn
b
^
)LORIMETER
WAST
E
I
a I
1 r
\J
*4L.
T
P B
P B
R R
Y Y
0 0
6 6
PROPORTIONING
PUMP
nl/min
2.9 WASH
2.9 SAMPLE o
SAMPLER
0.8 AIR 20/hr
2 = 1
1.2 DISTILLED
WATER
0.42 MIXED
REAGENT
2.00 WAoTr
RECORDER
50mm FLOW CELL
650-660 or 880nm FILTER
FIGURE 2 PHOSPHORUS MANIFOLD AA I
-------
to
WASH WATER
TO SAMPLER
0000
I
k HEATING WA
0 BATH WA
R 37 C
r
4
SI
k
^ oooo
,4
--*
£
-^
r
G G
0 0
BLACK
BLACK
0 W
W W
PROPORTIONING
1 miMD
nl/min
2.0 WASH
0.42 SAMPLE
0.32 AIR
0.32 DISTILLED
WATER
0.23 MIXED
REAGENT
.Qj-S- wnAQTr
0
SAMPLER
30/ hr
2«l
WASTED
RECORDER
DIGITAL
PRINTER
COLORIMETER
50mm FLOW CELL
650-660nm or
880nm FILTER
FIGURE 3 PHOSPHORUS MANIFOLD AAII
-------
RESIDUE, Total Filterable
(Dried at 180°C)
STORET NO. 70300
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The practical range of the determination is 10 mg/1 to 20,000 mg/1.
2. Summary of Method
2.1 A well-mixed sample is filtered through a standard glass fiber filter. The filtrate is
evaporated and dried to constant weight at 180°C.
2.2 If Residue, Total Non-Filterable is being determined, the filtrate from that
method may be used for Residue, Total Filterable.
3. Definitions
3.1 Filterable solids are defined as those solids capable of passing through a standard
glass fiber filter and dried to constant weight at 180°C.
4. Sample Handling and Preservation
4.1 Preservation of the sample is not practical; analysis should begin as soon as
possible.
5. Interferences
5.1 Highly mineralized waters containing significant concentrations of calcium,
magnesium, chloride and/or sulfate may be hygroscopic and will require
prolonged drying, desiccation and rapid weighing.
5.2 Samples containing high concentrations of bicarbonate will require careful and
possibly prolonged drying at 180°C to insure that all the bicarbonate is converted
to carbonate.
5.3 Too much residue in the evaporating dish will crust over and entrap water that
will not be driven off during drying. Total residue should be limited to about 200
mg.
6. Apparatus
6.1 Glass fiber filter discs, 4.7 cm or 2.2 cm, without organic binder, Reeve Angel
type 934-A, 984-H, Gelman type A, or equivalent.
6.2 Filter holder, membrane filter funnel or Gooch crucible adapter.
6.3 Suction flask, 500 ml.
6.4 Gooch crucibles, 25 ml (if 2.2 cm filter is used).
266
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6.5 Evaporating dishes, porcelain, 100 ml volume. (Vycor or platinum dishes may be
substituted).
6.6 Steam bath.
6.7 Drying oven, 180°C±2°C.
6.8 Desiccator.
6.9 Analytical balance, 200 g capacity, capable of weighing to 0.1 mg.
7. Procedure
7.1 Preparation of glass fiber filter disc: Place the disc on the membrane filter
apparatus or insert into bottom of a suitable Gooch crucible. While vacuum is
applied, wash the disc with three successive 20 ml volumes of distilled water.
Remove all traces of water by continuing to apply vacuum after water has passed
through. Discard washings.
7.2 Preparation of evaporating dishes: Heat the clean dish to 550±50°C for one hour
in a muffle furnace. Cool in desiccator and store until needed. Weigh immediately
before use.
7.3 Assemble the filtering apparatus and begin suction. Shake the sample vigorously
and rapidly transfer 100 ml to the funnel by means of a 100 ml graduated
cylinder. If total filterable residue is low, a larger volume may be filtered.
7.4 Filter the sample through the glass fiber filter and continue to apply vacuum for
about 3 minutes after filtration is complete to remove as much water as possible.
7.5 Transfer 100 ml (or a larger volume) of the filtrate to a weighed evaporating dish
and evaporate to dryness on a steam bath.
7.6 Dry the evaporated sample for at least one hour at 180±2°C. Cool in a desiccator
and weigh. Repeat the drying cycle until a constant weight is obtained or until
weight loss is less than 0.5 mg.
8. Calculation
8.1 Calculate filterable residue as follows:
(A-B)X 1000
Filt. residue, mg/1 =
C
where:
A = weight of dried residue + dish
B = weight of dish
C = volume of filtrate used
9. Precision and Accuracy
9.1 Precision data are not available at this time.
9.2 Accuracy data on actual sample cannot be obtained.
267
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RESIDUE, TOTAL NON-FILTERABLE
STORET NO. 00530
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The practical range of the determination is 10 mg/1 to 20,000 mg/1.
2. Summary of Method
2.1 A well-mixed sample is filtered through a standard glass fiber filter, and the
residue retained on the filter is dried to constant weight at 103-105°C.
2.2 The filtrate from this method may be used for Residue, Total Filterable.
3. Definitions
3.1 Non-filterable solids are defined as those solids which are retained by a standard
glass fiber filter and dried to constant weight at 103-105°C.
4. Sample Handling and Preservation
4.1 Non-homogeneous particulates such as leaves, sticks, fish, and lumps of fecal
matter should be excluded from the sample.
4.2 Preservation of the sample is not practical; analysis should begin as soon as
possible.
5. Interferences
5.1 Too much residue on the filter will entrap water and may require prolonged
drying.
6. Apparatus
6.1 Glass fiber filter discs, 4.7 cm or 2.2 cm, without organic binder, Reeve Angel
type 934-A or 984-H, Gelman type A, or equivalent.
6.2 Filter holder, membrane filter funnel or Gooch crucible adapter.
6.3 Suction flask, 500 ml.
6.4 Gooch crucibles, 25 ml (if 2.2 cm filter is used).
6.5 Drying oven, 103-105°C.
6.6 Desiccator.
6.7 Analytical balance, 200 g capacity, capable of weighing to 0.1 mg.
7. Procedure
7.1 Preparation of glass fiber filter disc: Place the disc on the membrane filter
apparatus or insert into bottom of a suitable Gooch crucible. While vacuum is
applied, wash the disc with three successive 20 ml volumes of distilled water.
268
-------
Remove all traces of water by continuing to apply vacuum after water has passed
through. Remove filter from membrane filter apparatus or both crucible and filter
if Gooch crucible is used, and dry in an oven at 103-105°C for one hour. Remove
to desiccator and store until needed. Weigh immediately before use.
7.2 Assemble the filtering apparatus and begin suction. Shake the sample vigorously
and rapidly transfer 100 ml to the funnel by means of a 100 ml volumetric
cylinder. If suspended matter is low, a larger volume may be filtered.
7.3 Carefully remove the filter from the membrane filter funnel assembly. Alterna-
tively, remove crucible and filter from crucible adapter. Dry at least one hour at
103-105°C. Cool in a desiccator and weigh. Repeat the drying cycle until a
constant weight is obtained or until weight loss is less than 0.5 mg.
8. Calculations
8.1 Calculate non-filterable residue as follows:
(A-B) X 1000
Non-filt. residue, mg/1 =
\^
where:
A = weight of filter + residue
B = weight of filter
C = ml of sample filtered
9. Precision and Accuracy
9.1 Precision data are not available at this time.
9.2 Accuracy data on actual samples cannot be obtained.
269
-------
RESIDUE, Total
STORE! NO. 00500
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The practical range of the determination is from 10 mg/1 to 20,000 mg/1.
2. Summary of Method
2.1 A well mixed aliquot of the test sample is quantitatively transferred to a
pre-weighed evaporating dish and evaporated to dryness at 103-105°C.
3. Definitions
3.1 Total Residue is defined as the sum of the homogenous suspended and dissolved
materials in a sample.
4. Sample Handling and Preservation
4.1 Samples should be analyzed as soon as practicable.
5. Interferences
5.1 Large, floating particles or submerged agglomerates (non-homogenous materials)
should be excluded from the test sample.
5.2 Floating oil and grease, if present, should be included in the sample and dispersed
by a blender device before aliquoting.
6. Apparatus
6.1 Evaporating dishes, porcelain, 90 mm, 100 ml capacity. (Vycor or platinum dishes
may be substituted and smaller size dishes may be used if required.)
7. Procedure
7.1 Heat the clean evaporating dish to 550±50°C for 1 hour in a muffle furnace. Cool,
desiccate, weigh and store in desiccator until ready for use.
7.2 Transfer a measured aliquot of sample to the pre-weighed dish and evaporate to
dryness on a steam bath or in a drying oven.
7.2.1 Choose an aliquot of sample sufficient to contain a residue of at least 25
mg. To obtain a weighable residue, successive aliquots of sample may be
added to the same dish.
7.2.2 If evaporation is performed in a drying oven, the temperature should be
lowered to approximately 98°C to prevent boiling and splattering of the
sample.
7.3 Dry the evaporated sample for at least 1 hour at 103-105°C. Cool in a desiccator
270
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and weigh. Repeat the cycle of drying at 103-105°C, cooling, desiccating and
weighing until a constant weight is obtained or until loss of weight is less than 4%
of the previous weight, or 0.5 mg, whichever is less.
8. Calculation
8.1 Calculate total residue as follows:
(A-B) X 1000
Total residue, mg/1 =
where:
A = weight df sample + dish
B = weight of dish
C =' volume of sample
9. Precision and Accuracy
9.1 Precision and accuracy data are not available at this time.
271
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RESIDUE, Volatile
STORET NO. Total 00505
Nonfilterable 00535
Filterable 00520
1. Scope and Application
1.1 This method determines the weight of solid material combustible at 550°C.
1.2 The test is useful in obtaining a rough approximation of the amount of organic
matter present in the solid fraction of sewage, activated sludge, industrial wastes,
or bottom sediments.
2. Summary of Method
2.1 The residue obtained from the determination of total filterable or non-filterable
residue is ignited at 550°C in a muffle furnace. The loss of weight on ignition is
reported as mg/1 volatile residue.
3. Comments
3.1 The test is subject to many errors due to loss of water of crystallization, loss of
volatile organic matter prior to combustion, incomplete oxidation of certain
complex organics, and decomposition of mineral salts during combustion.
3.2 The results should not be considered an accurate measure of organic carbon in the
sample, but may be useful in the control of plant operations.
3.3 The principal source of error in the determination is failure to obtain a
representative sample.
4. Sample Handling and Preservation
4.1 Preservation of the sample is not practical; analysis should begin as soon as
possible.
5. Precision and Accuracy
5.1 A collaborative study involving three laboratories examining four samples by
means of ten replicates showed a standard deviation of ±11 mg/1 at 170 mg/1
volatile residue concentration.
6. Reference
6.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
536, Method 224B (1971).
272
-------
SETTLEABLE MATTER
STORET NO. 50086
1. Scope and Application
1.1 This method is applicable to surface and saline waters, domestic and industrial
wastes.
1.2 The practical lower limit of the determination is about 1 ml/l/hr.
2. Summary of Method
2.! Settleable matter is measured volumetrically with an Irnhoff cone.
3. Comments
3.1 For some samples, a separation of settleable and floating materials will occur; in
such cases the floating materials are not measured.
3.2 Many treatment plants, especially plants equipped to perform gravimetric
measurements, determine residue non-filterable (suspended solids), in preference
to settleable matter, to insure that floating matter is included in the analysis.
4. Precision and Accuracy
4.1 Data on this determination is not available at this time.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
539, Method 224F, Procedure a (1971).
273
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SILICA, Dissolved
STORET NO. 00955
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 The working range of the method is approximately 2 to 25 mg silica/1. The upper
range can be extended by taking suitable aliquots; the lower range can be
extended by the addition of amirio-naphthol-sulfonic acid solution, as described
in the ASTM reference.
2. Summary of Method
2.1 A well-mixed sample is filtered through a 0.45 p membrane filter. The filtrate,
upon the addition of molybdate ion in acidic solution, forms a greenish-yellow
color complex proportional to the dissolved silica in the sample. The color
complex is then measured spectrophotometrically.
3. Comments
3.1 Excessive color and/or turbidity interfere. Correct by running blanks prepared
without addition of the ammonium molybdate solution.
4. Precision and Accuracy
4.1 Photometric evaluations by the amino-naphthoi-sulfonic acid procedure have an
estimated precision of ±0.10 mg/1 in the range from 0 to 2 mg/1 (ASTM).
4.2 Photometric evaluations of the silico-molybdate color in the range from 2 to 50
mg/1 have an estimated precision of approximately 4% of the quantity of silica
measured (ASTM).
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
303, Method 15IB (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 401, Method D859-68
(1973).
274
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SPECIFIC CONDUCTANCE
(A/mhos at 25°C)
STORET NO. 00095
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
2. Summary of Method
2.1 The specific conductance of a sample is measured by use of a self-contained
conductivity meter, Wheatstone bridge-type, or equivalent.
2.2 Samples are preferably analyzed at 25°C. If not, temperature corrections are
made and results reported at 25°C. ,:
3. Comments
3.1 Instrument must be standardized with KC1 solution before daily use.
3.2 Conductivity cell must be kept clean.
3.3 Field measurements with comparable instruments are reliable.
4. Precision and Accuracy
4.1 Forty-one analysts in 17 laboratories analyzed six synthetic water samples
containing increments of inorganic salts, with the following results:
Increment as
Specific Conductance
jumhos/cm
100
106
808
848
1640
1710
Precision as
Standard Deviation
I/mhos/ cm
7.55
8.14
66.1
79.6
106
119
Accuracy as
Bias,
%
-2.02
-0.76
-3.63
-4.54
-5.36
-5.08
Bias,
pmhos/cm
-2.0
-0.8
-29.3
-38.5
-87.9
-86.9
(FWPCA Method Study 1, Mineral and Physical Analyses.)
4.2 In a single laboratory (MDQARL), using surface water samples with an average
conductivity of 536 /umhos/cm at 25°C, the standard deviation was ±6.
275
-------
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Waste water, 13th Edition, p
323, Method 154(1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 128, Method
Dl 125-64 (1973).
276
-------
3.
4.
SULFATE
(Turbidimetric)
STORET NO. 00945
Scope and Application
1.1 This method is applicable to drinking and surface waters, domestic and industrial
wastes.
1.2 The method is suitable for all concentration ranges of sulfate; however, in order
to obtain reliable readings, use a sample aliquot containing not more than 40 mg
S04/l.
Summary of Method
2.1 Sulfate ion is converted to a barium sulfate suspension under controlled
conditions. The resulting turbidity is determined by a colorimeter or spectro-
photometer and compared to a curve prepared from standard sulfate solutions.
2.2 Suspended matter and color interfere. Correct by running blanks from which the
barium chloride has been omitted.
Comments
3.1 Proprietary reagents, such as Hach Sulfaver or equivalent, are acceptable.
Precision and Accuracy
4.1 Thirty-four analysts in 16 laboratories analyzed six synthetic water samples
containing exact increments of inorganic sulfate with the following results:
Increment as
Sulfate
mg/liter
8.6
9.2
110
122
188
199
Precision as
Standard Deviation
mg/liter
2.30
1.78
7.86
. 7.50
9.58
11.8
Accuracy as
Bias,
%
-3.72
-8.26
-3.01
-3.37
+0.04
-1.70
Bias,
mg/liter
-0.3
-0.8
-3.3
-4-1
+0.1
-3.4
(FWPCA Method Study 1, Mineral and Physical Analyses).
277
-------
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
334, Method 156C( 1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 425, Method B,
0516-68(1973).
278
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SULFATE
(Automated Chloranilate Method)
STORET NO. 00945
1. Scope and Application
1.1 This automated method is applicable to drinking and surface waters, domestic and
industrial wastes, in the range of 10 to 400 mg SO4 /I. Approximately 15 samples
per hour can be analyzed.
2. Summary of Method,
2.1 When solid barium chloranilate is added to a solution containing sulfate, barium
sulfate is precipitate'd, releasing the highly colored acid chloranilate ion. The color
intensity in the resulting chloranilic acid is proportional to the amount of sulfate
present.
3. Sample Handling and Preservation
3.1 No special requirements.
4. Interferences
4.1 Cations, such as calcium, aluminum, and iron, interfere by precipitating the
chloranilate. These ions are removed automatically by passage through an ion
exchange column.
5. Apparatus
5.1 Technicon AutoAnaiyzer consisting of:
5.1.1 Sampler I.
5.1.2 Continuous filter.
5.1.3 Manifold.
5.1.4 Proportioning pump.
5.1.5 Colorimeter equipped with 15 mm tubular flow cell and 520 nm filters.
5.1.6 Recorder.
5.1.7 Heating bath, 45°C.
5.2 Magnetic stirrer.
6. Reagents
6.1 Barium chloranilate: Add 9 g of barium chloranilate (BaC6Cl2O4) to 333 ml of
ethyl alcohol and dilute to 1 liter with distilled water.
6.2 Acetate buffer, pH 4.63: Dissolve 13.6 g of sodium acetate in distilled water. Add
6.4 ml of acetic acid and dilute to 1 liter with distilled water. Make fresh weekly.
279
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6.3 NaOH-EDTA solution: Dissolve 65 g of NaOH and 6 g of EDTA in distilled water
and dilute to 1 liter.
NOTE 1: This solution is also used to clean out manifold system at end of
sampling run.
6.4 Ion exchange resin: Dowex-50 W-X8, ionic form H+.
NOTE 2: Column is prepared by su6king a slurry of the resin into 12 inches of
"3/16-inch OD sleeving. This may be conveniently done by using a pipette and a
loose-fitting glass wool plug in the sleeve. The column, upon exhaustion, turns
red.
6.5 Stock solution: Dissolve 1.4790 g of oven-dried (105°C) Na2SO4 in distilled
water and dilute to 1 liter in a volumetric flask. 1.0 ml = 1.0 mg.
6.5.1 Prepare a series of standards by diluting suitable volumes of stock
solution to 100.0 ml with distilled water. The following dilutions
are suggested:
ml of Stock Solution Cone., mg/1
1.0 10
2.0 20
4.0 40
6.0 60
8.0 80
10.0 100
15.0 150
20.0 200
30.0 300
40.0 400
7. Procedure
7.1 Set up manifold as shown in Figure 1. (Note that any precipitated BaSO4 and the
unused barium chloranilate are removed by filtration. If any BaSO4 should come
through the filter, it is complexed by the NaOHEDTA reagent).
7.2 Allow both colorimeter and recorder to warm up for 30 minutes. Run a baseline
with all reagents, feeding distilled water through the sample line. Adjust dark
current and operative opening on colorimeter to obtain suitable baseline.
7.3 Place distilled water wash tubes in alternate openings in sampler and set sample
timing at 2.0 minutes.
280
-------
7.4 Place working standards in sampler in order of decreasing concentration.
Complete filling of sampler tray with unknown samples.
7.5 Switch sample line from distilled water to sampler and begin analysis.
8. Calculation
8.1 Prepare standard curve by plotting peak heights of processed standards against
known concentrations. Compute concentration of samples by comparing sample
peak heights with standard curve.
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL). using surface water samples at concentrations
of 39, 111, 188 and 294 mg SO4/1, the standard deviations were ±0.6, ±1.0, ±2.2
and ±0.8, respectively.
9.2 In a single laboratory (MDQARL) using surface water samples at concentrations
of 82 and 295 mg SO4/1, recoveries were 99% and 102%, respectively.
Bibliography
1. Barney, J. E., and Bertolocini, R. J., Anal. Chem., 29, 283 (1957).
2. Gales, M. E., Jr., Kaylor, W. H. and Longbottom, J. E., "Determination of Sulphate by
Automatic Colorimetric Analysis". Analyst, 93, 97 (1968).
281
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SULFATE
(Gravimetric)
STORET NO. 00945
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 This method is the most accurate method for sulfate concentrations above 10
mg/'l. Therefore, it should be used whenever results of the greatest accuracy are
required.
2. Summary of Method
2.1 Sulfate is precipitated as barium sulfate in a hydrochloric acid medium by the
addition of barium chloride. After a period of digestion, the precipitate is filtered,
washed with hot water until free of chloride, ignited, and weighed as BaSO4.
3. Comments
3.1 High results may be obtained for samples that contain suspended matter, nitrate,
sulfite and silica.
3.2 Alkali metal sulfates frequently yield low results. This is especially true of alkali
hydrogen sulfates. Occlusion of alkali sulfate with barium sulfate causes the
substitution of an element of lower atomic weight than barium in the precipitate.
Hydrogen sulfate of alkali metal acts similarly and decomposes when heated.
Heavy metals such as chromium and iron, cause low results by interfering with
complete precipitation and by formation of heavy metal sulfates.
4. Precision and Accuracy
4.1 A synthetic unknown sample containing 259 mg/1 sulfate, 108 mg/1 Ca, 82 mg/1
Mg, 3.1 mg/1 K, 19.9 mg/J Na, 241 mg/1 chloride, 250 jug/1 nitrite N, 1.1 mg/1
nitrate N and 42.5 mg/1 total alkalinity (contributed by NaHCO3), was analyzed
by the gravimetric method, with a relative standard deviation of 4.7% and a
relative error of 1.9% in 32 laboratories.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
331, Method 156 A (1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 425, Method A,
D516-68(1973).
283
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SULFIDE
(Titrimetric Iodine Method)
STORET NO. Total 00745
Dissolved 00746
1. Scope and Application
1.1 This method is applicable to the measurement of total and dissolved sulfides in
drinking, surface, and saline waters, domestic and industrial wastes.
1.2 Acid insoluble sulfides are not measured by the use of this test. (Copper sulfide is
the only common sulfide in this class).
1.3 This method is suitable for the measurement of sulfide in concentrations above 1
mg/1.
2. Summary of Method
2.1 Sulfides are stripped from the acidified sample with an inert gas and collected in a
zinc acetate solution. Excess iodine added to the zinc sulfide suspension reacts
with the sulfide under acidic conditions. Thiosulfate is used to measure unreacted
iodine to indicate the quantity of iodine consumed by sulfide.
3. Comments
3.1 Reduced sulfur compounds, such as sulfite, thiosulfate and hydrosulfite, which
decompose in acid may yield erratic results. Also, volatile iodine-consuming
substances will give high results.
3.2 Samples must be taken with a minimum of aeration. Sulfide may be volatilized by
aeration and any oxygen inadvertently added to the sample may convert the
sulfide to an unmeasurable form.
3.3 If the sample is not preserved with zinc acetate, the analysis must be started
immediately. Similarly, the measurement of dissolved sulfides must also be
commenced immediately.
4. Precision and Accuracy
4.1 Precision and accuracy for this method have not been determined.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewaters, 13th Edition, p
551-555, Method No. 228A (1971).
284
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SULFITE
STORET NO. 00740
1. Scope and Application
1.1 This method is applicable to drinking and surface waters, sewage and industrial
wastes.
1.2 The minimum detectable limit is 2-3 mg/1 SO3.
2. Summary of Method
2.1 An acidified sample containing an indicator is titrated with a standard potassium
iodide-iodate titrant to a faint permanent blue end point.
3. Comments
3.1 The temperature of the sample must be below 50°C.
3.2 Care must be taken to allow as little contact with air as possible. For example, do
not filter the sample and keep the buret tip below the surface of the sample.
3.3 Other oxidizable substances, such as organic compounds, ferrous iron and sulfide
are positive interferences. Nitrite gives a negative interference by oxidizing sulfite
when the sample is acidified; this is corrected by either using a proprietary
indicator which eliminates nitrite or by adding sulfamic acid. Copper and possibly
other heavy metals catalyze the oxidation of sulfite; EDTA is used to complex
metals.
3.4 A blank must be run.
4. Precision and Accuracy
4.1 Precision and accuracy data are not available at this time.
5. References
5.1 The procedure to be used for this determination is found in:
Standard Methods"for the Examination of Water and Wastewater, 13th Edition, p
337-338, Method 158(1971).
ASTM Standards, Part 23, Water; Atmospheric Analysis, p 436 Method D-1339
Method C (1973).
285
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TEMPERATURE
STORET NO. 00010
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
2. Summary of Method
2.1 Temperature measurements may be made with any good grade of mercury-filled
or dial type centigrade thermometer, or a thermistor.
3. Comments
3.1 Measurement device should be checked against a precision thermometer certified
by the National Bureau of Standards.
4. Precision and Accuracy
4.1 Precision and accuracy for this method have not been determined.
5. Reference
5.1 The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water and Wastewater, 13th Edition, p
348, Method 162(1971).
286
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THRESHOLD ODOR
(Consistent Series Method)
STORET NO. 60°C: 00086
Room Temp: 00085
1. Scope and Application
1.1 This method is applicable to the determination of threshold odor of drinking,
surface, and saline waters, domestic and industrial wastes.
1.2 Highly odorous ^ samples are reduced in concentration proportionately before
being tested. Thus, the method is applicable to samples ranging from nearly
odorless natural waters to industrial wastes with threshold odor numbers in the
thousands.
2. Summary of Method(')
2.1 The sample of water is diluted with odor-free water until a dilution that is of the
least definitely perceptible odor to each tester is found. The resulting ratio by
which the sample has been diluted is called the "threshold odor number" (T.O.).
2.2 People vary widely as to odor sensitivity, and even the same person will not be
consistent in the concentrations he can detect from day to day. Therefore, panels
of not less than five persons, and preferably 10 or more, are recommended to
overcome the variability of using one observer. (2)
2.2.1 As an absolute minimum, two persons are necessary: One to make the
sample dilutions and one to determine the threshold odor.
3. Sample Handling and Preservation
3.1 Water samples must be collected in glass bottles with glass or Teflon-lined
closures.
3.1.1 Plastic containers are not reliable for odor samples and must not be used.
3.2 Odor tests should be completed as soon as possible after collection of the sample.
If storage is necessary, collect at least 1000 ml of sample in a bottle filled to the
top. Refrigerate, making sure no extraneous odors can be drawn into the sample
as the water cools.
4. Interferences
4.1 Most tap waters and some waste waters are chlorinated. It is often desirable to
determine the odor of the chlorinated sample as well as of the same sample after
removal of chlorine. Dechlorination is achieved using sodium thiosulfate in exact
stoichiometric quantity.
287
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4.1.1 It is important to check a blank to which a similar amount of
dechlorinating agent has been added to determine if any odor has been
imparted. Such odor usually disappears upon standing if excess reagent
has not been added.
5. Apparatus
5.1 Odor-free glassware: Glassware must be freshly cleaned shortly before use, with
non-odorous soap and acid cleaning solution followed by rinsing with odor-free
water (6.1). Glassware used in odor testing should be reserved for that purpose
only. Rubber, cork, and plastic stoppers must not be used.
5.2 Constant temperature bath: A water bath or electric hotplate capable of
maintaining a temperature control of ±1°C for performing the odor test at 60°C.
The temperature bath must not contribute any odor to the odor flasks.
5.3 Odor flasks: Glass stoppered 500 ml (£ 32) Erlenmeyer flasks, or wide-mouthed
500 ml Erlenmeyer flasks equipped with Petri dishes as cover plates.
NOTE: Narrow-mouth vessels are not suitable for running odor tests. Potential
positive bias due to color and/or turbidity of water sample under observation can
be eliminated by wrapping odor flasks in aluminum foil, painting flasks with
non-odorous paint, or by using red actinic Erlenmeyer flasks.
5.4 Sample bottles: Glass bottles with glass or Teflon-lined closures.
5.5 Pipets, measuring: 10.0 and 1.0 ml graduated in tenths.
5.6 Graduated cylinders: 250, 200, 100, 50, and 25 ml.
5.7 Thermometer: 0-110°C (±1°C), chemical or metal stem dial type.
5.8 Odor-free water generator: See Figure 1.
6. Reagents
6.1 Odor-free water: Odor-free dilution water must be prepared as needed by
filtration through a bed of activated carbon. Most tap waters are suitable for
preparation of odor-free waters, except that it is necessary to check the filtered
water for chlorine residual, unusual salt concentrations, or unusually high or low
pH. All these may affect some odorous samples.
Where supplies are adequate, distilled water avoids these problems as a source of
odor-free water. A convenient odor-free water generator may be made as shown in
Figure 1. Pass tap or distilled water through the odor-free water generator at a
rate of 0.1 liter/minute. When the generator is first started, it should be flushed to
remove carbon fines before the odor-free water is used.
6.1.1 The quality of water obtained from the odor-free water generator should
be checked daily at the temperature tests are to be conducted (room
288
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temperature and/or 60°C). The life of the carbon wi!! vary with the
condition and amount of water filtered. Subtle odors of biological origin
are often found if moist carbon filters are permitted to stand idle
between test periods. Detection of odor in the water coming through the
carbon indicates a change of carbon is needed.
7. Procedure
7.1 Precautions: Selection of persons to make odor tests should be carefully made.
Extreme sensitivity is not required, but insensitive persons should not be used. A
good observer has a sincere interest in the test. Extraneous odor stimuli such as
those caused by smoking and eating prior to the test or through the use of scented
soaps, perfumes, and shaving lotions must be avoided. The tester should be free
from colds or allergies that affect odor-response. Frequent rests in an odor-free
atmosphere are recommended. The room in which the tests are to be conducted
should be free from distractions, drafts, and other odor. In certain industrial
atmospheres, a special odor-free room may be required, ventilated by air filtered
through activated carbon and maintained at a constant comfortable temperature
and humidity. For precise work a panel of five or more testers should be used.
The persons making the odor measurements should not prepare the samples and
should not know the dilution concentrations being evaluated. These persons
should have been made familiar with the procedure before participating in a panel
test. Always start with the most dilute sample to avoid tiring the senses with the
concentrated sample. The temperature of the samples during testing should be
kept within 1 degree of the specified temperature for the test.
7.2 Threshold measurement: The ratio by which the odor-bearing sample has to be
diluted with odor-free water for the odor to be just detectable by the odor test is
the "threshold odor number" (T.O.). The total volume of sample and odor-free
water used in each test is 200 ml. The proper volume of odor-free water is put
into the flask first; the sample is then added to the water. Table 1 gives the
dilutions and corresponding threshold numbers.
289
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2 HOLE
RUBBER STOPPER
W3W
f\rfff. '*Ji (( rlM
I in.
i^iii
GRANULAR
4 xlO-MESH
ACTIVATED
CARBON
PEA SIZE
GRAVEL
FIGURE 1. ODOR-FREE WATER GENERATOR
290
-------
Table 1
Threshold Odor Number
Corresponding to Various Dilutions
Sample Volume (ml) Threshold Odor
Diluted to 200 ml Number
200 1
100 2
50 4
25 8
12.5 16
6.3 32
3.1 64
1.6 128
0.8 256
7.3 Determine the approximate range of the threshold odor by:
7.3.1 Adding 200 ml, 50 ml, 12.5 ml, and 3.1 ml of the sample to separate 500
ml glass-stoppered Erlenmeyer flasks containing odor-free water to make
a total volume of 200 ml. A separate flask containing only odor-free
water serves as the reference for comparison. If run at 60°C, heat the
dilutions and the reference in the constant temperature bath at 60°C
(±1°C).
7.3.2 Shake the flask containing the odor-free water, remove the stopper, and
sniff the vapors. Test the sample containing the least amount of
odor-bearing water in the same way. If odor can be detected in this
dilution, more dilute samples must be prepared as described in (7.3.3). If
odor cannot be detected in the first dilution, repeat the above procedure
using the sample containing the next higher concentration of the
odor-bearing water, and continue this process until odor is clearly
detected.
7.3.3 If the sample being tested requires more extensive dilution than is
provided by Table 1, an intermediate dilution is prepared from 20 ml of
sample diluted to 200 ml with odor-free water. Use this dilution for the
291
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threshold determination. Multiply the T.O. obtained by ten to correct
for the intermediate dilution. In rare cases more than one tenfold
intermediate dilution step may be required.
7.4 Based on the results obtained in the preliminary test, prepare a set of dilutions
using Table 2 as a guide. One or more blanks are inserted in the series, in the
vicinity of the expected threshold, but avoiding any repeated pattern. The
observer does not know which dilutions are odorous and which are blanks. He
smells each flask in sequence, beginning with the least concentrated sample and
comparing with a known flask of odor-free water, until odor is detected with
utmost certainty.
Table 2
Dilutions for Various Odor Intensities
Sample Volume in Which Odor First Noted
200ml 50ml 12.5ml 3.1ml
Volume (ml)
200
100
50
25
12.5
of Sample
100
50
25
12.5
6.3
to be Diluted
50
25
12.5
6.3
3.1
to 200 ml
(Intermediate
Dilution
See 7.3.3)
7.5 Record the observations of each tester by indicating whether odor is noted (+
sign) in each test flask.
For example:
ml sample
diluted to 200 ml 12.5 0 25 0 50 100 200
Response + + + +
8. Calculations
8.1 The threshold odor number is the dilution ratio at which odor is just detectable.
In the example above (7.5), the first detectable odor occurred when 25 ml sample
was diluted to 200 ml. Thus, the threshold is 200 divided by 25, equals 8. Table 1
lists the threshold numbers that correspond to common dilutions.
292
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8.2 Anomalous responses sometimes occur; a low concentration may he called
positive and a higher concentration in the series may be called negative. In such a
case, the threshold is designated as that point of detection after which no further
anomalies occur.
For instance:
ml sample
diluted to 200 ml 6.3 12.5 0 25 SO 100
Response + + + +
threshold
8.3 Calculations of panel results to find the most probable average threshold are best
accomplished by appropriate statistical methods. For most purposes, the
threshold of a group can be expressed as the geometric mean (G.M.) of the
individual thresholds. The geometric mean is calculated in the following manner:
8.3.1 Obtain odor response as outlined in Procedure and record results.
For example:
Table 3
Sample Odor Series
ml of Odor-
free Water
188
175
200
150
200
100
0
ml of
Sample
12.5
25
0
50
0
100
. 200
Observer Response*
1 2-3 4
'____
_ e +
_ _ _ _
e +
+ + e e
+ + + . +
5
e
+
+
-I-
*Circled plus equals threshold level.
293
-------
8.3.2 Obtain individual threshold odor numbers from Table 1.
Observer T.O.
1 4
2 8
3 2
4 2
5 8
8.3.3 The geometric mean is equal to the nth root of the product of n
numbers. Therefore:
4X8X2X2X8= 1024
5 log 1024 3.0103
and Vl024 = = =0.6021
5 5
and anti-log of 0.6021 = 4 = T.O.
9; Precision and Accuracy
9.1 Precision and accuracy data are not available at this time.
9.2 A threshold number is not a precise value. In the case of the single observer, it
represents a judgment at the time of testing. Panel results are more meaningful
because individual differences have less influence on the result. One or two
observers can develop useful data if comparison with larger panels has been made
to check their sensitivity. Comparisons of data from time to time or place to place
should not be attempted unless all test conditions have been carefully
standardized and some basis for comparison of observer intensities exists.
Bibliography
1. Standard Methods, 13th Edition, Amer. Public Health Asso., New York, N.Y., p 248,
Method 136(1971).
2. ASTM, Comm E-18, STP 433, "Basic Principles of Sensory Evaluation"; STP 434,
Manual on Sensory Testing Methods; STP 440, "Correlation of Subjective-Objective
Methods in the Study of Odors and Taste"; Phil., Pennsylvania (1968).
3. Baker, R A., "Critical Evaluation of Olfactory Measurement". Jour. WPCF, 34, 582
(1962).
294
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TURBIDITY
STORET NO. 00076
1. Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters in the range of
turbidity from 0 to 40 nephelometric turbidity units (NTU).
NOTE 1: NTU's are considered comparable to the previously reported Formazin
Turbidity Units (FTU) and Jackson Turbidity Units (JTU).
2. Summary of Method,
2.1 The method is based upon a comparison of the intensity of light scattered by the
sample under defined conditions with the intensity of light scattered by a
standard reference suspension. The higher the intensity of scattered light, the
higher the turbidity. Readings, in NTU's, are made in a nephelometer designed
according to specifications outlined in Apparatus 5. A standard suspension of
Formazin, prepared under closely defined conditions, is used to calibrate the
instrument.
2.1.1 Formazin polymer is used as the turbidity reference suspension for water
because it is more reproducible than other types of standards previously
used for turbidity standards.
3. Sample Handling and Preservation
3-1 Samples taken for turbidity measurements should be analyzed as soon as possible.
Preservation of samples is not recommended.
4. Interferences
4.1 The presence of floating debris and coarse sediments which settle out rapidly will
give low readings. Finely divided air bubbles will affect the results in a positive
manner.
4.2 The presence of true color, that is the color of water which is due to dissolved
substances which absorb light, will cause turbidities to be low, although this effect
is generally not significant with finished waters.
5. Apparatus
5.1 The turbidimeter shall consist of a nephelometer with light source for illuminating
the sample and one or more photo-electric detectors with a readout device to
indicate the intensity of light scattered at right angles to the path of the incident
light. The turbidimeter should be so designed that little stray light reaches the
detector in the absence of turbidity and should be free from significant drift after
a short warm-up period.
295
-------
5.2 The sensitivity of the instrument should permit detection of turbidity differences
of 0.02 unit or less in waters having turbidities less than 1 unit. The instrument
should measure from 0 to 40 units turbidity. Several ranges will be necessary to
obtain both adequate coverage and sufficient sensitivity for low turbidities.
5.3 The sample tubes to be used with the available instrument must be of clear,
colorless glass. They should be kept scrupulously clean, both inside and out, and
discarded when they become scratched or etched. They must not be handled at all
where the light strikes them, but should be provided with sufficient extra length,
or with a protective case, so that they may be handled.
5.4 Differences in physical design of turbidimeters will cause differences in measured
values for turbidity even though the same suspension is used for calibration. To
minimize such differences, the following design criteria should be observed:
5.4.1 Light source: Tungsten lamp operated at not less than 85% of rated
voltage or more than rated voltage.
5.4.2 Distance traversed by incident light and scattered light within the sample
tube: Total not to exceed 10 cm.
5.4.3 Angle of light acceptance of the detector: Centered at 90° to the
incident light path and not to exceed ±30° from 90°.
5.4.4 Maximum turbidity to be measured: 40 units.
5.5 The Hach Turbidimeter, Model 2100 and 2100 A, is in wide use and has been
found to be reliable; however, other instruments meeting the above design criteria
are acceptable.
6. Reagents
6.1 Turbidity-free water: Pass distilled water through a 0.45ju pore size membrane
filter if such filtered water shows a lower turbidity than the distilled water.
6.2 Stock turbidity suspension:
Solution 1: Dissolve 1.00 g hydrazine sulfate, (NH2)2 -HjSC^, in distilled water
and dilute to 100 ml in a volumetric flask.
Solution 2: Dissolve 10.00 g hexamethylenetetramine in distilled water and dilute
to 100 ml in a volumetric flask.
In a 100 ml volumetric flask, mix 5.0 ml Solution 1 with 5.0 ml Solution 2. Allow
to stand 24 hours at 25 ± 3°C, then dilute to the mark and mix.
6.3 Standard turbidity suspension: Dilute 10.00 ml stock turbidity suspension to 100
ml with turbidity-free water. The turbidity of this suspension is defined as 40
units. Dilute portions of the standard turbidity suspension with turbidity-free
water as required.
296
-------
6.3.1 A new stock turbidity suspension should be prepared each month. The
standard turbidity suspension and dilute turbidity standards should be
prepared weekly by dilution of the stock turbidity suspension.
7. Procedure
7.1 Turbidimeter calibration: The manufacturer's operating instructions should be
ST.
followed. Measure standards on the turbidimeter covering the range of interest. If
the instrument is already calibrated in standard turbidity units, this procedure will
check the accuracy of the calibration scales. At least one standard should be run
in each instrument range to be used. Some instruments permit adjustments of
sensitivity so that scale values will correspond to turbidities. Reliance on a
manufacturer's solid scattering standard for setting overall instrument sensitivity
for all ranges is not an acceptable practice unless the turbidimeter has been shown
to be free of drift on all ranges. If a pre-calibrated scale is not supplied, then
calibration curves should be prepared for each range of the instrument.
7.2 Turbidities less than 40 units: Shake the sample to thoroughly disperse the solids.
Wait until air bubbles disappear then pour the sample into the turbidimeter tube.
Read the turbidity directly from the instrument scale or from the appropriate
calibration curve.
7.3 Turbidities exceeding 40 units: Dilute the sample with one or more volumes of
turbidity-free water until the turbidity falls below 40 units. The turbidity of the
original sample is then computed from the turbidity of the diluted sample and the
dilution factor. For example, if 5 volumes of turbidity-free water were added to 1
volume of sample, and the diluted sample showed a turbidity of 30 units, then the
turbidity of the original sample was 180 units.
7.3.1 The Hach Turbidimeters, Models 2100 and 2100A, are equipped with 5
separate scales: 0-0.2, 0-1.0, 0-100, and 0-1000 NTU. The upper scales
are to be used only as indicators of required dilution volumes to reduce
readings to less than 40 NTU.
NOTE 2: Comparative work performed in the MDQAR Laboratory
indicates a progressive error on sample turbidities in excess of 40 units.
297
-------
8. Calculation
8.1 Multiply sample readings by appropriate dilution to obtain final reading.
8.2 Report results as follows:
NTU Record to Nearest:
0.0-1.0 0.05
1-10 0.1
10-40 1
40-100 5
100-400 10
400-1000 50
>1000 100
9. Precision and Accuracy
9.1 In a single laboratory (MDQARL), using surface water samples at levels of 26, 41,
75 and 180 NTU, the standard deviations were ±0.60, ±0.94, ±1.2 and ±4.7 units,
respectively.
9.2 Accuracy data is not available at this time.
ft U. S; GOVERNMENT PWNTDIG OFFICE : 1974 625-714/67
298
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APPENDIX G
HANDBOOK FOR ANALYTICAL QUALITY CONTROL
IN WATER AND WASTEWATER LABORATORIES
For
U.S. ENVIRONMENTAL PROTECTION AGENCY
Technology Transfer
By
ANALYTICAL QUALITY CONTROL LABORATORY
National Environmental Research Center
Cincinnati, Ohio
June 1972
-------
ABSTRACT
One of the fundamental responsibilities of management is the establishment of a continuing
program to insure the reliability and validity of analytical laboratory and field data gathered
in water treatment and wastewater pollution control activities.
This handbook is addressed to laboratory directors, leaders of field investigations, and other
personnel who bear responsibility for water and wastewater data. Subject matter of the
handbook is concerned primarily with quality control for chemical and physical tests and
measurements. Sufficient information is offered to allow the reader to inaugurate, or to
reinforce, a program of analytical quality control which will emphasize early recognition,
prevention and correction of factors leading to breakdowns in the validity of data.
111
-------
ACKNOWLEDGEMENT
This handbook was prepared by the Analytical Quality Control Laboratory of the United
States Environmental Protection Agency. The contributions of the following individuals in
preparing the handbook are gratefully acknowledged.
D. G. Ballinger
R. L. Booth
M. R. Midgett
R. C. Kroner
J. F. Kopp
J. J. Lichtenberg
J. A. Winter
R. C. Dressman
J. W. Eichelberger
J. E. Longbottom
Inquiries regarding material contained in the handbook should be made to Environmental
Protection Agency, National Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio 45268.
-------
TABLE OF CONTENTS
Chapter Page
ABSTRACT iii
ACKNOWLEDGEMENT v
TABLE OF CONTENTS vii
LIST OF FIGURES ix
LIST OF TABLES xi
1 IMPORTANCE OF QUALITY CONTROL !-!
1.1 General 1-1
1.2 Quality Control Program 1-1
1.3 Analytical Methods 1-2
1.4 References 1-3
2 LABORATORY SERVICES 2-1
2.1 General 2-1
2.2 Distilled Water 2-1
2.3 Ammonia-Free Water 2-4
2.4 Carbon Dioxide-Free Water 2-4
2.5 Ion-Free Water 2-4
2.6 Compressed Air 2-4
2.7 Electrical Services 2-5
2.8 References 2-5
3 INSTRUMENTAL QUALITY CONTROL 3-1
3.1 Introduction 3-1
3.2 Analytical Balances 3-2
3.3 pH Meters 3-4
3.4 Conductivity Meters 3-7
3.5 Turbidimeters 3-11
3.6 Spectrophotometers 3-11
3.7 Organic Carbon Analyzer 3-23
3.8 Selective Ion Electrodes 3-28
3.9 References 3-29
4 GLASSWARE 4-1
4.1 General 4-1
4.2 Types of Glassware 4-1
4.3 Volumetric Analyses 4-2
4.4 Federal Specifications for Volumetric Glassware 4-4
4.5 Cleaning of Glass and Porcelain 4-6
4.6 Special Cleaning Requirements 4-7
4.7 Disposable Glassware 4-8
4.8 Specialized Glassware 4-8
4.9 Fritted Ware 4-9
4.10 References 4-11
vn
-------
REAGENTS, SOLVENTS AND GASES 5-1
5.1 Introduction 5-1
5.2 Reagent Quality 5-1
5.3 Elimination of Determinate Errors 5-4
5.4 References 5-7
CONTROL OF ANALYTICAL PERFORMANCE 6-1
6.1 Introduction 6-1
6.2 Precision and Accuracy 6-1
6.3 Evaluation of Daily Performance 6-4
6.4 Quality Control Charts 6-5
6.5 References . 6-18
DATA HANDLING AND REPORTING 7-1
7.1 Introduction 7-1
7.2 The Analytical Value 7-1
7.3 Report Forms 7-5
7.4 STORET-Computerized Storage and Retrieval
of Water Quality Data 7-9
7.5 SHAVES-A Consolidated Data Reporting
and Evaluation System 7-11
7.6 References 7-11
SPECIAL REQUIREMENTS FOR TRACE ORGANIC ANALYSIS 8-1
8.1 Introduction 8-1
8.2 Discrete Bottled Samples 8-1
8.3 Carbon Adsorption Samples 8-2
8.4 Glassware 8-2
8.5 Reagents and Chemicals 8-2
8.6 Common Analytical Operations 8-3
8.7 Gas-Liquid Chromatography 8-4
8.8 Qualitative Analysis 8-7
8.9 Quantitative Analysis 8-8
8.10 Thin-Layer Chromatography 8-9
8.11 Column Chromatography 8-10
8.12 References 8-10
SKILLS AND TRAINING 9-1
9.1 General 9-1
9.2 Skills 9-2
9.3 Training 9-4
vui
-------
LIST OF FIGURES
Figure No. Page
3-1 Analytical Balance 3-3
3-2 pH Meter 3-5
3-3 Conductivity Meter 3-8
3-4 Turbidimeter 3-12
3-5 Spectrophotometer 3-13
3-6 Atomic Absorption Unit 3-19
3-7 Device for Reproducible Positioning of Burner Height 3-22
3-8 Organic Carbon Analyzer 3-27
3-9 Selective Ion Meter 3-30
4-1 Titration Bench 4-3
4-2 Example of Markings on Glassware 4-6
6-1 Essentials of Control Chart ' 6-5
6-2 Effects of a and j3 Levels on Standard Control Chart 6-8
6-3 Laboratory Quality Control Charts 6-10
7-1 Example of Bench Sheet 7-7
7-2 Example of Summary Review Sheet 7-8
7-3 Example of STORE! Report Form 7-10
IX
-------
LIST OF TABLES
Table No. Page
2-1 Water Purity 2-1
2-2 Comparison of Distillates from Glass and Metal Stills 2-2
3-1 Instruments Commonly Used in Water and Wastewater Analysis 3-1
3-2 pH Values of NBS Standards from 0 to 30° C 3-6
3-3 Performance Characteristics of Typical pH Meter 3-7
3-4 Electrical Conductivity of Potassium Chloride
Reference Solutions 3-iO
3-5 Design Features of Some Common Spectrophotometers 3-15
3-6 Design Features of Some Common Atomic
Absorption Instruments 3-25
4-1 Tolerances for Volumetric Glassware 4^5
4-2 Fritted Ware Porosity 4-9
4-3 Cleaning of Filters 4-10
6-1 Precision Data on River Water Samples for Phosphorus
AutoAnalyzer Method 6-2
6-2 Accuracy Data on River Water Samples for Phosphorus
AutoAnalyzer Method 6-3
6-3 Factors for Computing Control Chart Lines 6-12
9-1 Skill-Time Rating of Standard Analytical Operations ' 9-3
XI
-------
Chapter 1
IMPORTANCE OF QUALITY CONTROL
1.1 General
The role of the analytical laboratory is to provide qualitative and quantitative data to be
used in decision making. To be valuable, the data must accurately describe the character-
istics or the concentration of constituents in the sample submitted to the laboratory. In
many cases, an approximate answer or incorrect result is worse than no answer at all,
because it will lead to faulty interpretations.
Decisions made using water and wastewater data are far-reaching. Water quality standards
are set to establish satisfactory conditions for a given water use. The laboratory data define
whether that condition is being met, and whether the water can be used for its intended
purpose. If the laboratory results indicate a violation of the standard, action is required on
the part of pollution control authorities. With the present emphasis on legal action and
social pressures to abate pollution, the analyst should be aware of his. responsibility to
provide laboratory results that are a reliable description of the sample. Furthermore, the
analyst must be aware that his professional competence, the procedures he has used, and the
reported values may be used and challenged in court. To satisfactorily meet this challenge,
the laboratory data must be backed up by an adequate program to document the proper
control and application of all of the factors which affect the final result.
In wastewater analyses, the laboratory data define the treatment plant influent, the status
of the steps in the treatment process, and the final load imposed upon the water resources.
Decisions on process changes, plant modification, or even the construction of a new facility
may be based upon the results of laboratory analyses. The financial implications alone are
significant reasons for extreme care in analysis.
Research investigations in water pollution control rest upon a firm base of laboratory data.
The final result sought can usually be described in numerical terms. The progress of the
research and the alternative pathways available are generally evaluated on the basis of
laboratory data. The value of the research effort will depend upon the validity of the
laboratory results.
1.2 Quality Control Program
Because of the.importance of laboratory analyses and the resulting actions which they
produce, a program to insure the reliability of the data is essential. It is recognized that all
analysts practice quality control to varying degrees, depending somewhat upon their train-
ing, professional pride, and awareness of the importance of the work they are doing. How-
ever, under the pressure of daily workload, analytical quality control may be easily
neglected. Therefore, an established, routine control program applied to every analytical test
is important in assuring the reliability of the final results.
The quality control program in the laboratory has two primary functions. First, the program
should monitor the reliability (truth) of the results reported. It should continually provide
an answer to "How good (tru'e) are the results submitted?" This phase may be termed
"measurement of quality." The second function is the control of quality in order to meet
1-1
-------
the program requirements for reliability. For example, the processing of spiked samples is
the measurement of quality, while the use of analytical grade reagents is a control measure.
Just as each analytical method has a rigid protocol, so the quality control associated with
that test must also involve definite required steps to monitor and assure that the result is
correct. The steps in quality control will vary with the type of analysis. For example, in a
titration, standardization of the titrant on a frequent basis is an element of quality control.
In an instrumental method, the check-out of instrument response and the calibration of the
instrument in concentration units is also a quality control function. Ideally, all of the
variables which can affect the final answer should be considered, evaluated, and controlled.
This handbook considers the factors which go into creating an analytical result, and provides
recommendations for the control of these factors in order to insure that the best possible
answer is obtained. A program based upon these recommendations will give the analyst and
his supervisor confidence in the reliability and the representative nature of the sample
characteristics being reported.
Without exception, the final responsibility for the reliability of the analytical results sub-
mitted rests with the Laboratory Director.
1.3 Analytical Methods
In general, the widespread use of an analytical method indicates that it is a reliable means of
analysis, and this fact tends to support the validity of the test result reported. Conversely,
the use of a little-known technique forces the data user to place faith in the judgement of
the analyst. When the analyst uses a "private" method, or one not commonly accepted in
the field, he must stand alone in defining both his choice of the method and the result
obtained.
The need for standardization of methods within a single laboratory is readily apparent.
Uniform methods between cooperating laboratories are also important in order to remove
the methodology as a variable in comparison or joint use of data between laboratories.
Uniformity of methods is particularly important when laboratories are providing data to a
common data bank, such as STORET*, or when several laboratories are cooperating in joint
field surveys. A lack of standardization of methods raises doubts as to the validity of the
results reported. If the same constituent is measured by different analytical procedures
within a single laboratory, or in several laboratories, the question is raised as to which
procedure is superior, and why the superior method is not used throughout.
The physical and chemical methods used should be selected by the following criteria:
a. The method should measure the desired constituent with precision and accuracy
sufficient to meet the data needs in the presence of the interferences normally
encountered in polluted waters.
b. The procedure should utilize the equipment and skills normally available in the
average water pollution control laboratory.
*STORET is the acronym used to identify the computer-oriented U.S. Environmental
Protection Agency Water Quality Control Information System for STOrage and RETrieval
of data and information.
1-2
-------
c. The selected methods should be in use in many laboratories or have been sufficiently
tested to establish their validity.
d. The method should be sufficiently rapid, to permit routine use for the examination
of large numbers of samples.
The use of EPA methods in all EPA laboratories provides a common base for combined data
between Agency programs. Uniformity throughout EPA lends considerable support to the
validity of the results reported by the Agency.
Regardless of the analytical method used in the laboratory,the specific methodology should
be carefully documented. In some water pollution reports it is customary to state that
Standard Methods (1) have been used throughout. Close examination indicates, however, that
this is not strictly true. In many laboratories, the standard method has been modified
because of recent research or personal preferences of the laboratory staff. In other cases the
standard method has been replaced with a better one. Statements concerning the methods
used in arriving at laboratory data should be clearly and honestly stated. The methods used
should be adequately referenced and the procedures applied exactly as directed.
Knowing the specific method which has been used, the reviewer can apply the associated
precision and accuracy of the method when interpreting the laboratory results. If the
analytical methodology is in doubt, the data user may honestly inquire as to the reliability
of the result he is to interpret.
The advantages of strict adherence to accepted methods should not stifle investigations
leading to improvements in analytical procedures. In spite of the value of accepted and
documented methods, occasions do arise when a procedure must be modified to eliminate
unusual interference, or to yield increased sensitivity. When modification is necessary, the
revision should be carefully worked out to accomplish the desired result. It 'is advisable to
assemble data using both the regular and the modified procedure to show the superiority of
the latter. This useful information can be brought to the attention of the individuals and
groups responsible for methods standardization. For maximum benefit, the modified
procedure should be rewritten in the standard format so that the substituted procedure may
be used throughout the laboratory for routine examination of samples. Responsibility for
the use of a non-standard procedure rests with the analyst and his supervisor, since such use
represents a departure from accepted practice.
In field operations, the problem of transport of samples to the laboratory, or the need to
examine a large number of samples to arrive at gross values will sometimes require the use of
rapid field methods yielding approximate answers. Such methods should be used with
caution, and with a clear understanding that the results obtained do not compare in relia-
bility with those obtained using standard laboratory methods. The fact that "quick and
dirty" methods have been used should be noted, and the results should not be reported along
with more reliable laboratory-derived analytical information. The data user is entitled to
know that approximate values have been obtained for screening purposes only, and that the
results do not represent the customary precision and accuracy obtained in the laboratory.
1.4 References
1. Standard Methods for the Examination of Water and Wastewater, 13th Edition, Amer-
ican Public Health Association, New York (1971).
1-3
-------
CHAPTER 2
LABORATORY SERVICES
2.1 General
Quality control of laboratory analyses involves consideration and control of the many
variables which affect the production of reliable data. The quality of the laboratory services
available to the analyst must be included among these variables. An abundant supply of
distilled water, free from interferences and other undesirable contaminants, is an absolute
necessity. An adequate source of clean, dry, compressed air is needed. Electrical power for
routine laboratory7 use and voltage-regulated sources for delicate electronic instrumentation
must be provided. This chapter, therefore, will be devoted to describing methods of
maintaining the quality of these services, as used in laboratory operations.
2.2 Distilled Water
Distilled or demineralized water is used in the laboratory for dilution, preparation of reagent
solutions, and final rinsing of glassware. Ordinary distilled water is usually not pure. It may
be contaminated by dissolved gases and by materials leached from the container in which it
has been stored. Volatile organics distilled over from the feed water may be present, and
non-volatile impurities may occasionally be carried over by the steam, in the form of a
spray. The concentration of these contaminants is usually quite small, and distilled water is
used for many analyses without further purification. However, it is highly important that
the still, storage tank, and any associated piping be carefully selected, installed, and main-
tained in such a way as to assure minimum contamination.
Water purity has been defined in many different ways, but one generally accepted definition
states that high purity water is water that has been distilled and/or deionized so that it will
have a specific resistance of 500,000 ohms (2.0 micromhos conductivity) or greater. This
definition is satisfactory as a base to work from, but for more critical requirements, the
breakdown shown in Table 2-1 has been suggested to express degrees of purity (1).
Table 2-1
WATER PURITY
Degree of Purity Maximum Approximate Concentration
Conductivity of Electrolyte
(micromhos/cm) (mg/1)
Pure 10 2-5
Very Pure 1 0.2-0.5
Ultrapure 0.1 0.01-0.02
Theoretically Pure 0.055 0.00
2-1
-------
Source
All-Glass Still
Metal Still
Zn
9
B
12
13
Fe
1
2
Mn Al
<1 4
<1 <5
Cu
5
11
Ni
<2
Properly designed metal stills from reputable manufacturers offer a convenient and reliable
source of distilled water. These stills are usually constructed of copper, brass, and bronze.
All surfaces that contact the distillate should be heavily coated with pure tin to prevent
metallic contamination. The metal storage tank should be of sturdy construction, have a
tight-fitting cover, and a filter in the air vent to remove airborne dust, gases, and fumes.
For special purposes, an all-glass distillation unit may be preferable to the metal still. These
stills are usually smaller, and of more limited capacity than the metal stills. An actual
comparison in which the distillates from an all-glass still and a metal still were analyzed
spectrographically for certain trace metal contaminants is shown below in Table 2-2.
Table 2-2
COMPARISON OF DISTILLATES FROM GLASS AND METAL STILLS
Element and Concentration (Micrograms/1)
Pb
<2
26
It can be seen that the all-glass still produced a product which had substantially lower
contamination from zinc, copper, and lead. Other analyses have indicated the same general
relationship, except that a boron concentration of 100 micrograms/liter was found in water
from the all-glass still on one occasion. This-was probably related to the length of time the
distillate had remained in the glass storage reservoir.
All stills require periodic cleaning to remove solids which have been deposited from the feed
water. Hard water and high dissolved solids content promote scale formation in the evapor-
ator, and cleaning frequency will thus depend on the quality of the feed water. The boiler of
an all-glass still should be drained daily and refilled with clean water. Build-up of scale is
easily detected, and the boiler and condenser coils should be cleaned at frequent intervals.
Metal stills usually incorporate a constant bleeder device which retards scale formation to
some extent. However, these units should still be dismantled and cleaned at regular intervals.
Cleaning should always be in accordance with the manufacturer's instructions.
Pre-treatment of the incoming feed water will often improve still performance and raise the
quality of the distillate. For example, preliminary softening of hard water removes calcium
and magnesium prior to distillation. This reduces scale formation in the boiler and conden-
ser, thereby reducing maintenance service. These softeners employ the ion exchange
principle using a sodium chloride cycle, and are relatively inexpensive to operate. A carbon
filtration system, installed at the feed water intake, will remove organic materials which
might subsequently be carried over in the distillate. If trace concentrations of ions are a
major concern, the distillate may be passed through a mixed-bed ion exchanger.
At least two commercially manufactured systems are available for production of high purity
water by ion exchange. The Millipore Super-Q System (Millipore Corp., Bedford, Mass.),
consists essentially of disposable cartridges for prefiltration, organic absorption, deioniza-
tion and Millipore filtration. The company claims it can produce 10 megohm water,
containing no particulate matter larger than 0.45 micron in size, from tap water, at the rate
of 20 gallons per hour. Continental Water Conditioning Corp., El Paso, Texas, advertises a
2-2
-------
system which can be tailored to the needs of the customer. Performance specifications
include minimum flow rates of 45 gallons per hour and total dissolved solids of less than 0.1
mg/1 when required.
Specific conductance is a rapid and simple measurement for determining the inorganic
quality of distilled water. Stills of the types previously discussed are capable of producing a
distillate with a specific conductance of less than 2.0 micromhos at 25°C. This is equivalent
to 0.5-1.0 mg/1 of ionized material. Frequent checks should be made to determine that
optimum performance is being maintained. A purity meter installed between the still and
the storage reservoir will monitor the conductivity of the distillate, in terms of the equiva-
lent in mg/1 of sodium chloride. If the reading on the meter begins to rise above the present
limit of conductivity, effective action should be taken to eliminate the source of contamin-
ation. Organic quality is more difficult to monitor, but the total organic carbon determin-
ation is a simple and rapid test for organic contaminants.
A piping system for delivering distilled water to the area of use within the laboratory is a
convenient and desirable feature. In this case, special care should be taken that the quality
of the water is not degraded between the still and the point of use. Piping may be of tin,
tin-lined brass, stainless steel, plastic, or chemically resistant glass, depending on the quality
of the water desired and on available funds. Tin is best, but is also very expensive. As a
compromise, plastic pipe, or glass pipe with Teflon* O-rings at all connecting joints is
satisfactory for most purposes. The glass pipe has an obvious advantage when freedom from
trace amounts of organic materials is important.
When there is no piped-in supply, distilled water will probably be transported to the
laboratory and stored in polyethylene or glass bottles of about 5-gallon capacity. If stored in
glass containers, distilled water will gradually leach the more soluble materials from the glass
and cause an increase in dissolved solids. Therefore, only borosilicate-free glass containers
should be used. Polyethylene bottles contain organic plasticizers, and traces of these
materials may be leached from the container walls. These are of little consequence, except
in some organic analyses. Rubber stoppers often used in storage containers contain leachable
materials, including significant quantities of zinc. This is usually no problem, since the water
is not in direct contact with the stopper. However, the analyst should be aware of the
potential for contamination, especially when the supply is not replenished by frequent use.
The delivery tube may consist of a piece of glass tubing which extends almost to the bottom
of the bottle, and which is bent downward above the bottle neck, with a three-to-four-foot
piece of flexible tubing attached for mobility. Vinyl tubing is preferable to latex rubber,
because it is less leachable. However, a short piece of latex tubing may be required at the
outlet for better control of the pinchcock. The vent tube in the stopper should be protected
against the entrance of dust.
Ordinary distilled water is quite adequate for many analyses, including the determination of
major cations and anions. Certain needs may require the use of double- or even triple-
distilled water. Redistillation from an alkaline permanganate solution can be used to obtain
a water with low organic background. When determining trace organics by solvent extrac-
tion and gas chromatography, distilled water with sufficiently low background may be
extremely difficult to obtain. In this case, pre-extraction of the water with the solvent used
in the respective analysis may be helpful in eliminating undesirable peaks in the blank.
'Trademark of E.I. duPont de Nemours & Co.
2-3
-------
Certain analyses require special treatment or conditioning of the distilled water, and these
will now be discussed.
2.3 Ammonia-Free Water
Removal of ammonia can be accomplished by shaking ordinary distilled water with a strong
cation exchanger, or by passing distilled water through a column of such material. For
limited volumes of ammonia-free water, use of the Quikpure (Box 254, Chicago, 111.) 500-ml
bottle is highly recommended. The ion-free water described below is also suitable for use in
the determination of ammonia.
2.4 Carbon-Dioxide-Free Water
Carbon-dioxide-free water may be prepared by boiling distilled water for 15 minutes and
cooling to room temperature. As.an alternative, distilled water may be vigorously aerated
with a stream of inert gas for a period sufficient to achieve saturation and CO2 removal.
Nitrogen is most frequently used. The final pH of the water should lie between 6.2 and 7.2.
It is not advisable to store CO2-free water for extended periods.
2.5 Ion-Free Water
A multi-purpose high purity water, free from trace amounts of the common ions, may be
conveniently prepared by slowly passing distilled water through an ion-exchange column
containing one part of a strongly acidic cation-exchange resin in the hydroxyl form. Resins
of a quality suitable for analytical work must be used. Ion-exchange cartridges of the
research grade, available from scientific supply houses, have been found satisfactory. By
using a fresh column and high quality distilled water, a water corresponding to the ASTM
designation for referee reagent water (2) (maximum 0.1 mg/1 total matter and maximum
conductivity of 0.1 micromho) can be obtained. This water is suitable for use in the
determination of ammonia, trace metals, and low concentrations of most cations and
anions. It is not suited to some organic analyses, however, because this treatment adds
organic contaminants to the water by contact with the ion-exchange materials.
2.6 Compressed Air
The quality of compressed air required in the laboratory is usually very high, and special
attention should be given to producing and maintaining clean air until it reaches the outlet.
Oil, water, and dirt are undesirable contaminants in compressed air, and it is important to
install equipment which generates dry, oil-free air. When pressures of less than 50 psi are
required, a rotary-type compressor, using a water seal and no oil, eliminates any addition of
oil which would subsequently have to be removed from the system. Large, horizontal,
water-cooled compressors will usually be used when higher pressures are required.
Compression heats air, thus increasing its tendency to retain moisture. An aftercooler is
therefore necessary to remove water. Absorption filters should be used at the compressor to
prevent moisture from entering the piping system. Galvanized steel pipe with threaded,
malleable-iron fittings, or solder-joint copper tubing should be used for piping the air to the
laboratory.
When the compressed air entering the laboratory is of low quality, an efficient filter should
be installed between the outlet and the point of use to trap oil, moisture, and other
contaminants. As an alternative, high quality compressed air of the dry grade is commercially
available in cylinders when no other source exists.
2-4
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2.7 Electrical Services
An adequate electrical system is indispensable to the modern laboratory. This involves
having both 115- and 230-volt sources in sufficient capacity for the type of work that must
be done. Requirements for satisfactory lighting, proper functioning of sensitive instruments,
and operation of high current devices must be considered. Any specialized equipment may
present unusual demands on the electrical supply.
Due to the special type of work, requirements for a laboratory lighting system are quite
different from those in other areas. Accurate readings of glassware graduations, balance
verniers, and other measuring lines must be made. Titration endpoints, sometimes involving
subtle changes in color or shading, must be observed. Levels of illumination, brightness,
glare, and location of light sources should be controlled to facilitate ease in making these
measurements and to provide maximum comfort for the employees.
Spectrophotometers, flame photometers, atomic absorption equipment, emission spectro-
graphs, gas chromatographs, etc. have complicated electronic circuits which require
relatively constant voltage to maintain stable, drift-free instrument operation. If the voltage
to these circuits varies, there is a resulting change in resistance, temperature, current,
efficiency, light output, and component life. These characteristics are interrelated, and one
cannot be changed without affecting the others. Voltage regulation is therefore necessary to
eliminate these conditions.
Many instruments have built-in voltage regulators which perform this function satisfactorily.
In the absence of these, a small, portable, constant-voltage transformer should be placed in
the circuit between the electrical outlet and the instrument. Such units are available from
Sola Basic Industries, Elk Grove Village, 111., and are capable of supplying a constant output
of 118 volts from an input which varies between 95 and 130 volts. When requirements are
more stringent, special transformer-regulated circuits can be used to supply constant voltage.
Only the instrument receiving the regulated voltage should be operated from such a circuit
at any given time. These lines are in addition to, and separate from the ordinary circuits
used for operation of equipment with less critical requirements.
Electrical heating devices provide desirable heat sources, and should offer continuously
variable temperature control. Hot plates and muffle furnaces wired for 230-volt current will
probably give better service than those which operate on 115 volts, especially if the lower
voltage circuit is only marginally adequate. Water baths and laboratory ovens with
maximum operating temperatures of about 200° C perform well at 115 volts. Care must be
taken to ground all equipment which could constitute a shock hazard. The three-pronged
plugs which incorporate the ground are best for this purpose.
2.8 References
1. Applebaum, S. B., and Crits, G. J., "Producing High Purity Water." Industrial Water
Engineering, Sept./Oct. 1964.
2. 1968 Book of ASTM Standards, Part 23, Atmospheric Analysis, pp. 225-6, American
Society for Testing and Materials, 1916 Race Street, Philadelphia, Pa. 19103.
2-5
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CHAPTERS
INSTRUMENTAL QUALITY CONTROL
3.1 Introduction
The modern analytical laboratory depends very heavily upon instrumentation. This
. statement may be completely obvious, but it should be remembered that the exceptional
emphasis on electronic equipment has really begun since the development of the transistor
and the computer. Analytical instrumentation, to a certain extent, is always in the
development stage, with manufacturers continually redesigning and upgrading their
products, striving for miniaturization, better durability and sensitivity, and improved
automation. The net result to laboratory supervisors and staff members is a bewildering
stream of advertising brochures, announcements, and catalogues of newly available
equipment. Consequently, the selection and purchase of analytical equipment is, at all
times, beset with uncertainty.
Table 3-1 lists the instruments most commonly used for water and wastewater analysis.
These represent basic equipment used in routine work and should be the subject of careful
consideration before purchase. Further, operation and maintenance of these devices ought
to be a primary consideration in production of satisfactory data. Obviously, a fundamental
understanding of instrument design will assist the analyst in the correct use of the
instrument and in some cases aid in detecting instrumental failures.
In the pages that follow an attempt is made to discuss basic instrument design and to offer
some remarks whenever possible about desirable instrumental features.
Table 3-1
INSTRUMENTS COMMONLY USED
IN WATER AND WASTEWATER ANALYSIS
Analytical Balance
Potentiometer (pH meter)
Conductivity meter
Turbidimeter
Spectrophotometers
a. Visual
b. Ultraviolet
c. Infrared
d. Atomic absorption
Total Carbon Analyzer
Gas Chromatographs
Miscellaneous
a. Temperature devices (ovens, water
baths, etc.)
b. Recorders
c. Selective Ion Electrodes
3-1
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3.2 Analytical Balances
The most important piece of equipment in any analytical laboratory is the analytical
balance (See Figure 3-1). It bears the same relationship to accuracy of measurements
produced by a laboratory as the Greenwich standard clock has to international
time-keeping. If the balance is not accurate all data related to weight-prepared standards will
contain the same degree of error. The balance, therefore, should be the most protected and
cared-for instrument in the laboratory. Unfortunately, care of the balance is frequently
overlooked.
There are many fine balances on the market designed to meet a variety of needs such as
sensitivity, speed weighing, batch weighing, etc. Types of balances include general purpose,
micro-, electro-, semi-analytical, analytical and other special purpose instruments. Each type
of balance has its own place in the scheme of laboratory operation but the analytical
balance is by far the most important in the production of reliable data.
Most analytical balances in use today in well equipped laboratories are of the "single pan"
variety. Single-pan capacities range from 80 grams to the popular 200-gram models with
sensitivities from 0.01 to 1 mg. Features of single-pan balances include mechanical lifting
and substitution of weights, digital readout of weights, and mechanical zeroing of the empty
balance. The advantage of the single-pan balance over the old "two-pan" balance is in
greatly increased weighing speed and improved weighing accuracy because of mechanical
weight handling. With all the design improvements, however, the modern analytical balance
is still a fragile instrument, subject to shock, temperature and humidity changes,
mishandling and various other insults. Some of the precautions to be observed in
maintaining and prolonging the dependable life of a balance are as follows:
a. Analytical balances should be mounted on a heavy shock-proof table, preferably
one with adequate working surface and a suitable drawer for storage of balance
accessories; balance level should be checked frequently and adjusted when
necessary.
b. Balances should be located away from laboratory traffic, protected from sudden
drafts and humidity changes.
c. Balance temperatures should be equilibrated with room temperature; this is
especially important if building heat is shut off or reduced during non-working
hours.
d. When not in use, the beam should be raised from the knife edges, the weights
returned to the beam, objects such as weighing dish removed from the pan, and the
slide door closed.
e. Special precautions should be taken to avoid spillage of corrosive chemicals on the
pan or inside the balance case; the interior of balance housing should be kept
scrupulously clean.
f. Balances should be checked and adjusted periodically by a company service man or
balance consultant; if service is not available locally, follow the manufacturer's
instructions as closely as possible.
3-2
-------
OJ
Figure 3-1. ANALYTICAL BALANCE
-------
g. The balance should be operated at all times according to the manufacturer's
instructions.
Standardized weights to be used in checking balance accuracy, and that meet U.S. Bureau of
Standards specifications, may be purchased from various supply houses. A very complete set
of directions for checking the performance of a balance is contained in Part 30 of ASTM
Standards (1).
Since all analytical balances of the 200-gram capacity have about the same specifications
with reference to sensitivity, precision, convenience, and price, and since these specifications
are suitable for normal weighing requirements in water and wastewater laboratories, it is safe
to assume that there is no clear preference for a certain model, and selection will probably
be made on the basis of service availability.
3.3 pH Meters
The concept of pH as a means of expressing the degree of effective acidity or alkalinity as
contrasted with total acidity or alkalinity was developed by Sorenson hi 1909. It was not
until about 1940 that commercial instruments were developed for routine laboratory
measurement of pH.
A basic pH meter (See Figure 3-2) consists of a voltage source, amplifier, and readout
device, either scale or digital. Certain additional refinements produce varying performance
characteristics between models. Some models incorporate expanded scales for increased
readability and solid state circuitry for operating stability and extreme accuracy. All
instruments of recent design also include temperature adjustment and slope adjustment to
correct for asymmetric potential of glass electrodes. Other features are scales that facilitate
use of selective ion electrodes, recorder output, and interfacing with complex data handling
systems.
In routine analytical work, the glass electrode is used as the indicator and the calomel
electrode as the reference. Glass electrodes have a very fast response time in highly buffered
solutions. However, accurate readings are obtained slowly in poorly buffered samples, and
particularly when changing from buffered to unbuffered samples, as after standardization.
Electrodes, both glass and calomel, should be well rinsed with distilled water after each
reading, and should be rinsed or dipped several times into the next test sample before the
final reading is taken. Weakly buffered samples should be stirred during measurement. Glass
electrodes should not be allowed to become dry during periods of inactivity. When not in
use they should be immersed in distilled water.
The first step in standardization of the instrument is done by immersing the glass and
calomel electrodes into a buffer of known pH, setting the meter scale or needle to the pH of
the buffer and adjusting the proper controls to bring the circuit into balance. The
temperature compensating dial should be set at the sample temperature. The pH of the
standard buffer should be within about two pH units of the sample. For best accuracy, the
instrument should be calibrated against two buffers that bracket the pH of the samples.
The presence of a faulty electrode is indicated by failure to obtain a reasonably correct
value for the pH of the second reference buffer solution after the meter has been
standardized with the first. A cracked glass electrode will often yield pH readings that are
essentially the same for both standards. The response of electrodes may also be impaired by
-------
Figure 3-2. pH METER
-------
failure to maintain the KC1 level in the calomel electrode, or by certain specific materials
such as oily substances and precipitates that may coat the surfaces. A faulty condition can
be recognized from the check with the two buffer solutions. If either of these conditions
should occur the electrode can probably be restored to normal by an appropriate cleaning
procedure. Complete and detailed cleaning methods are given in Part 23 of ASTM Standards
(2).
Because of the asymmetric potential of the glass electrode most pH meters are built with a
"slope adjustment" which enables the analyst to correct for slight electrode errors that
occur when standardization is performed at two different pH levels. Exact details of slope
adjustment and slope check may vary with different models of instruments. The slope
adjustment must be made whenever electrodes are changed, subjected to vigorous cleaning,
or refilled with fresh electrolyte. The slope adjustment feature is highly desirable and
recommended for consideration when purchasing a new meter.
Most pH meters now available are built with transistorized circuits rather than vacuum tubes
which greatly reduces warm-up time and increases stability of the meter. Also, many
instruments are designed with a switching circuit so that the conventional 0-14 scale may be
used to read a single pH unit. The "expanded-scale" feature allows for more accurate
readings and may be of definite value when the meter is used for potentiometric titrations.
It is of dubious value, however, in routine analytical work', since readings more percise than
±0.1 pH are seldom required.
Solid state circuitry has led to improved design of compact instruments suitable for field
work. Field-type instruments are generally battery-powered, and require more maintenance
and more frequent standardization than laboratory instruments.
Standard buffer solutions, covering a range of pH, may be purchased from almost any
chemical supply house and are completely satisfactory for routine use. Table 3-2 below gives
a list of NBS buffers (easily made in the laboratory) and the resulting pH at several different
temperatures.
Table 3-2
pH VALUES OF NBS STANDARDS FROM 0 - 30°C
0.05M
Temp. Potassium
°C Tetroxalate
0
10
15
20
25
30
1.67
1.67
1.67
1.68
1.68
1.69
Potassium
Acid Tar-
trate (Sat.
at 25°C)
3.56
3.55
0.025 M
0.05 M Potassium 0.01 M
Potassium Dihydrogen Phosphate Sodium
Acid + 0.025 M Sodium Tetra-
Phthalate Dihydrogen Phosphate borate
4.01 6.98 9.46
4.00 6.92 9.33
4.00 6.90 9.27
4.00 6.88 9.22
4.01 6.86 9.18
4.01 6.85 9.14
3-6
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Some idea of the effect of temperature on pH may be obtained by observing temperature vs
pH of various buffers shown in the table. Theoretically, the potential response of the
electrode system changes 0.20 mV per pH unit per degree centigrade. Since all pH meters
measure potential but read out in pH. a variable compensation is used. A rough rule of
thumb is that temperature compensation is about 0.05 pH units per 5 degree increase in
temperature.
Typical performance data of a conventional expanded scale pH meter is shown in Table 3-3
below.
Table 3-3
PERFORMANCE CHARACTERISTICS OF TYPICAL pH METER
Range
Smallest scale division
Accuracy
Reproducibility
Temp, compensation
Input impedance
Power requirements
Dimensions
Normal Scale
0 to 14 pH
±1400 mv
.0.1 pH
10 mv
±0.05 pH
±5 mv
±0.02 pH
±2 mv
0 to 100°C (manual
or automatic)
>1014
115/220V, 50/60 Hz
12'/2" w x 11" d x 8>/4" h
Expanded Scale
1 pH
±100 mv
0.005 pH
0.5 mv
±0.002 pH
±2% of reading
±0.002 pH
±0.2 mv
|13
3.4 Conductivity Meters
Solutions of electrolytes conduct an electric current by the migration of ions under the
influence of an electric field. For a constant applied EMF, the current flowing between
opposing electrodes immersed in the electrolyte will vary inversely with the resistance of the
solution. The reciprocal of the resistance is called conductance and is expressed in reciprocal
ohms or mhos. For natural water samples where the resistance is high, the usual reporting
unit is in micromhos. Figure 3-3 shows a typical conductivity meter.
The passage of direct current through an electrolyte causes changes in the electrolyte, hence,
to prevent polarization, it is necessary to use an alternating current or current pulses of
short duration when measuring conductivity. Originally, conductivity meters were built
using a rapidly alternating current of low intensity in the audio range, and were equipped
3-7
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00
Figure 3-3. CONDUCTIVITY METER
-------
with balance. For obvious reasons, the earpiece has been replaced by other devices, .such as
the cathode ray tube commonly known as the "magic eye". Practically all conductivity
meters on the present market use some variation of the "magic eye" for indicating solution
conductivity, and include a stepping switch for varying resistances in steps of 10X. The
instruments are therefore capable of reading conductivities from about 0.1 ^ mhos to about
250,000 V- mhos.
The sensing element for a conductivity measurement is the conductivity cell, which
normally consists of two thin plates of platinized metal, rigidly supported with a very
precise parallel spacing. For protection, the plates are mounted inside a glass tube, with
openings in the side walls and submersible end for access of sample. Variations in designs
have included use of hard rubber and plastics for protection of the cell plates. Glass may be
preferable, in that the plates may be visually observed lor cleanliness and possible damage,
but the more durable encasements have the advantage of greater protection and reduced cell
breakage. One manufacturer offers a cell containing circular carbon plates embedded in an
epoxy-type plastic. Reversing the usual procedures, the sample is poured into the cell. The
cell is particularly attractive because of its ruggedness and the fact that it can be cleaned
without changing the cell factor.
Special precaution is taken with the arrangement of lead wires from the plates. Since stray
electrolytic and capacitive current will pass between them, thereby distorting the bridge
balance, the wires must be properly separated, usually by use of a non-conducting bead or
collar on one of the lead wires.
In routine use, cells should be frequently examined to insure that (a) platinized coating of
plates is intact, (b) plates are not coated with suspended matter, (c) plates are not bent,
distorted, or misaligned, and (d) lead wires are properly spaced.
Temperature has a pronounced effect on the conductance of solutions, and must be
corrected when results are reported. The specified temperature for reporting data used by
most analytical groups (and all EPA laboratories) is 25°C. Data correction may be
accomplished by adjusting sample temperatures to 25°C.,or by use of mathematical or
electronic adjustment. Adjustment of sample temperature is the preferred system, because
of the empirical nature of the mathematical correction. However, investigative work in the
Analytical Quality Control Laboratory, and at the upper Ohio Basin Office, EPA, has shown
that acceptable data is obtained if conductivity readings taken at stream-side are
electronically temperature-corrected to 25° C.
Instrumental troubles are seldom encountered with conductivity meters because of the
design simplicity. When troubles occur they are usually in the cell, and for most accurate
work the following procedures should be used:
a. Standardize the cell and establish a cell factor by measuring the conductivity of a
standard potassium chloride solution (standard conductivity tables may be found in
various handbooks).
b. Rinse the cell by repeated immersion in distilled water.
c. Again, immerse the cell in the sample several times before obtaining a reading.
d. If the meter is equipped with a "magic eye", determine the maximum width of the
3-9
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shadow at least twice, by approaching the endpoint from a low reading upward, and
from a high reading downward.
Because the cell constants are subject to slow change, even under ideal conditions, and
sometimes to more rapid change under adverse conditions, it is recommended that the cell
constant be periodically established. Table 3-4, below, can be used for this operation.
Table 3-4
ELECTRICAL CONDUCTIVITY
OF POTASSIUM CHLORIDE REFERENCE SOLUTIONS
Solution Normality Method of Preparation Temp. (°C) Conductivity Q mhos)
A 0.1 7.4365 g KCI/1 0 7,138
at 20°C 18 11,167
25 12,856
B 0.01 0.7440 g KCI/1 0 773
at 20°C 18 1,220
25 1,408
C 0.001 Dilute 100 ml of B
to 1 1 at20°C 25 147
K1+K2
For instruments reading in mhos, calculate the cell constant as follows:
L =
1,000,000 xKx
where
L = cell constant
K. = Conductivity, in ^ mhos/cm, of theKC1 solution
at the temperature of measurement
K2 = conductivity, in ^ mhos, at the same temperature,
of the distilled water used to prepare the reference
solution
Kv = measured conductance, in mhos
.A.
Conductivity equipment which has generally been found to be reliable for laboratory work
includes the YSI #31 (Yellow Springs Instrument Co., Box 279, Yellow Springs, Ohio
45387), the Lab-Line Mark IV #1100 and MC3 #11025 (Lab-Line Instruments Inc., 15th &
Bloomingdale Aves., Melrose Park, 111. 60160), and Industrial Instruments #RC 16B2 and
RC-18 (Industrial Instruments Inc., 89 Commerce Road, Cedar Grove, NJ. 07009).
The YSI #31 is particularly suitable for routine lab work because of the brilliance of the
magic eye. The Lab-Line meters provide the sturdy cell previously mentioned. The
Industrial Instruments #RC-18 is designed for extreme accuracy but the number of dial
settings required to obtain a single reading do not recommend it for routine analysis. A
recent instrument survey conducted by the Methods and Performance Activity of the
Analytical Quality Control Laboratory showed that a preponderance of EPA Laboratories
used the Industrial Instruments RC 16B2.
3-10
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3.5 Turbidimeters
Instruments for the measurement of turbidity have traditionally employed principles of
design related to transmission or reflectance of light. The lack of a primary standard for
turbidity, however, has resulted in a complete absence of uniformity among the available
instruments. Further, the Jackson Candle Turbidimeter, which does not depend upon the
use of a primary standard, is a primitive instrument, subject to many interferences, and the
measurements generally are not reproducible.
Recent investigations (3) have resulted in the design of an instrument which has been
adopted by the Environmental Protection Agency as a standard. The specifications for the
instrument are described elsewhere (4). Presently, the Hach Turbidimeter. Model 2100,
(Box 907, Ames, Iowa 50010) is the only turbidity measuring device manufactured which
meets these specifications. Figure 3-4 shows this instrument.
The Hach Turbidimeter Model 21 00 employs, for standardization, a suspension of formazin,
especially used because of its stability and uniform particle size. For calibration purposes,
the formazin is permanently embedded in a cylinder of lucite, the cylinder duplicating the
size and shape of the sample cuvette. Although the instrument is designed with a series of
scales ranging 0-1. 0-10, 0-100 and 0-1000 .1CU, it has been recommended that turbidity
readings in excess of 40 JCU be rejected (3). Correct use of the meter therefore requires that
samples containing turbidities in excess of 40 JCU be diluted to a value below this level and
the results multiplied by the proper dilution factor.
For production of data with maximum accuracy and precision the following precautions
should be observed:
a. Protect the lucite standard from nicks, scratches and fingerprints.
.. . ~_ ^. -. ~ ,,
H cation Wi luc IUL/ILC.
c. Use a well mixed sample in the sample cuvette; do not take readings until finely
dispersed bubbles have disappeared.
d. Dilute samples containing excess turbidity to some value below 40 JCU; take
reading, and multiply results by correct dilution factor.
3.6 Speetrophotometers
Since a large portion of routine quantitative measurements are performed colorirnetrically.
the spectrophotometer (See Figure 3-5) is usually the workhorse of any analytical
laboratory. Indeed, the versatility of the instrument, and the number of demands imposed
upon it. have resulted in a large variety of designs and price ranges. A systematic listing and
detailed discussion of all instrumental types would be beyond the scope of this chapter. As a
matter of convenience and practicality, therefore, spectrophotometers are discussed
separately as visible, ultraviolet, infrared, and atomic absorption instruments.
A spectrophotometer is an instrument for measuring an amount of light or radiant energy
transmitted through a solution, as a function of wave-length. A spectrophotometer differs
3-11
-------
I J
Figure 3-4. TURBIDIMETER
-------
Figure 3-5. SPECTROPHOTOMETER
-------
from a filter photometer in that it uses continuously variable, and more nearly
monochromatic, bands of light. Filter photometers are relatively insensitive, and lack the
versatility of spectrophotometers. They are used most profitably where a single method can
be designed to fit the instrument.
The essential parts of a spectrophotometer are:
a. A source of radiant energy, usually a tungsten filament bulb,
b. The Monochromator, a device for isolating narrow bands of light,
c. Cells (cuvettes), for holding the colored solution under investigation, and
d. The photodetector, a device to detect and measure the radiant energy passing
through the sample solution.
Each of the essential features listed, especially the monochromator and the photodetector
system, vary in design principles from one instrument to another. Table 3-5 shows some of
the features of the more commonly used spectrophotometers.
3.6.1. Visible Range
Desirable features on a visible-range spectrophotometer are determined by the anticipated
use of the instrument. Simple, limited programs requiring use of only a few parameters at
gross concentrations, can probably be supported by an inexpensive, but reliable instrument,
such as the B&L Spectronic 20. (See Table 3-5.) On the other hand, if a laboratory program
requires a wide variety of measurements on diverse samples at very low concentrations, a
more versatile instrument may be needed. One of the prime considerations would be
adaptability to various cell sizes, at least from 1.0 to 5.0 cm. Many spectrophotometers now
available are satisfactory for water quality analyses.
As shown in Table 3-5, which lists only a few of the available spectrophotometers, higher
priced instruments strive for versatility, including interchangeable sources, detectors, and
cells. Complete information on instrument specifications can be found in the publication
Industrial Research (Beverly Shores, Ind. 46301), Nov. 20, 1969.
3.6.2. Ultraviolet Range
An ultraviolet spectrophotometer is similar in design to a visual range instrument,
differences being in the light source and the optics. The light source is a hydrogen or
deuterium discharge lamp which emits radiations in the UV portion of the spectrum,*
generally from about 200 m^ to the low visible region. The optical system, if of the prism
type, must be constructed of UV-transparant material, usually quartz. Sample cells must
also be constructed of quartz or other UV-transparent material. If a grating system is used in
an UV system, the grating may be specially cut (blazed) in the UV region for greater
sensitivity. A number of UV spectrophotometers are available: the Beckman DU, the
Hitachi, the Bausch and Lomb, the Gary, the Hilger and the Leeds & Northrup.
3.6.3. Infrared Range
A number of instrumental changes are required in the construction of spectrophotometers
3-14
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Table 3-5
DESIGN FEATURES OF SOME COMMON SPECTROPHOTOMETERS
Beckman B
Beckman Du
Bausch & Lomb
Spectronic 20
Coleman 101
Hitachi
Light Source
Optical
System
Slits
Range
Detector
Sample Cells
Readout
Attachment
Tungsten Bulb from
CV Transformer
Glass Prism
Variable
320-1 000 mp
Interchangeable
Phototubes 320-625
and 625-1 000 m^
1.0 to 5.0cm
Meter Scale,
%T, O.D.
Test-tube adapter
flame photometry
Tungsten Bulb,
CV Transformer
Hydrogen Lamp
Quartz Prism
Variable
200-1 000 mil
Interchangeable
Phototubes 200-625
and 625-lOOOmpi
1.0,5.0, 10.0cm
Null Point Potentiometer,
%T, O.D.
UV Adapter with H2
lamp flame photometry
Tungsten Bulb,
Reg. Transformer
Grating, 600/mm
Fixed, band width
20 m^
325-1000 m^
Interchangeable
Phototubes 325-625
and 625-1 000 m/i
1.1, 1.6,2.2cm
Meter Scale %T, O.D.
Interchangeable test-
tube adapters, digital
readout, reflectance
adapter
Tungsten Bulb,
CV Transformer
Hydrogen (Deuterium)
Lamp
Grating, 600/mm
Fixed, band width
10m/i
220-900 mM,
using both lamps
Hitachi Dual Range
Phototube, 220-900 m^
1.0,5.0,20.0cm
Meter Scale, %T, O.D.
Recorder jack or
digital readout
Tungsten Bulb
Hydrogen (Deuterium)
Lamp
Grating, 1440/mm,
Blazed at 200 m/x
Variable
1 95-950 mM , using
both lamps
Dual Range Phototube
1 95-950 mM
1 .0 to 1 0.0 cm,
micro cells
Meter Scale, %T, O.D.
Atomic absorption
reflectance, fluorescence
emission, flow thru
cells
Marketed as Perkin-Elmer J139 or Coleman #139-001
3-15
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for measurements in the infrared (IR) region. Modifications are needed, because optical
materials, such as glass and quartz, absorb radiant energy in the IR region and ordinary
photocells do not respond.
Most IR spectrophotometers use front-surfaced mirrors to eliminate the necessity for
radiant energy to pass through quartz, glass, or other lens materials. The mirrors are usually
parabolic to facilitate gathering the diffuse IR energy. Instruments must be protected from
high humidities and water vapor to avoid deterioration of the optical system, and also to
avoid extraneous absorption bands in the IR.
The energy or light source for an IR instrument may be a Nernst glower or a Globar. Either
source has certain characteristics that recommend it for use, but the Globar is more
commonly used because it has a more stable emission and it is more rugged. Receiving or
detection units may be a thermocouple, a bolometer, thermistor, or a photoconductor cell.
Of all the IR instruments available, two are in relatively common use in EPA laboratories.
The Perkin-Elmer Infracord is a low cost model designed for routine work with simplicity of
operation. The Perkin Elmer #621 (723-G Main Ave., Norwalk, Conn., 06852) is a far more
sophisticated instrument, designed for basic research.
3.6.4 Proper Use of Spectrophotometers
The manufacturer's instructions for proper use should be followed in all cases. Several
safeguards against misuse of the instruments, however, are mandatory.
Instruments should be checked for wavelength alignment. If a particular colored solution is
to be used at a closely specified wavelength, considerable loss of sensitivity can be
encountered if the wavelength control is misaligned. In visual instruments, an excellent
reference point is the maximum absorption for a dilute solution of potassium permanganate,
which has a dual peak at 526 m// and 546m//. On inexpensive grating instruments, which
possess less resolution than the prism instruments, the permanganate peak appears at 525 to
550 m/i as a single flat-topped spike.
For both UV and IR instruments, standard absorption curves for many organic materials
have been published so that reference material for standard peaks is easily available.
Standard films of styrene and other transparent plastics are available for IR wavelength
checks.
Although most instruments contain built-in transformers for stabilization of the electronic
circuits, an exterior, high capacity, constant-voltage transformer is recommended for general
laboratory control. A number of controlled-voltage outlets in the laboratory are especially
desirable in industrial areas or in buildings containing heavy, electrically operated equipment
where voltage surges on adjacent power lines are apt to be frequent. An unstable voltage is
frequently indicated by a flickering needle on the meter. The flickering behavior may be
intermittent or it may occur at certain times of the day when heavy machinery in the area
may be starting or stopping.
Too much emphasis cannot be placed on care of absorption cells. All cells should be kept
scrupulously clean, free of scratches, fingerprints, smudges and evaporated film residues.
Matched cells should be checked to see that they are equivalent by placing portions of the
3-17
-------
same solution in both cells and taking several readings of the %T or OD values. If a cell is
mismatched it should be discarded or reserved for rough work. (Directions for cleaning cells
are detailed in Chapter 4).
Generally speaking, trained technicians may operate any of the spectrophotometers
successfully. However, interpretation of data from both the UV and IR instruments
becomes increasingly complex, and requires more training and specialization. IR interpreta-
tion requires special training, and because of the special techniques of sample preparation,
instrument operation, and interpretation of absorption curves, mere compliance with the
operations manual is not sufficient.
3.6.5. Atomic Absorption
There are a number of differences in the basic design and accessories for atomic absorption
equipment that require consideration before purchase and during subsequent use. These
choices concern the light source, nebulizer burners, optical systems, readout devices, and
mode conversion. Some of these choices are not readily obvious, and require that the
purchaser or user be familiar with the types and numbers of samples to be analyzed and the
specific elements to be measured before a choice is made. For a program analyzing a wide
variety of samples for a number of elements at varying concentrations, an instrument of
maximum versatility would be required. A typical atomic absorption unit is shown in Figure
3-6.
3.6.5.1 Lamp Mounts
A basic design feature of atomic absorption spectrometers is the convenience of the hollow
cathode (HC) lamp changeover system. Some instruments provide for as many as six lamps
in a rotating turret, all electronically stabilized and ready for use by simply rotating the
lamp turret. Other instruments provide for use of only one lamp at a time in the lamp
housing, and require manual removal and replacement whenever more than one element is
to be measured. A quick changeover system is desirable, especially if a number of lamp
changes are needed during a period of operation. Conversely, if lamp changes are infrequent,
multi-lamp mounts do not represent a great convenience.
For optimal use of the instrument, certain precautions should be observed. After the proper
lamp has been selected the hollow cathode current should be adjusted according to the
manufacturer's recommendations and allowed to electronically stabilize (warm up) before
use. This requires approximately 15 minutes. During this period, the monochromator may
be positioned at the correct wavelength and the proper slit width selected. For those
instruments employing a multi-lamp turret, a warm-up current is provided to those lamps
not in use, thereby minimizing the warm-up period when the turret is rotated. In a
single-lamp instrument, the instability exhibited during warm-up is minimized by the use of
a double-beam optical system.
3.6.5.1.1 Single Element and Multi-Element Lamps
As an adjunct to single-lamp mounts, HC lamps using from two to as many as six elements
in combination are available, thereby increasing the versatility of the AA spectrometer. For
instance, a single-lamp instrument such as the Perkin-Elmer may analyze for elements with
only one lamp substitution, whereas a six-lamp turret such as the Jarrell-Ash (590 Lincoln
Street, Waltham, Mass. 02154), using multi-element lamps could run 18 or more elements.
3-18
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Figure 3-6. ATOMIC ABSORPTION UNIT
-------
Multi-element lamps are considerably cheaper per element than single-element lamps, but
the savings may not realized if the lamps are not used strategically, because all the elements
in the cathode deteriorate when the lamp is used, regardless of which element is measured.
The deterioration phenomena results from the different volatilities of metals used in the
cathode. One metal volatilizes (sputters) more rapidly than the others and redeposits upon
the cathode causing an increase in surface area of that metal, and decreasing the exposed
area of the other cathode metals. Thus, with continual use, a drift in signal will be noted
with at least one metal increasing and the other (or others) decreasing. If one can ignore the
dubious cost savings of multi-element lamps, use of single-element lamps would result in
more precise and accurate data.
The individual line intensities of an element in a multi-element HC lamp will usually be less
than that of a lamp containing a pure cathode of the same element. This is because each
element must now share the discharge energy with all other elements present. However, this
reduction should not affect the output by a factor of more than 1/2 to 1/6, depending on
the combination and number of elements combined. The output can be even greater in some
multi-element lamps because alloying may permit a higher operating current than the pure
cathode itself. All HC lamps have life expectancies which are related to the volatility of the
cathode metal, and for this reason, the manufacturer's recommendations for amperage at
which the lamp is operated should be closely followed.
A recent advance in HC lamp design, the high-intensity lamp, promises increased sensitivity
for some elements. It is also predicted that the newly designed lamps will be used in atomic
fluorescence techniques, with significant gains in sensitivity for metals analysis.
Recent improvements in design and manufacture of hollow cathode lamps have resulted in
lamps with more constant output and a longer life. Under normal conditions a HC lamp may
be expected to operate satisfactorily for several years. At one time, hollow cathode lamps
were guaranteed for a minimum amp^hour period. This has been changed, however, to a
90-day warranty. It is good practice to date newly purchased lamps and inspect immediately
upon receipt. Operating current and voltage will be indicated on the lamp and should not be
exceeded during use. An increase in background noise and/or a loss of sensitivity are signs of
lamp deterioration.
3.6.5.2 Burner Types
The most difficult and inefficient step in the AA process is converting the metal in the
sample from an ion or a molecule to the neutral atomic state. It is the function of the
atomizer and the burner to produce the desired neutral atomic condition of the elements.
With minor modifications burners are the same as those used for flame photometry.
Basically there are two different types of burners. They are the total-consumption or
surface-mix burner, and the laminar-flow or pre-mix burner. There are many variations of
these two basic types, such as the Boling, the high-solids, the turbulent-flow, the tri-flame,
nitrous-oxide burner and many others. As one might expect, there are many similarities
among the various burners, the different names resulting from the different manufacturers.
The element being determined and the type of sample solution dictate the type of burner to
be used.
Generally, all types and makes of burners can be adjusted laterally, rotationally and
3-20
-------
vertically for selection of the most sensitive absorbing area of the flame for the specific
element sought. The vertical adjustment is probably the most important since the position
of greatest sensitivity varies from element to element.
Burner height is of utmost importance in changing from one element to another. Certain
instruments are provided with a vernier adjustment for reproducing burner-height settings,
but many are not. Figure 3-7 shows a simply designed device used in the Analytical Quality
Control Laboratory for reproducing exact burner height. The gauge is positioned on the
burner and the height determined from the light beam striking the calibrated scale. The
point at which the beam strikes the gauge is recorded for future use.
3.6.5.3 Single-Beam and Double- (Split) Beam instruments
There is a great deal of existing uncertainty among instrument users about the relative
merits of single-beam and double-beam instruments. Neither system is the final answer.
With a single-beam instrument the light beam from the source passes directly through the
flame to the detector. In a double-beam system the light from the source is divided by a
beam splitter into two paths. One path, the reference beam, goes directly to the detector.
The second path, the sample beam, goes through the flame to the detector. The chopper
alternately reflects and passes each beam, creating two equal beams falling alternately upon
the detector. If the beams are equal they cancel the alternate impulses reaching the detector
and no signal is generated. If the beams are different, the resulting imbalance causes the
detector to generate an a.c. signal which is amplified and measured. Any difference between
the reference and sample beam is measured as a direct function of absorbed light. The
advantage of the double-beam design, therefore, is that any variations in the source are of
reduced importance, and smaller dependence is placed upon the stability of the power
supply. However, stabilization of the power supply can eliminate the apparent need for the
split-beam system. Further, a beam splitter requires use of additional mirrors or optical
accessories that cause some loss of radiant energy. Neither system, however, compensates
for variation in flame intensity.
A single-beam system does not monitor source variations but offers certain other
advantages. It allows use of low-intensity lamps, smaller slit settings and smaller gain. As a
consequence, the single-beam instrument, properly designed, is capable of operating with
lower noise, better signal-to-noise ratio and therefore better precision and improved
sensitivity. Because the simplified optical system conserves radiant energy, especially in the
shorter wavelengths, it facilitates operation in the low wavelength range. With this
advantage, it should be possible to obtain better sensitivity for those elements with strong
resonance lines below 350 m/z and even those slightly below 300 m^.
3.6.5.4. Readout Devices
Early models of the AA instrument offered only a meter, calibrated in percentage
absorption. In the surge of competitive design, more sophisticated readout devices were
built into or offered as accessories to various models. At the present time any desired
readout method may be obtained with almost any instrument. Less expensive designs still
provide meters with conventional needle indicators. More costly instruments offer any
combination of built-in digital sealers, calibrated in concentration, external digital printout
in concentration, typewriter printout or typewriter with punch tape. Even inexpensive
instruments are built with recorder interfacing.
3-21
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SMALL CENTIMETER GAUGE
CIRCULAR
BASE
W
BEAM FROM HOLLOW CATHODE LAMP
-T
0
Figure 3-7 DEVICE FOR REPRODUCIBLE POSITIONING OF BURNER HEIGHT
3-22
-------
Choice of a readout system is predicated largely upon laboratory needs and availability of
budget. In general, any step toward complete automation is desirable but the degree of
automation should be compatible with the laboratory program.
3.6.5.5 Miscellaneous Accessories
A number of instruments contain a mode selector, making an instrument usable for either
flame absorption or flame emission. The conversion to flame emission is a desirable feature
since certain elements are more amenable to analysis by this method.
Automatic sample changers are offered for almost all instruments on the market, and as has
been previously stated, any automation feature is desirable. However, unless a laboratory
program performs a large number of repetitious measurements daily, an automatic sample
changer would not be required. As a practical measure, other commonly used sample-
changing devices not expressly designed for AA use, can easily be interfaced with almost any
AA instrument.
3.6.5.6 Instrument Choice
Table 3-6 summarizes some of the design features of various commercially available
instruments. Since many of the features are common to most models, the basic choice
appears to be between double-beam and single-beam instruments. On this basis, at this
writing, it is probable that the most widely used instrument in the EPA is the Perkin-Elnxer
303, one of two double-beam instruments on the market.
The Instrumentation Laboratories Model 153 (113 Hartwell Ave., Lexington, Mass. 02173)
has a number of desirable features. In addition to features listed in the table, it includes
push-button ignition, push-button wavelength scan, fail-safe solenoids to prevent improper
flame settings, fail-safe flame monitoring, absorption integrator at selected time intervals,
visual "peaking" meters, curve correction and several other features. The new Perkin-Elrner
Model 403 also offers most of these features in addition to very sophisticated data handling
accessories.
3.7 Organic Carbon Analyzer
A number of devices designed to measure the organic content of aqueous samples have
appeared on the market within the last five years. The oldest, or first of these instruments, is
the Dow Beckman Carbonaceous Analyzer (Figure 3-8). The apparatus measures organic
carbon as carbon, by oxidizing a very small sample at a temperature of 900°, in a stream of
oxygen, converting all organically bound carbon to carbon dioxide, which is then measured
by a Golay-type thermal detector. The instrument is able to detect about 20 mg. of carbon.
A recent modification of the original Dow Beckman Carbonaceous Analyzer employs a
dual-combustion-tube system operating at different temperatures to distinguish between
total carbon and inorganic carbon. Organic carbon is found by difference.
Another such instrument, also developed by Dow Chemical Company, is marketed by the
Fisher Scientific Company (Instrument Division, 711 Forbes Ave., Pittsburgh, Pa. 15219) as
the "Aquarator". The principle of operation is similar to the Dow Beckman Carbonaceous
Analyzer, except that carbon dioxide fed through the combustion tube is reduced to carbon
monoxide and measured in an infrared detector. The reaction does not measure carbon, per
se, but reducing materials. The results can be equated with the COD test, but the instrument
3-23
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is best suited for industrial wastes and sewage. It lacks the sensitivity needed for COD
measurements on relatively clean waters. The lower limit of measurement is about 40 mg/1
COD.
A third instrument developed by Dow researchers is franchised to Ionics Corporation (165
Grove Street, Watertown, Mass. 02172) and sold as the "TOD Analyzer". This instrument is
also similar in principle to the carbon analyzer, except that the device measures oxygen in
and out of the combustion tube. Depletion of oxygen is correlated with oxygen demand of
the sample. However, the reaction of oxygen with sulfur and nitrogen is not stoichiometric,
and results for some samples may be questionable. A malfunction of the sample injection
system appears to be the main problem with the instrument to date. An evaluation study
conducted by the Hudson Delaware Basins Office, EPA, indicates a very good potential use
for the instrument.
Another device, designed by Union Carbide Corporation (Ionics Inc., 65 Grove St.,
Watertown, Mass. 02172) is similar to the original Dow Beckman Analyzer except that the
combustion tube contains a coil of heated palladium wire. An aqueous sample injected into
the combustion tube is decomposed to hydrogen and oxygen through the catalytic action of
the palladium. Carbon from organic matter combines with the water-produced oxygen to
form carbon dioxide, with a final infrared measurement. Results obtained are theoretically
identical to the Dow Beckman Analyzer, but the repeated failure of the sample injection
system has prevented evaluation of the apparatus as a laboratory instrument. It is being
routinely used, however, as a continuous monitoring system for total carbon.
To date the only instrument which has been successfully demonstrated and wruch meets the
needs of EPA laboratories is the Dow Beckman Carbonaceous Analyzer or its successor, the
Model 915. Operating instructions for this device are detailed elsewhere (4).
Precautions to be observed in use of the Dow Beckman Carbonaceous Analyzer or the DB
Model 915 are detailed below:
a. All inorganic carbon present in the sample as carbonate or bicarbonate must be
removed prior to analysis, or accounted for, when using the Model 915.
b. Particulate matter in the sample should not be larger than the opening of the
hypodermic needle used to take the sample. If necessary the sample should be
homogenized by some means designed to reduce the particle size.
c. The instrument should be preconditioned with repeated injections of distilled water
to obtain stable operating conditions before actual sample injection.
d. Furnace temperature and oxygen flow should be maintained at proper settings
during period of operation.
e. Perform periodic maintenance of instrument as follows:
1. repack combustion tube with fresh asbestos,
2. clean and dry micro filter,
3. clean and dry infrared cell,
4. recharge reference cell with nitrogen.
Need for replacement of asbestos packing is indicated by a loss of sensitivity and wide
3-24
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Table 3-6.
DESIGN FEATURES OF SOME COMMON ATOMIC ABSORPTION INSTRUMENTS
Design Feature
Model Number
Source Arrange-
ment
Optical System
Mode
Detector
Detector
Substitution
Readout
Range
Inst. Labs
153
Turret-6
Dual double-beam,
beam, grating,
single pass
Absorption and
emission
Photomultiplier
Yes-Photodiode
in Channel B
Direct concentra-
tional readout
with curve
corrector
1 90-800 mfi
Jarrell-Ash
82-500
Turret-6
Single-beam,
a.c., grating,
multipass
Absorption and
emission
Photomultiplier
Yes
Meter readout
plug-in recorder
1 90-800 m/*
Norelco-Unicam
SP-90
Turret-3
Single-beam
a.c., prism
Absorption and
emission
Photomultiplier
Yes
Meter readout
or recorder
1 90-770 mil
198-850 with
special photo tube
Perkin-Elmer
403
Single Lamp
Double-beam,
grating
Absorption and
emission
Photomultiplier
Digital concen-
tration readout
1 90-800 m//
Varian
AA4
Turret-4
Single-beam,
grating
Absorption and
emission
Photomultiplier
Yes
Meter readout
Hewlett-Packard
5960 A
Turret-6
Single-beam,
a.c., interference
filter
Absorption only
Photomultiplier
Not given
Meter readout
1 90-800 mil
Bausch & Lomb
AC2-20
Turret-3
Single-beam,
a.c., grating,
single-pass
Absorption and
emission plus UV
visible spectro-
photometry
Photomultiplier
Not given
Meter readout
1 90-800 m/i
Beckman
979
Turret-3
Single-beam,
grating, multi-
pass
Absorption, emission,
and regular spectro-
photometric analysis
Photomultiplier
Not given
Meter-plug-in
recorder
1 90-770 m/^ ,
190-&52 mM optional
Slit
Accessories
Special
Adjustable
Data printer,
sample changer,
curve correction
2 elements simul-
taneously using
interference filters,
push button
operation
Adjustable
Digital concen-
tration readout,
sample changer
Adjustable
Plug-in electronics,
automatic sample
changer with log
recorder
Adjustable
Typewriter
readout, sample
changer
Adjustable
Digital printer
with curve
correction,
sample changer
Fixed
Recorder readout
Adjustable and fixed Adjustable
5-pass system plug-in electronics
Paper punch tape 186-1000
with typewriter to
produce a typed
report, push
button operation
Recorder readout,
B&L concentrational
readout, sample
changer
Push-button opera- Dialable filters for
tion for one of six work above 560
preset determinations
Digital readout,
data printer,
sample changer
3-pass system
3-25
-------
U)
Figure 3-8. ORGANIC CARBON ANALYZER
-------
peaks. Unsteady baseline and excessive noise is caused by a dirty or partly clogged micro
filter. An accumulation of moisture in the infrared cell is indicated by loss of sensitivity and
excessive noise.
3.8 Selective ion Electrodes
In recent years a variety of ion-selective electrodes have been commercially available, and
show great promise as fast and efficient tools for in situ monitoring and for laboratory
analysis of all types of samples. A list of ions susceptible to analysis is indicative of the
interest and progress being made in this field. Electrodes, or "probes" as they are popularly
called, are available for measurement of monovalent cations, sulfate, nitrate, perchlorate,
and a number of others. Dissolved oxygen probes should also be included in this list,
although they are not technically selective ion probes. There have been a number of reviews
concerning the theory and application of probes, most recently a concise article by Rechnitz
(5).
Selective ion probes generally measure what they claim to measurespecific ion activity.
They do not measure concentrations of un-ionized materials. For example, the probe
designed to measure divalent cations promises to measure total hardness as a function of
calcium and magnesium. However, since the probe does not respond to un-ionized calcium
and magnesium, it does not accurately measure total hardness. As a consequence of this
deficiency, much investigation is being carried out to devise means of determining total
concentration of the constituent sought.
"Methods for Chemical Analysis of Water and Wastes" (4) outlined procedures for use of
two probes, fluoride and DO. The chloride probe is also used on the automatic monitoring
devices and will be listed as an approved procedure in future editions of EPA. Methods.
Evaluation of additional ion selective electrodes for cyanide, ammonium, and sulfide ions is
now underway, and will be reported in the near future. Various techniques for use of the
probes are reviewed by Rise-man (6).
Personnel in water pollution laboratories are encouraged to investigate the use of selective
ion probes as a means of reducing analytical work and improving data quality. At the
present time, however, only the fluoride. DO, and chloride probes are recommended for
routine use in data collection.
A basic question relating to the use of selective ion probes is the number of standards
required to prepare a standard curve. It is generally agreed that the more standards used for
the preparation of a colorimetric curve, the more reliable the data resulting from use of the
curve. On the other hand, only one or two points are normally used in standardization of a
meter for measuring pH, conductivity or DO. In the conversion of a colorimetric procedure
such as fluoride to a probe-type measurement, the tendency is to prepare a millivolt vs
concentration curve using the usual six to ten standards. Probe manufacturers insist that
only one or two points are needed since the linearity of response has been established and
only the slope of the line must be known. The alternatives are:
a. Take readings in distilled water at 0 and at some concentration approximating
concentration of sample; establish slope of line.
b. Take readings in distilled water at 0 and two concentrations bracketing expected
concentration of unknowns to establish curve or slope.
3-28
-------
c. Take readings at 0 and at decade concentrations as 1, 10, 100, etc., for standard
curve.
d. Take reading of sample, add known increment of measured constituent and read
again; establish proportionality factor.
The method for the use of the fluoride electrode specifies use of multiple standards in the
range between 0 to 2 mg/liter, because this system has supplied very precise data when
compared to the colorimetric methods using the same set of standards. The system of
incremental addition appears to have considerable merit since the electrode response is
established in the presence of possible interferences. At the present time no single procedure
for standardizing probe response has been adopted by a majority of users.
When a selective ion electrode appears to be malfunctioning, the same check system may be
used as for a faulty glass pH electrode. It is unlikely, however, that the electrode will be
cracked; it will probably be dry, or insufficiently filled with the necessary solution. The
piobe assembly and instructions for refilling customarily accompany the item when shipped
by the manufacturer and said instructions should be followed by the user.
Selective ion probes are available from several manufacturers including Beekman, Corning,
Coleman and Orion. The Orion organization (11 Blackstone St., Cambridge, Mass. 02139) is
the .largest producer in the field and offers not only selective ion probes but a sizable
complement of electronic equipment for use with the probes. Figure 3-9 illustrates an Orion
selective ion meter.
Dissolved oxygen probes and meters of various designs have been offered by a large number '
of manufacturers including Weston & Stack, Beekman, Jarrell-Ash, Union Carbide, Yellow
Springs Instrument, Delta Scientific, and others.
3.9 References
1. 1968 Book of ASTM Standards, Part 30; Testing Single Arm Balances, pp. 1071-84:
American Society for Testing and Materials, 1916 Race Street, Philadelphia, Pa. 19103.
2. 1968 Book of ASTM Standards, Part 23; Test for pH of Industrial Waste Water, pp.
292-3; American Society for Testing and Materials, 1916 Race Street, Philadelphia. Pa.
19103.
3. Black, A. P., and Hannah, S. A., "Measurement of Low Turbidities," JAWWA,_57, 901
(1965).
4. Methods for Chemical Analysis of Water and Wastes, EPA, Analytical Quality Control
Laboratory, 1971.
5. Rechnitz. G. A., "Ion Selective Electrodes," Chemical Engineering News, p. 146. June
12, 1967.
6. Rise-man, Jean M., "Measurement of Inorganic Water Pollutants by Specific Ion
Electrode," American Laboratory, p. 32. July 1969.
3-29
-------
O.)
OJ
O
Figure 3-9. SELECTIVE ION METER
-------
CHAPTER 4
GLASSWARE
4.1 General
The measurement of trace constituents in water demands methods capable of maximum
sensitivity. This is especially true for metals and trace organics such as pesticides, as well as
for the determination of ammonia and phosphorus. In addition to sensitive methods,
however, there are other areas that require special consideration. One such area is that of the
cleanliness of laboratory glassware. Obviously, the very sensitive analytical systems are more
sensitive to errors resulting from the improper use or choice of apparatus, as well as to
contamination effects due to an improper method of cleaning the apparatus. The purpose of
this chapter is to discuss the kinds of glassware available, the use of volumetric ware, and
various cleaning requirements.
4.2 Types of G lassware
Laboratory vessels serve three functions: storage of reagents, measurement of solution
volumes and confinement of reactions. For special purposes, vessels made from materials
such as porcelain, nickel, iron, aluminum, platinum, stainless steel, and plastic may be
employed to advantage. Glass, however, is the most widely used material of construction.
There are many grades and types of glassware from which to choose, ranging from student
grade to others possessing specific properties such as resistance to thermal shock, alkali, low
boron content, and super strength. Soft-glass containers are usually relatively soluble, and
therefore are not recommended for general use, especially for storage of reagents. The
mainstay of the modem analytical laboratory is a highly resistant borosilicate glass, such as
that manufactured by Corning Glass Works under the name "Pyrex" or by Kimble Glass Co.
as "Kirr.ax". This glassware is satisfactory for all analyses included in "Methods for
Chemical Analysis of Water and Wastes (1)."
Depending on the particular manufacturer, various trade names are used for specific brands
possessing special properties such as resistance to heat, shock, alkalies, etc. Examples of
some of these follow:
a. Kimax- or Pyrex-brand glass is a relatively inert all-purpose borosilicate glass.
b. Vycor-brand glass is a silica glass (96%) made to withstand continuous temperatures
up to 900°C and can be down-shocked in ice water without breakage.
c. Corning brand glass is claimed to be 50 times more resistant to alkalies than
conventional ware and practically boron free (max. 0.2%).
d. Ray-Sorb- or Low-Actinic-brand glass is for use with light-sensitive material.
e. Corex-brand labware is harder than conventional borosilicates, better able to resist
clouding and scratching.
The use of plastic vessels, containers and other apparatus made of Teflon, polyethylene,
polystyrene and polyproplylene has increased markedly over recent years. Some of these
materials, such as Teflon, are quite expensive; however, Teflon stopcock plugs have
4-1
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practically replaced glass plugs in burets, separatory funnels, etc. because lubrication to
avoid sticking or "freezing" is not required. Polyprolylene, a methylpentene polymer, is
available as laboratory bottles, graduates, beakers and even volumetric flasks. It is crystal
clear, shatter-proof, autoclavable and chemically resistant.
Some points to consider in choosing glassware and/or plasticware are:
a. Generally, the special types of glass listed above are not required to perform the
analyses given in "Methods for Chemical Analysis of Water and Wastes" (1).
b. Unless instructed otherwise, borosilicate or polyethylene bottles are to be used for
the storage of reagents and standard solutions.
c. Certain dilute metal solutions may plate out on glass container walls over long
periods of storage. Thus, dilute metal standard solutions are prepared fresh at the
time of analysis.
d. For some operations, disposable glassware is entirely satisfactory. One example is
the use of disposable test tubes as sample containers for use with the Technicon
Automatic Sampler.
e. Plastic bottles of polyethylene and/or Teflon have been found satisfactory for the
shipment of water samples. Strong mineral acids (such as sulfuric acid) and organic
solvents will readily attack polyethylene and are to be avoided.
f. Borosilicate glassware is not completely inert, particularly to alkalies; therefore,
standard solutions of silica, boron and the alkali metals are usually stored in
polyethylene bottles.
For additional information the reader is referred to the catalogs of the various glass and
plastic manufacturers. These catalogs contain a wealth of information as to specific
properties, uses, sizes, etc.
4.3 Volumetric Analyses
By common usage, accurately calibrated glassware for precise measurements of volume has
become known as volumetric glassware. This group includes volumetric flasks, volumetric
pipets and accurately calibrated burets. Less accurate types of glassware including graduated
cylinders, serological and measuring pipets also have specific uses in the analytical
laboratory, when exact volumes are unnecessary. A typical laboratory glassware setup is
shown in Figure 4-1.
The precision of volumetric work depends in part upon the accuracy with which volumes of
solutions can be measured and there are certain sources of error which must be carefully
considered. The volumetric apparatus must be read correctly; that is, the bottom of the
meniscus should be tangent to the calibration mark. There are other sources of error,
however, such as changes in temperature which result in changes in the actual capacity of
glass apparatus and in the volume of the solutions.The capacity of an ordinary glass flask of
1000 ml volume increases 0.025 ml per degree rise in temperature, but if made of
borosilicate glass the increase is much less. One thousand ml of water or of most 0.1 N
solutions increases in volume by approximately 0.20 ml per 1°C increase at room
4-2
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Figure 4-1. TITRATION BENCH
-------
temperature. Thus solutions must be measured at the temperature at which the apparatus
was calibrated. This temperature (usually 20° C) will be indicated on all volumetric ware.
There may also be errors of calibration of the apparatus; that is, the volume marked on the
apparatus may not be the true volume. Such errors can be eliminated only by recalibrating
the apparatus or by replacing it.
Volumetric apparatus is calibrated "to contain" or "to deliver" a definite volume of liquid.
This will be indicated on the apparatus with the letters "TC" (to contain) or "TD" (to
deliver). Volumetric flasks are calibrated to contain a given volume. They are available in
various shapes and sizes ranging from 1- to 2000-ml capacity.
Volumetric pipets are calibrated to deliver a fixed volume. The usual capacities are 1 thru
100 ml although micro-pipets are also available. In emptying volumetric pipets, they should
be held in a vertical position and the outflow should be unrestricted. The tip of the pipet is
kept in contact with the wall of the receiving vessel for a second or two after the free flow
has stopped. The liquid remaining in the tip is not removed; this is most important.
Measuring and serological pipets should also be held in a vertical position for dispensing
liquids; however, the tip of the pipet is only touched to the wet surface of the receiving
vessel after the outflow has ceased. For those pipets where the small amount of liquid
remaining in the tip is to be blown out and added, indication is made by a frosted band near
the top. The band is usually located far enough down so that it will not touch the
technician's lips when liquid is being drawn up or blown out.
Burets are used to deliver definite volumes. The more common types are usually of 25- or
50-ml capacity, graduated to tenths of a milliliter, and are provided with stopcocks. For
precise analytical methods in microchemistry, micro-burets are also used. Micro-burets
generally are of 5- or 10-ml capacity, graduated in hundredths of a milliliter division.
Automatic burets with reservoirs are also available ranging in capacity from 10 to 100 ml.
Reservoir capacity ranges from 100 to 4000 ml.
General rules in regard to the manipulation of a buret are as follows: Do not attempt to dry
a buret which has been cleaned for use, but rinse it two or three times with a small volume
of the solution with which it is to be filled. Do not allow alkaline solutions to stand in a
buret, because the glass will be attacked, and the stopcock, unless made of Teflon, will tend
to freeze. A 50-ml buret should not be emptied faster than 0.7 ml per second, otherwise too
much liquid will adhere to the walls and as the solution drains down, the meniscus will
gradually rise, giving a high false reading. It should be emphasized that improper use of
and/or reading of burets can result in serious calculation errors.
In the case of all apparatus for delivering liquids, the glass must be absolutely clean so that
the film of liquid never breaks at any point. Careful attention must be paid to this fact or
the required amount of solution will not be delivered. The various cleaning agents and their
use are described later.
4.4 Federal Specifications for Volumetric Glassware
Circular 602 of the National Bureau of Standards, "Testing of Glass Volumetric Apparatus",
describes the Federal Specifications for volumetric glassware. The National Bureau of
Standards no longer accepts stock quantities of volumetric apparatus from manufacturers or
dealers for certification and return for future sale to consumers. This certification service is
-------
still available, but apparatus will be tested only when submitted by the ultimate user, and
then only after an agreement has been reached with the Bureau concerning the work to be
done.
Consequently, the various glass manufacturers have discontinued the listing of NBS-certified
ware. In its place catalogue listings of volumetric glass apparatus which meet the Federal
Specifications are designated as Class A and all such glassware is permanently marked with a
large "A". These NBS specifications are listed in Table 4-1. The ware in question includes
the usual burets, volumetric flasks and volumetric pipets.
Table 4-.1
TOLERANCES FOR VOLUMETRIC GLASSWARE
(Abridged from National Bureau of Standards Data, 1941)
Capacity (ml)
less than and including Limit of error (ml)
Graduated Flasks
25 0.03
50 0.05
100 0.08
200 0.10
250 0.11
300 0.12
500 0.15
LOOO 0.30
2,000 0.50
Transfer pipets
2 0.006
5 0.01
10 0.02
25 0.025
30 0.03
50 0.05
100 0.08
200 0.10
Burets1
5 0.01
10 0.02
30 0.03
50 0.05
100 0.10
Limits of error are of total or partial capacity. Customary practice is to test the capacity at 5 intervals.
4-5
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In addition to the "A" marking found on calibrated glassware and the temperature at which
the calibration was made, other markings also appear. These include the type of glass, such
as Pyrex, Corex, Kimax, etc., the stock number of the particular item, and the capacity of
the vessel. If the vessel contains a ground-glass connection, this will also be included along
with the TD or TC symbol. An example of the markings usually found on volumetric ware is
shown in Figure 4-2.
PYREX GLASS co. KIMAX
USA * TYPE
J lfi*~
A *
500 ml 10.20ml *r-
TP ^floP f
\ \l L\l W *
un t^Rfln <
STANDARD
TAPER
SIZE
TO
CONTAIN
STOCK
* 19 X
> A
' H
> Rfifl
' JUU r
> TP °fl
' 1 V CU
...» un ?
J
nl
°C
ROR
Figure 4-2. EXAMPLE OF MARKINGS ON GLASSWARE
Class A glassware need not be recalibrated before use. However, if it should become
necessary to calibrate a particular piece of glassware, directions may be found in texts (2) on
quantitative analysis.
4.5 Cleaning of Glass and Porcelain
The method of cleaning should be adapted to both the substances that are to be removed,
and the determination to be performed. Water-soluble substances are simply washed out
with hot or cold water, and the vessel is finally rinsed with successive small amounts of
distilled water. Other substances more difficult to remove may require the use of a
detergent, organic solvent, dichromate cleaning solution, nitric acid or aqua regia (25
percent v/v cone. HNO3 in cone. HC1), In all cases it is good practice to rinse a vessel with
tap water as soon as possible after use. Material allowed to dry on glassware is much more
difficult to remove.
Volumetric glassware, especially burets, may be thoroughly cleaned by a mixture containing
the following: 30 g sodium hydroxide, 4 g sodium hexametaphosphate (trade name,
Calgon), 8 g trisodium phosphate, and 1 liter water. A gram or two of sodium lauryl sulfate
or other surfactant will improve its action in some cases. This solution should be used with a
buret brush.
Dichromate cleaning solution (chromic acid) is a powerful cleaning agent; however, due to
its destructive nature upon clothing and upon laboratory furniture, extreme care must be
taken when using this mixture. If any of the solution is spilled, it must be cleaned up
4-6
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immediately. Chromic acid solution may be prepared in the laboratory by adding 1 liter of
concentrated sulfuric acid slowly, with stirring, to 35 ml saturated sodium dichromate
solution. This mixture must be allowed to stand for approximately 15 minutes in the vessel
which is being 'cleaned and may then be .returned to a storage bottle. Following the
chromic-acid wash, the vessels are rinsed thoroughly with tap water, then with small
successive portions of distilled water. Fuming nitric acid acts more rapidly, but is
disagreeable to handle. In either case, when the acid becomes dilute, the cleaning mixture is
no longer effective. A mixture of concentrated sulfuric and fuming nitric acids is even more
efficient but is also hazardous to use. A persistent greasy layer or spot may be removed by
acetone or by allowing a warm solution of sodium hydroxide, about 1 g per 50 ml of water,
to stand in the vessel for 10-15 minutes; after rinsing with water, dilute hydrochloric acid,
and water again, the vessel is usually clean. Alcoholic potassium hydroxide is also effective
in removing grease. To dry glass apparatus, rinse with acetone and blow or draw air through
it.
4.6 Special Cleaning Requirements
Absorption cells, used in spectrophotometers, should be kept scrupulously clean, free of
scratches, fingerprints, smudges and evaporated film residues. The cells may be cleaned with
detergent solutions for removal of organic residues, but should not be soaked for prolonged
periods in caustic solutions because of the possibility of etching. Organic solvents may be
used to rinse cells in which organic materials have been used. Nitric acid rinses are
permissible, but dichromate solutions are not recommended because of the adsorptive
properties of dichromate on glass. Rinsing and drying of cells with alcohol or acetone before
storage is a preferred practice. Matched cells should be checked to see that they are-
equivalent by placing portions of the same solution in both cells and taking several readings
of the transmittance (%T) or optical density (OD) values. If a cell is mismatched it should be
discarded or reserved for rough work.
For certain determinations, especially trace metals, the glassware should also be rinsed with
a 1:1 nitric acid-water mixture. This operation is followed by thoroughly rinsing with tap
water and successive portions of distilled water. This may require as many as 12-15 rinses,
especially if chromium is being determined. The nitric acid rinse is also especially important
if lead is being determined.
Glassware to be used for phosphate determinations should not be washed with detergents
containing phosphates. This glassware must be thoroughly rinsed with tap water and
distilled water. For ammonia and Kjeldahl nitrogen, the glassware must be rinsed with
ammonia-free water (See Chapter 2).
Glassware to be used in the determination of trace organic constituents in water, such as
chlorinated pesticides, should be as free as possible of organic contaminants. A chromic acid
wash of at least 15 minutes is necessary to destroy these organic residues. Rinse thoroughly
with tap water, and finally with distilled water. Glassware may be dried for immediate use
by rinsing with redistilled acetone. Otherwise glassware may be oven dried or drip dried.
Glassware should be stored immediately after drying to prevent any accumulation of dust.
Store inverted or with mouth of glassware covered with foil.
Bottles to be used for the collection of samples for organic analyses should be rinsed
successively with chromic acid cleaning solution, tap water, distilled water, and finally
several times with redistilled solvent (e.g., acetone,- hexane, petroleum ether, chloroform).
4-7
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Caps are washed with detergent, rinsed with tap water, distilled water and solvent. Liners are
treated in the same way as the bottles and are stored in a sealed container.
4.7 Disposable Glassware
When the risk of washing a pipet for reuse becomes too great, as in the case of use with
toxic materials, or when the cost of washing glassware becomes prohibitive, disposable
pipets may be the answer, provided they meet the necessary specification. Various types are
available including bacteriological, serological and micro-dilution pipets. Disposable glass-
ware generally is made of soft glass.
4.8 Specialized Glassware
The use of vessels and glassware fitted with standard-taper, ground-glass, and ball-and-socket
joints has increased because of certain advantages such as less leakage and fewer freezeups.
Standard-taper, interchangeable ground joints save time and trouble in assembling apparatus.
They are precision-ground with tested abrasives to insure an accurate fit and freedom from
leakage. Ball and socket joints increase flexibility of operation and eliminate the need for
exact alignments of apparatus. Symbols and their meaning as applied to standard joints,
stoppers and stopcocks are shown below.
4.8.1. Standard Taper (J)
^ is the symbol used to designate interchangeable joints, stoppers and stopcocks, complying
with the requirements of Commercial Standard CS-21, published by the National Bureau of
Standards. All mating parts are finished to a 1:10 taper.
The size of a particular piece appears after the appropriate symbol. Due primarily to the
greater variety of apparatus equipped with J fittings, a number of different types of
identifications are used, as follows:
a. For jointsa two-part number, as J 24/40, with 24 being the approximate diameter
in mm at the large end of the taper, and 40 the axial length of taper, also in mm.
b. For stopcocksa single number, as J 2, with 2 mmbeing the approximate diameter
of the hole or holes through the plug.
c. For bottles-a single number, as J 19, with 19 mm being the appropriate diameter
at top of neck. However, there are differences in dimensions between the bottle and
flask stoppers.
d. For flasks, etc. - a single number, as J 19, with 19 mm being the appropriate
diameter of the opening at top of neck.
4.8.2 Spherical Joints (J)
5 is the designation for spherical (semi-ball) joints complying with CS-21. The complete
designation of a spherical joint also consists of a two-part number, as 12/2, with 12 being
the approximate diameter of the ball and 2 the bore of the ball and the socket, also in mm.
4-8
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4.8.3 Product Standard (g)
£. is a new symbol. It will appear in a forthcoming NBS Product Standard for stopcocks with
Teflon plugs, with the mating surfaces being finished to a 1:5 taper. As with J stopcocks, a
single number is used. Thus, £ 2 means a Teflon stopcock with a hole of approximately
2-mm diameter in the plug.
4.9 Fritted Ware
For certain laboratory operations the use of Fritted Ware for filtration (as in total dissolved
solids and suspended solids determinations), gas dispersion, absorption, and/or extractions
may be of an advantage.
There are six different porosities of Fritted Ware available, so that precipitates varying in
size can be filtered at maximum speed with no sacrifice or retentivity. Porosity is controlled
in manufacture, and discs are individually tested and graded into these classifications. The
extra-coarse and coarse porosities are held toward the maximum pore diameter as listed. The
medium, fine, very fine, and ultra-fine are held toward the minimum pore diameter as listed
in Table 4-2.
Table 4-2
FRITTED WARE POROSITY
Porosity
Grade
Extra Coarse
Coarse
Medium
Fine
Very Fine
Ultra-Fine
Designation
M
F
VF
UF
Pore Size
(Microns) Principal Uses
170-220 Coarse filtration. Gas
dispersion, washing,
absorption.
40-60 Coarse filtration. Gas
dispersion, washing,
absorption.
10-15 Filtration and extraction.
4-5.5 Filtration and extraction.
2-2.5 General bacterial filtration.
0.9-1.4 General bacterial filtration.
Pore sizes are determined by the method specified in ASTM E 128, "Maximum
Diameter and Permeability of Rigid Porous Filters for Laboratory Use." (3)
Pore
4-9
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4.9.1 Recommended Procedures for Maximum Filter Life
a. New Filters. Wash new filters by suction with hot hydrochloric acid, followed by a
water rinse.
b. Pressure Limits. The maximum safe differential pressure on a disc is 15 pounds per
square inch.
c. Thermal Shock. Fritted ware has less resistance to thermal shock than non-porous
glassware. Hence, excessive, rapid temperature changes and direct exposure to a
flame should be avoided. Heating in a furnace to 500°C may be done safely,
provided the heating and cooling are gradual. Dry ware may be brought to constant
weight by heating at 105-110° C.
Never subject a damp filter of ultra-fine porosity to a sudden temperature change. Steam
produced in the interior may cause cracking.
4.9.2 Cleaning of Used Filters
In many cases, precipitates can be removed by rinsing with water, passed through from the
underside, with the pressure not exceeding 15 pounds per square inch. The suggestions that
follow in Table 4-3 will be helpful in dealing with material that will not be removed by the
reverse water-wash. The use of strong alkalies, strong hydrofluoric acid and phosphoric acid
should be avoided. Also, scratching of the surfaces will weaken the discs.
Material
Albumen
Aluminous and
siliceous residues
Copper or iron oxides
Fatty materials
Mercuric sulfide
Organic matter
Silver chloride
Table 4-3
CLEANING OF FILTERS
Removal Agent
Hot ammonia or hydrochloric acid
2% Hydrofluoric acid followed by con-
centrated sulfuric acid. Rinse immediately
with water until no trace of acid can be
detected.
Hot hydrochloric acid plus potassium
chlorate.
Carbon tetrachloride
Hot aqua regia
Hot concentrated cleaning solution, or hot
concentrated sulfuric acid with a few drops
of sodium nitrite.
Ammonium or sodium hyposulfite.
4-10
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4.10 References
1. "Methods for Chemical Analysis of Water and Wastes," EPA, Analytical Quality Control
Laboratory, 1971.
2. Willare, H. H., and Furman, N. H., Elementary Quantitative AnalysisTheory and
Practice, D. Van Nostrand Co., Inc., New York (1947).
3. 1968 Book of ASTM Standards, Part 30, American Society for Testing and Materials,
1916 Race Street, Philadelphia, Pa. 19103.
4-11
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Chapter 5
REAGENTS, SOLVENTS, AND GASES
5.1 Introduction
The objective of this chapter is to provide general information and suggestions that will
serve to keep the analyst conscious of his responsibilities in analytical quality control, as
they relate to reagents, solvents and gases. While the material presented here will assist the
analyst in producing high quality data, it is by no means complete. !t is incumbent on the
analyst to obtain details of special precautions required to insure proper selection,
preparation, and storage of reagents, solvents and gases from the descriptions of individual
methods.
5.2 Reagent Quality
Chemical reagents, solvents, and gases are available in a wide variety of grades of purity,
ranging from technical grade to various "ultra pure" grades. The purity of these materials
required in analytical chemistry varies with the type of analysis. The parameter being
measured and the sensitivity and specificity of the detection system are important factors in
determining the purity of the reagents required. For many analyses, e.g., most inorganic
analyses, analytical reagent grade is satisfactory. Other analyses, e.g., trace organic and
radiological, frequently require special "ultra pure" reagents, solvents, and gases. In methods
where the purity of reagents is not specified it is intended that analytical reagent grade be
used. Reagents of lesser purity than that specified by the method should not be used. The
labels on the container should be checked and the contents examined to verify that the
purity of the reagents meets the needs of the particular method involved. The quality of
reagents, solvents, and gases required for the various classes of analyses: inorganic, metals,
radiological, and organic, are discussed below.
Reagents must always be prepared and standardized with the utmost of care and technique,
against reliable primary standards. They must be restandardized or prepared fresh as often as
required by their stability. Stock and working standard solutions must be checked regularly
for signs of deterioration, e.g., discoloration, formation of precipitates, and concentration.
Standard solutions should be properly labeled as to compound, concentration, solvent, date,
and preparer.
Primary standards must be obtained from a reliable source, pretreated, e.g., dried, under
specified conditions, accurately prepared in calibrated volumetric glassware, and stored in
containers that will not alter the reagent. A large number of primary standards are available
from the National Bureau of Standards (NBS). A complete listing of available standards is
given in NBS Special Publication 260 (1). Primary standards may also be obtained from
many chemical supply companies. Suppliers for special quality reagents, solvents, and gases
are noted in later discussions of the various classes of analyses. Reagents and solvents of all
grades are available from many chemical supply houses.
There is some confusion among chemists as to the definition of the terms ANALYTICAL
REAGENT GRADE, REAGENT GRADE, and ACS ANALYTICAL REAGENT GRADE. A
review of the literature and chemical supply catalogs indicates that the three terms are
synonymous. Hereafter, in this document, the term ANALYTICAL REAGENT GRADE
5-1
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(AR) will be used. It is intended that AR chemicals and solvents shall conform to the
current specifications of the Committee on Analytical Reagents of the American Chemical
Society (2).
The ASTM Manual on Industrial Water and Industrial Waste Water (3), Part 23 of ASTM
Standards (4), and "Standard Methods for the Examination of Water and Wastewater" (5)
devote separate chapters to problems related to preparation, standardization, and storage of
reagents. The information provided therein is particularly appropriate to inorganic
determinations. The type of volumetric glassware to be used, the effect of certain reagents
on glassware, the effect of temperature on volumetric measurements, purity of reagents,
absorption of gases and water vapor from the air, standardization of solutions, instability,
and need for frequent standardization of certain reagents are among the topics discussed. It
is recommended that the analyst become thoroughly familiar with these publications.
5.2.1 General Inorganic Analyses
In general, AR-grade reagents and solvents are satisfactory for inorganic analyses. Primary
standard reagents must, of course, be used for standardizing all volumetric solutions.
Commercially prepared reagents and standard solutions are very convenient and may be
used when it is demonstrated that they meet the method requirements. All prepared
reagents must be checked for accuracy.
The individual methods specify the reagents that require frequent standardization, or other
special treatment, and the analyst must follow through with these essential operations. To
avoid waste, the analyst should prepare a limited volume of such reagents, depending on the
quantity required over a given period of time. Examples and brief discussions of the kind of
problems that occur are given under Paragraph 5.3, "Elimination of Determinate Errors".
As far as possible, distilled water used for preparation of reagent solutions must be free of
measurable amounts of the constituent to be determined. Special requirements for distilled
water are given in Chapter 3 of this manual and in individual method descriptions.
Compressed gases, such as oxygen and nitrogen, used for total organic carbon determination
may be of commercial grade.
5.2.2 Metals Analyses
All standards used for atomic absorption and emission spectroscopy should be of
spectroquality. It is recommended that other reagents and solvents also be of
spectroquality, although AR grade is sometimes satisfactory. Standards may be prepared by
the analyst in the laboratory or prepared, spectrographically standardized materials may be
purchased commercially. Standards required for determination of metals in water are not
generally available from the National Bureau of Standards.
Analytical reagent grade nitric and hydrochloric acids must be specially prepared by ,
distillation in borosilicate glass and diluted with deionized distilled water. All other reagents
and standards are also prepared in deionized water.
In general, fuel and oxidant gases used for atomic absorption can be of commercial grade.
Air supplied by an ordinary laboratory compressor is quite satisfactory, if adequate pressure
is maintained and necessary precautions are taken to filter oil, water, and possible trace
5-2
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metals from the line. For certain determinations, e.g., aluminum, reagent-grade nitrous
oxide is required.
5.2.3 Radiological Analyses
The great sensitivity of radioactive counting instruments requires that scintillation grade
reagents and solvents, or equivalent, be used for all radioactivity determinations. Some of
the reagents, for example, strontium carbonate and yttrium oxide carriers used for the
determination of strontium 90 and yttrium 90, must be stable, that is free of radioactivity.
Barium sulfate, used for coprecipitation of radium must be free from all traces of radium.
These reagents and solvents are commercially available from chemical supply houses.
Calibrated standard sources of specific radioactive materials with known count and date of
counting are available from various suppliers. No single company supplies all standards.
Gases used for radioactive counting must be of high purity and extra dry. Gases such as
helium and air are aged for about 30 days to allow radioactive background to decay. All
gases are checked for background before use. Some cylinders contain inherent radioactivity
which is imparted to the gas. When this background is above normal, the gas should not be
used for radioactivity determinations.
5.2.4 Organic Chemical Analyses
The minimum purity of reagents and solvents that can be used for organic analyses is AR
grade. Reference grade standards should be used whenever available. Special note should be
taken of the assay of standard materials. Owing to the great sensitivity (nanogram and
subnanogram quantities) of gas chromatography (GC), which is often used to quantitate
organic results, much greater purity is frequently required (6). The specificity of some GC
detectors requires that reagents and solvents be free of certain classes of compounds. For
example, analyses by electron capture require that reagents and solvents be free of
electronegative materials that would interfere with the determination of specific compounds
in the sample. Similarly, use of the flame photometric detector requires that reagents and
solvents be free from sulfur and/or phosphorus interference. Pesticide quality solvents are
available from several sources. These are often satisfactory for many organic GC
determinations. However, the contents of each container must be checked to assure its
suitability for the analyses. Similarly, all analytical reagents and other chemicals must also
be checked routinely.
The quality of gases required for GC determinations varies somewhat with the type of
detector. In general, the compressed gases are a prepurified dry grade. Grade A helium from
the U.S. Bureau of Mines has always been satisfactory. The Dphrmann nitrogen-detection
system requires the use of ultra-pure hydrogen for satisfactory results. The use of
molecular-sieve, carrier-gas filters and drying tubes is required on combustion gases. They
are recommended for use on all other gases. It is recommended that the analyst familiarize
himself with an article by Burke (7) on practical aspects of gas chromatography.
All reagents, solvents, and adsorbents used for thin-layer chromatography must be checked
to be certain that there are no impurities present that will react with the chromogenic
reagent or otherwise interfere with subsequent qualitative or quantitative determinations.
Glass-backed layers prepared in the laboratory or precoated layers supplied by a
manufacturer may be used. However, precoated layers are more difficult to scrape.
5-3
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Therefore, it is recommended that layers prepared in the laboratory be used when zones are
to be scraped in order to recover isolated compounds. Plastic-backed layers are generally
unsatisfactory for this type of analysis.
Adsorbents most commonly used for column chromatographic clean-up of sample extracts
are Florisil, silica gel, and alumina. These must be pre-activated according to the method
specifications and checked for interfering constituents.
l
5.3 Elimination of Determinate Errors
In order to produce high quality analytical data, determinate errors must be eliminated or at
least minimized. For purposes of this discussion, we assume that a competent analyst and
reliable equipment, in optimum operating condition, are available. Thus, determinate errors
that might result from an inexperienced or careless analyst and poor equipment are
eliminated. The remaining sources of error are the reagents, solvents, and gases that are used
throughout the analyses. The quality of these materials, even though they are AR grade or
better, may vary from one source to another, from one lot to another, and even within the
same lot. Therefore, the analyst must predetermine that all of these materials are free of
interfering substances under the conditions of the analyses. To do this he must have a
regular check program. Materials that do not meet requirements are replaced or purified so
that they can be used.
5.3.1 Reagent Blank
The first step the analyst must take is to determine the background or blank of each of the
reagents and solvents used in a given method of analysis. The conditions for determining the
blank must be identical to those used throughout the analysis, including the detection
system. If the reagents and solvents contain substances that interfere with a particular
determination, satisfactory reagents and solvents must be found. Where possible and
practical, they should be treated so that they can be used.
5.3.2 Method Blank
After determining the individual reagent or solvent blanks, the analyst must determine the
method blank to see if the cumulative blank interferes with the analyses. The method blank
is determined by following the procedure step by step, including all of the reagents and
solvents, in the quantity required by the method. If the cumulative blank interferes with the
determination, steps must be taken to eliminate or reduce the interference to a level that
will permit this combination of solvents and reagents to be used. If the blank cannot be
eliminated, the magnitude of the interference must be considered when calculating the
concentration of specific constitutents in the samples being analyzed.
A method blank should be determined whenever an analysis is made. The number of blanks
to be run is determined by the method of analysis and the number of samples being analyzed
at a given time. In some methods, such as the AutoAnalyzer procedures, the method blank
is automatically and continuously compensated for since a continuous flow of the reagents
passes through the detector. In other procedures, such as the gas chromatographic
determination of pesticides, a method blank is run with each series of samples analyzed.
Usually this is one blank for every nine samples.
5-4
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5.3.3 Elimination of Interferences and Other Sources of Error
Procedures for eliminating or at least minimizing impurities that produce specific
interferences or high general background, vary with the reagent and method involved. These
procedures may include: recrystallization, precipitation, distillation, washing with an
appropriate solvent, or a combination of these. Examples of procedures used for various
types of analyses are given below. For complete information, the analyst should consult the
individual methods.
5.3.3.1 General Inorganic Analyses
Analytical reagent grade chemicals and solvents usually present no interference problems in
inorganic analyses. However, some reagents do not always meet methods requirements. An
example is potassium persulfate .used in phosphorus and nitrogen determinations. This
reagent is frequently contaminated with ammonia. Therefore, it is routinely purified by
passing air through a heated water solution of the reagent. The purified potassium persulfate
is recovered by recrystallization.
A problem more commonly encountered in inorganic analyses is the rapid deterioration of
the standard reagents and other ingredients. To minimize or eliminate this problem some
reagents, for example, ferrous ammonium sulfate, must be standardized daily. Others, such
as sodium thiosulfate used for dissolved oxygen determination, may require a substitute
reagent, e.g., phenyl arsene oxide. Solid phenol which readily oxidizes and acquires a
reddish color can be purified by distillation. Starch indicator used for idiometric titrations
may be prepared for each use or preserved by refrigeration, or by addition of zinc chloride
or other suitable compounds.
5.3.3.2 Metals Analyses
In general, spectrograde chemicals, solvents, and gases present no interference problems in
atomic absorption or emission spectrographic determinations. However, standards which do
not meet the requirements of the method are sometimes obtained. Ordinarily, no effort is
made to purify them. They are simply replaced by new reagents of sufficient purity. Some
reagents may form precipitates on standing. Such reagents will reduce the accuracy of
quantitative analyses and should not be used.
5.3.3.3 Radiological Analyses
In general, reagents that do not meet the purity requirements for radiological
determinations are replaced with reagents that are satisfactory. However, in some instances
(for example, barium sulfate used for coprecipitation of radium), it may be necessary to
carry out repeated recrystallization to remove all forms of radium, and reduce the
background count to a useable level. In some instances, solvents that do not meet
requirements may be distilled to produce adequate purity. In some cases, gases having
background counts may be useable after aging as described earlier. If not, they should be
replaced with gases that are satisfactory.
5.3.3.4 Organic Analyses
Many AR-grade chemicals and solvents, and at times pesticide quality solvents, do not meet
the specifications required for the determination of specific organic compounds. Impurities
5-5
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that are considered trace, or insignificant, for many analytical uses, are often present in
greater quantities than the organic constituents being measured. Coupled with the
several-hundred-fold concentration of the sample extract that is usually required, such
impurities can cause very significant interferences in trace organic analyses. Reagents and
solvents found to be unsatisfactory, under the conditions of the analyses, must be replaced
or cleaned up so that they are useable. Some useful clean-up procedures are:
a. Washing the inorganic reagents with each solvent that the reagent contacts during
the analysis,
b. Washing the adsorbents, such as silica gel G and Florisil, with the solvents that are
used for a specified column or thin-layer chromatographic procedure,
c. Pre-extracting distilled water with solvents used for the particular analysis involved,
d. Pre-extracting aqueous reagent solutions with the solvents involved,
e. Redistilling solvents in all-glass systems using an efficient fractionating column,
f. Recrystallizing reagents and dyes used in colorimetric or thin-layer determinations,
If the reagents and solvents thus produced are not of sufficient purity, they should be
replaced.
Dirty gases (quality less than specified) are particularly troublesome in gas chromatographic
analyses. They may reduce the sensitivity of the detector, and produce a high or noisy
baseline. If this occurs, the cylinder should be replaced immediately. Similarly, if cylinders
of compressed gases are completely emptied in use, the end volumes of the gas may produce
a similar and often more severe effect. Oils and water may get into the system and foul the
detector. When this occurs the system must be dismantled and cleaned. Overhaul of the
detector may be required. To reduce chances of this, it is recommended that all gas
cylinders be replaced when the pressure falls to 100-200 psi. Filter driers are of little help in
coping with this type of contamination.
5.3.4 Storing and Maintaining Quality of Reagents and Solvents
Having carried out the tasks of selecting, preparing, and verifying the suitability of reagents,
solvents, and gases, the analyst must properly store them to prevent contamination and
deterioration prior to their use. Borosilicate glass bottles with ground glass stoppers are
recommended for most standard solutions and solvents. Plastic containers, e.g.,
polyethylene, are recommended for alkaline solutions. Plastic containers must not be used
for reagents or solvents intended for organic analyses. However, plastic containers may be
used for reagents not involved with organic analyses if they maintain a constant volume, and
it is demonstrated that they do not produce interferences and do not absorb constitutents
of interest. It is important that all containers be properly cleaned and stored prior to use.
(Refer to Chapter 4 for details).
Standard reagents and solvents must always be stored according to the manufacturer's
directions. Reagents or solvents that are sensitive to the light should be stored in dark
bottles and/or stored in a cool, dark place. It is particularly important to store materials
used for radiological determinations in dark bottles, since photoluminescence will produce
5-6
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high background if light sensitive detectors are used for counting. Some reagents require
refrigeration.
Adsorbents for thin-layer and column chromatography are stored in the containers that they
are supplied in, or according to the requirements of individual methods. Activated carbon,
used for collection of samples for organic analyses, must be stored and processed in areas
protected from atmospheric and other sources of contamination (8).
The analyst should pay particular attention to the stability of the standard reagents.
Standards should not be kept longer than recommended by the manufacturer, or in the
method. Some standards are susceptible to changes in normality due to absorption of gases
or water vapor from the air. Provisions for minimizing this effect are given in Part 23 of
ASTM Standards (4).
The concentration of the standards will change as a result of evaporation of solvent. This is
especially true of standards prepared in volatile organic solvents. Therefore, the reagent
bottles should be kept stoppered, except when actually in use. The chemical composition of
certain standards may change on standing. Certain pesticides, for instance, will degrade if
prepared in acetone that contains small quantities of water. Thus, it is essential that working
standards be frequently checked to determine changes in concentration or composition.
Stock solutions should be checked before preparing new working standards from them.
5.4 References
1. National Bureau of Standards, Special Publication 260, "Standard Reference Materials",
July 1969.
2. "Reagent Chemicals, American Chemical Society Specifications", American Chemical
Society, Washington, D.C.
3. ASTM Special Technical Publication No. 148-H, "Manual on Industrial Water and
Industrial Waste Water", 2nd Edition, p. 869 (1965).
4. 1968 Book of ASTM Standards, __Part 23; p. 897: American Society for Testing and
Materials, 1916 Race Street, Philadelphia, Pa. 19103.
5. Standard Methods for the Examination of Water and Wastewater, 13th Edition,
American Public Health Association, New York (1971).
6. "FWPCA Method for Chlorinated Hydrocarbon Pesticides in Water and Waste Water",
Federal Water Pollution Control Administration, Analytical Quality Control
Laboratory, November 1969.
7. Burke, J., JAOAC, 48, 1037 (1965).
8. Breidenbach, A. W., et al, "The Identification and Measurement of Chlorinated
Hydrocarbon Pesticides in Surface Waters", Publication WP-22, Federal Water Pollution
Control Administration, Washington, D.C. (November 1966).
5-7
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Chapter 6
CONTROL OF ANALYTICAL PERFORMANCE
6:1 Introduction
This chapter is limited to a discussion of the control of analytical performance in the
laboratory. It is assumed that a valid sample has been properly taken, preserved, and
delivered to the laboratory for analyses; that the laboratory analyses were done according to
currently-recognized methods; and that the recording and reporting of subsequent
laboratory results were done in a systematic, uniform, and permanent fashion (See Chapter
7). It must be recognized (and practiced!), however, that quality control begins with the
sample collection and does not end until the resulting data are reported. The laboratory
control of analytical performance is but one vital link in obtaining valid data. A continuous
rapport and conscientious use of quality control between field sampling, laboratory
analyses, and management decisions are necessary to insure this validity.
Earlier chapters have discussed such key elements as laboratory services, instrumentation,
glassware, reagents, solvents, and gases; the reader should refer to these sections to
determine the necessary specifications and requirements required for quality control. On the
assumption that these variables are under control, that a single method is being used, and
that the complete system is initially under control, what should be done in the evaluation of
daily performance to document that valid data are being produced? First, valid precision
and accuracy data should be available on the method and analyst. Thereafter, systematic
daily checks are required to show that reproducible results are being obtained, and that the
methodology is actually measuring what is in the sample. These items are discussed in detail
in the following sections.
6.2 Precision and Accuracy
Precision refers to the reproducibility among replicate observations. In an Analytical Quality
Control Program, it is determined, not on reference standards, but by the use of actual
water samples which cover a range of concentrations and a variety of interfering materials
usually encountered by the analyst. Obviously, such data should not be collected until the
analyst is thoroughly familiar with the method, and has obtained a reproducible standard
curve. For colorimetric analyses, the initial standard curve should include a blank and a
series of at least eight standards encompassing the full concentration range to be used for
routine sample analyses. Subsequently, at least two standards (a high and a low) should be
analyzed to verify the original standard curve. For other measurements, such as pH,
conductivity, turbidity, etc., instruments should be standardized according to
manufacturer's instructions (See Chapter 3) and sound, scientific practice.
There are a number of different methods available for the determination of precision. One
method that has been successfully employed by experienced AutoAnalyzer users, and can
be adapted to many other analytical instrumentation and chemical procedures, is described
as follows:
a. Four separate concentration levels should be studied, including a low concentration
near the sensitivity level of the method, two intermediate concentrations, and a
concentration near the upper limit of application of the method.
6-1
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b. Seven replicate determinations should be made at each of the concentrations tested.
c. To allow for changes in instrument conditions, the precision study should cover at
least two hours of normal laboratory operation.
d. In -order to permit the maximum interferences in sequential operation, it is
suggested that the samples be run in the following order: high, low, intermediate,
intermediate. This series is then repeated seven times to obtain the desired reph'ca-
tion.
e. The precision statement should include a range of standard deviations over the
tested range of concentration. Thus, four standard deviations will be obtained over
a range of four concentrations, but the statement should contain only the extremes
of standard deviations and concentrations studied.
An example of data generated from such an approach is shown in Table 6-1.
Table 6-1
PRECISION DATA ON RIVER WATER SAMPLES FOR PHOSPHORUS
AUTOANALYZER METHOD
Cone., (mg P/l)
Sample Kanawha Klamath Arkansas Big Souix
1
2
3
4
5
6
7
Avg.
s
0.05
0.06
0.06
0.06
0.06
0.06
0.06
0.059
0.004
0.10
0.10
0.10
0.11
0.11
0.11
-
0.105
0.005
0.48
0.48
0.49
0.48
0.48
0.48
-
0.482
0.004
0.62
0.62
0.62
0.63
0.62
0.62
0.62
0.621
0.004
The resulting precision statement would read as follows:
"In a single laboratory, using surface water samples at concentrations of 0.06 and 0.62
mg P/l, the standard deviation was ±0.004 (Analytical Quality Control Laboratory)."
Thus, the statement contains the number of laboratories involved, the type of samples, the
concentrations used, the resulting standard deviation (s) and the reference source.
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Accuracy refers to a degree of difference between observed and known, or actual, values.
Again, accuracy should be determined on actual water samples routinely analyzed, and
preferably, on the same series as those used in the precision determinations. The method
employed by experienced Auto Analyzer users consists of the following key steps:
a. Known amounts of the particular constituent should be added to actual samples at
concentrations where the precision of the method is satisfactory. It is suggested
that amounts be added to the low-concentration sample, sufficient to double that
concentration, and that an amount be added to one of the intermediate
concentrations, sufficient -to bring the final concentration in the sample to
approximately 75% of the upper limit of application of the method.
b. Seven replicate determinations at each concentration should be made.
c. Accuracy should be reported as the percent recovery at the final concentration of
the spiked sample. Percent recovery at each concentration should be the mean of
. the seven replicate results.
Data were obtained with this approach by using two of the water samples previously used in
the precision study reported in Table 6-1 (Kanawha and Arkansas Rivers). They are reported
in Table 6-2.
Table 6-2
ACCURACY DATA ON RIVER WATER SAMPLES FOR PHOSPHORUS
AUTOANALYZER METHOD
Cone., (mgP/1)
: Kanawha Arkansas
Sample (Added 0.06 mg/lP) (Added 0.3 mg/1 P)
1 0.105 0.74
2 0.105 0.75
3 0.105 0.75
4 0.110 0.73
5 0.110 0.74
6 0.110 0.75
7 0.105 0.75
Avg. 0.107 0.74
% Recovery T °-107 1 [ W "I
L 0.059 + 0.06 > 100 = 90 |_ 0.48 + 0.30 J ^ 100 = 95
6-3
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Again, in order to contain the key elements, the accuracy statement would read as follows:
"In a single laboratory, using surface water samples at concentrationsofO.il and 0.74
mg P/l, recoveries were 90% and 95%, respectively (Analytical Quality Control
Laboratory)".
Once collected and documented, these precision and accuracy data may be used in a number
of ways. Two important examples are: (1) They present clearcut evidence that the analyst in
question is indeed capable of analyzing the water samples for that particular parameter.
That is, he has the standard method under control, and is capable of generating valid data;
and (2) the data can be used in the evaluation of daily performance in reference to replicate
samples, spiked standards.and samples, and in the preparation of quality control charts.
As observed, the above methods can be adapted to other chemical procedures and analytical
instruments. They have been used on manual titration methods for such parameters as
alkalinity, chloride, and hardness; on general inorganic instruments such as pH,
conductivity, selective ion, and turbidity meters; and on the Beckman Carbonaceous
Analyzer. Other instruments, such as atomic absorption and flame emission
spectrophotometers, could also be evaluated by these methods; however, radiological
instrumentation and gas chromatography systems (See Chapter 8) require special
techniques.
6.3 Evaluation of Daily Performance
Once valid precision and accuracy data are available on the method and the analyst,
systematic daily checks are necessary to insure that valid data are being generated. First of
all, verification of the originally-constructed standard curve is mandatory. As previously
noted, at least two standards (a high and a low) should be analyzed routinely along with a
blank to determine that comparable operating conditions exist. If the data do not
substantiate such control, the analyst must systematically trouble-shoot his system until the
problem is corrected.
In order to document that reproducible results are being obtained (i.e., precision of the
method), it is necessary to run replicate samples. Although frequency of such replicate
analyses is, by nature, dependent on such factors as the original precision of the method, the
reliability of the instrumentation involved, and the experience of the analyst, good
laboratory technique is to run duplicate analyses at least ten percent of the time. The
resulting data should agree favorably with the known precision of the method. If they do
not, the system is not under control, and results are subject to question.
Concurrently, quality control should include assurance that the daily system is actually
measuring what is in the sample (i.e., accuracy of the method). Although it is far preferable
to have obtained values check with known or actual values, it should be recognized that
inaccuracy does not destroy the value of data if the degree and precision of the error is
known and taken into account. In order to account for background contamination and/or
sample interferences, and as a matter of routine practice, spiked samples should be used in
addition to standards. As in the case of duplicate sample analyses, good laboratory
technique dictates that spiked samples be run at least ten percent of the time.
Thus, daily control of analytical performance in the laboratory requires approximately
15-20 percent of the analyst's time. Considering the elapsed time and combined efforts of
6-4
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skilled personnel that are represented in a final laboratory result, this is a comparatively
small price to pay for, not a "number", but a valid concentration value.
A most convenient way of recording the obtained precision and accuracy data is through the
preparation of quality control charts. Plotting of said data systematically answers the
question as to whether the laboratory analyses are under control, and is useful in observing
developing trends of positive or negative bias. Because of its importance in documenting the
quality control being practiced daily in the laboratory, the construction and uses of quality
control charts are treated as a separate topic in the next section.
A broader and somewhat different form of evaluation of daily performance may be made
through routine participation in interiaboratory round-robin studies. Samples analyzed in
such a cooperative program should be treated as part of the routine sample load. In so
doing, the analyst is able to compare his individual performance against other laboratory
personnel, and to have a reliable measure of the particular method's capabilities. In many
respects such samples can be regarded as reputable "blind samples"; a necessary ingredient
in the quality control of laboratory results.
6.4 Quality Control Charts
Quality control charts were originally developed for the control of production processes
where large numbers of items were being manufactured and inspected on an essentially
continuous basis. As shown in Figure 6-1, a control chart consists of a graphical chart with the
vertical scale plotted in units of the test result and the horizontal scale in units of time or
sequence of results. The upper and lower control limits shown on the chart are used as
criteria for action, or for judging the significance of variations between duplicate samples.
The central line represents the average or the standard value of the statistical measure being
plotted.
"c3
Upper Control Limit
Central (Average)
Lower Control Limit
Time or Order of Results
Figure 6-1 ESSENTIALS OF CONTROL CHART
As observed in the previous section on the evaluation of daily performance, daily precision
and accuracy data can be plotted by means of these quality control charts to determine if
valid, questionable, or invalid data are being generated from day to day. There are several
techniques available for actually constructing quality control charts and plotting subsequent
data.JTwo currently in use are the Shewhart technique (1,2) and the CuSum technique (3).
In both techniques, precision control charts are constructed from duplicate sample
6-5
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analyses, whereas, accuracy control charts are constructed from spiked samples or
standards data generated in monitoring recovery efficiencies. At least 15 to 20 sets of
duplicate and 15 to 20 sets of spiked sample data from an in-control process are necessary
for the initial construction. A system is initially said to be in control when the standard
deviation and recovery efficiency data for a given parameter are comparable to those
obtained by other experienced laboratories. It is also necessary that the initial and
subsequent sets of data be obtained under normal laboratory operation conditions, that the
same analyst or group of analysts run the analysis, and that the same analytical method is
used.
6.4.1 Cumulative-Summation (CuSum) Quality Control Charts
There are various systems currently available for plotting data in the form of cumulative
sum charts (4). One system that has been in continuous use within EPA Region VI is that of
Harkins and Crowe (3). It has proved most useful in monitoring the validity of data
generated by a contracting laboratory and is currently being used routinely to daily record
intra-laboratory performance in technical operations. The following material has been
excerpted from their manual (3), in order to accurately describe the construction and use of
these charts:
6.4.1.1 Construction of CuSum Quality Control Charts
The control charts are derived from three basic calculations:
a. Standard deviations (Sd) of the differences between duplicates or, in the case of
spiked or standard samples, between the known quantity and the quantity
obtained.
b. The upper control limit (UL)
c. The lower control limit (LL)
Prior to these calculations, two decisions must be made:
a. The a and 0 levels
b. The allowable variability levels
Mathematical Equations
di2--
'd ITT
S2 = - = Variance of the differences
Sd = ~~\J Sd =. Standard deviation of the differences
S2, = (.8Sd)2 = .64 S2 (estimates a J )
S2 = (1.2Sd)2 =1.44 S2 (estimates a 2 )
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2 log.
UL(M) =
1 1
logp
1
S2
i
LL(M) =
21ogp
+M
K TH
_!__ 1
o2 e2
Where: UL(M)
LL(M)
di
N
§2
c2
= upper limit at M sets of samples
= lower limit at M sets of samples
= the difference between the i set of duplicates or spiked
samples
= the total number of sets of duplicates or spiked samples
used to construct the control charts
= minimum amount of variation allowed in the system
= maximum amount of variation allowed in the system
a = percent (decimal fraction) of time you are willing to judge
the procedure out of control when it is in control
j3 = percent (decimal fraction) of time you are willing to judge
the procedure in control when it is out of control
M = number of sets of duplicates or spiked samples used in
calculating the value to be plotted on the chart
By definition, a is the probability of judging the process to be out of control when in fact, it
is in control. It is recommended that a be chosen to lie between the boundaries of .05 and
.15, that is, the laboratory personnel are willing to stop the laboratory process somewhere.
between 5 and 15% of the time, judging it to be out of control, when in fact, it is in control.
If the cost of examining a process to determine the reason or reasons for being out of
control is considerable, then it may be desirable to choose a Iowa. Likewise, if the cost is
negligible, it may be desirable to choose a larger a value, and thus stop the process more
frequently. (See Figure 6-2)
6-7
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On the other hand, 0 is defined as the probability of judging the process to be in control
when it is not. Again, it is recommended that 0 be chosen to lie between the values of .05
and .15; thus, the laboratory personnel are willing to accept out of control data somewhere
between 5.and 15% of the time. The economic considerations used for choosing a are also
applicable to the choice of B. (See Figure 6-2.)
It is also essential to set maximum and minimum allowable variability levels. It is necessary
to specify a value for the minimum and maximum amount of variation that will be
allowable in the system. These minimum and maximum amounts are referred to as ° Q
and a j respectively. The values used should be based on a knowledge of the variation in
the procedure under consideration. However, if such knowledge is not available, the values
« 99 2
may be arbitrarily set at o = (a .20a) and a = (a + .20 a ) .
LABORATORY IDENTITY CONTROL CHART
PARAMETER - METHOD
DATE
RANGE OF CONCENTRATION
a and 0 LEVELS
STANDARD DEVIATION
UPPER CONTROL LIMIT EQUATION
LOWER CONTROL LIMIT EQUATION
Sample Set No. (M)
Figure 6-2. EFFECT OF a AND 0 LEVELS ON STANDARD CONTROL CHART
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6.4.1.2 Use of CuSum Control Charts
Once the control charts are constructed, and prior to their use, consideration must be given
to the number of duplicate analyses to be conducted during a series of samples; likewise, the
same decision must be made on spiked or standard samples.
In considering the number of duplicate and spiked sample analyses to be conducted in a
series of samples, it is necessary to weight the consequences when the data go out of
control. The consequences of this situation are reanalyzing a series of samples or discarding
the questionable data obtained. The samples to be reanalyzed are those lying between the
last in-control point and the present out-of-control point. A realistic frequency for running
duplicate and spiked samples would be every fifth sample; however, economic consideration
and experience may require more or less frequent duplicate and spiked sample analyses.
Once the frequency of duplicate and spiked samples has been determined, it is' then
necessary to prepare spiked or standard samples in concentrations relative to the
concentration of the control charts, which should be similar to those of the environmental
samples. These spiked or standard samples must be intermittently dispersed among the series
of samples to be analyzed and without the analyst's knowledge of concentration. Similarly,
duplicate samples must be intermittently dispersed throughout the series of samples to be
analyzed, and ideally, without the analyst's knowledge; however, this is sometimes very
difficult to accomplish.
The results of the duplicate and spiked sample analyses should be calculated immediately
upon analyzing the samples to allow for early detection of problems that may exist in the
laboratory. An example of these calculations follows:
Duplicate
Sample No. Results
M No. 1 No. 2 Difference (di) di2 S (di2)
1 5.4 5.2 .2 .04 .04
2 4.8 4.7 .1 . .01 .05
3 6.1 5.8 .3 .09 .14
Upon plotting the summation or 2(di2), one of three possibilities can occur (See Figure
6-3):
a. Out of control on the upper limit
When data goes out of control on the upper limit the following steps should be
taken:
1. Stop work immediately
2. Determine problems
(a) Precision control chart
(1) The analyst
(2) Nature of the sample
(3) Glassware contamination
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SAMPLE SET NO.
ANALYSIS IN CONTROL
NO PROBLEMS:
CONTINUE ANALYSIS
SAMPLE SET NO.
ANALYSIS OUT OF CONTROL
UPPER LIMIT
PROCEDURES:
1. STOP ANALYSIS
2. LOCATE PROBLEM
3. CORRECT PROBLEM
4. RERUN SAMPLES
5. START CHART AT SAMPLE
SET NO. 1.
SAMPLE SET NO.
ANALYSIS OUT OF CONTROL
LOWER LIMIT
INCREASED EFFICIENCY OR
FALSE REPORTING
PROCEDURES:
1. CONTINUE ANALYSIS
2. CONSTRUCT NEW CHART
WITH RECENT DATA
3. OBSERVE ANALYST
SAMPLE SET NO.
ANALYSIS OUT OF CONTROL
UPPER LIMIT
CONTINUOUS ERROR TREND
PROCEDURES:
SAME AS ABOVE BUT STOP
ANALYSIS WHEN TREND IS
DETECTED.
Figure 6-3. LABORATORY QUALITY CONTROL CHARTS
6-10
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(b) Accuracy control chart
(1) The analyst
(2) Glassware contamination
(3) Contaminated reagents
(4) Instrument problems
(5) Sample interference with the spiked material
3. Rerun samples represented by that sample set number, including additional
duplicate and spiked samples.
4. Begin plotting at sample No. 1 on chart.
b. In control within the upper and lower limit lines
When data continuously fall in between the upper and lower control limits, the
analyses should be continued until an out-of-control trend is detected.
c. Out of control on the lower limit
When data fall out of control on the lower limit, the following steps should be
taken:
1. Continue analyses unless trend changes
2. Construct new control charts on recent data
3. Check analyst's reporting of data
6.4.2 Shewhart Quality Control Charts
Dr, Walter A. Shewhart of Eel! Telephone Laboratories developed the basic theory of
control charts in the 1920's. His book on statistical quality control (1) grew out of this
original work. Since then, industrial acceptance of these control chart concepts and other
statistical techniques have refined and quantitated the quest for quality in manufacturing.
Although originally developed for control of production processes when large numbers of
articles were being manufactured and inspected on an essentially continuous basis, these
same concepts have been readily adapted to laboratory operations where the analyst
produces comparatively fewer results on an intermittent basis.
As in the CuSum approach, precision control charts are prepared from data resulting from
duplicate sample analyses and accuracy control charts from duplicate spiked standards or
samples. Once the control charts are constructed, however, data are plotted as individual
values rather than cumulative sums.
Certain constants (factors) are also involved in the preparation of Shewhart Charts.
Depending upon how the data are grouped, what the size of each grouping is, arid what
control limit formulation is being calculated, Table 6-3 will serve as a basic reference
point:
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Table 6-3
FACTORS FOR COMPUTING CONTROL CHART LINES (5, 6)
Observations in Factor Factor
Subgroup (n) A, D,
2 1.88 3.27
3 1.02 2.58
4 0.73 2.28
5 0.58 2.12
6 0.48 2.00
7 0.42" 1.92
8 0.37 1.86
Inherent in the Shewhart approach is recognition of the basic assumption that variations
exist in every method. That is, no procedure is so perfect, so unaffected by its environment,
that it will always give exactly the same assay value or product. Where such situations seem
to exist, either the device used to measure the process is not sensitive enough or the person
making the measurements is not performing properly. For our purposes, the recorded
difference between paired samples should never be less than one-half the minimum
detectable limit of the parameter under consideration. In the following outlines for
preparing precision and accuracy control charts, nitrate data were used to develop the
examples. Therefore the minimum values shown are 0.05 (one-half the observed minimum
detectable limit of 0.1 mg/1 as N).
6.4.3 Precision Control Charts
These charts are developed by collecting data for many samples, a minimum of 15 to 20,
run in duplicate under assumed controlled conditions. Once these data have been generated,
preferably over an extended period of laboratory time, the following steps should be
followed to construct the control chart:
a. List the range (R) for each set of samples. That is, the absolute value of the
difference between each set of duplicate samples.-
6-12
-------
Note: The following ranges were observed in the nitrate data:
0.1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.1
0.05
0.05
0.05
0.30
0.10
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
b. Calculate the average range (R) by summing the list of R values and dividing by the
number of sets of duplicates:
SR
R = ij _
R =^11- 0.06
35
c. Calculate the Upper Control Limit (UCL) on the range according to the formula:
UCLR = D4R,
where D4 is a constant dependent on the number of units in the subgroup. In this
case, since two observations are in the subgroup, d4 = 3.27 (see Table 6-3).
UCLR = 3.27 R = 0.20
d. Calculate the Upper Warning Limit (UWL) on the range according to the formula:
UWLR = 2/3 (D4R-R)+R,
which for duplicate samples reduces to
UWLR = 2.51 R
UWLR = 0.15
This UWL corresponds to the 95% confidence level.
6-13
-------
e. Now graph R, UWLR, and UCLR in the following manner:
R
0.3
0.2
0.1
0
UCLR = 0.20
UWLR= 0.15
R = 0.06
12345
Order of Results
(e.g., duplicate sample sets)
f. The above precision control chart for nitrates is now complete, and can be used to
plot R values on subsequent duplicate samples to determine if the system is in
control, out of control (plotted R value beyond the UCL), and/or to detect any
trends developing within the system.
1. In this example, a trend has developed between duplicate sample sets 4 thru 7.
Although the system is not out of control, all variables in the procedure should
be checked in an attempt to stop this obvious trend before the UCL is reached.
R
0.3
0.2
0.1
X
UCLR = 0.20
UWLR = 0.15
X
R = 0.06
XXX
345678
Order of Results
10
2. In this example, the system has clearly gone out of control between duplicate
samples 3 and 4. At this point, the system can be stopped and all variables in
the system checked, or another set of duplicate samples can be run to verify
the observed difference. Once the system has been corrected, all samples
between set 3 and 4 should be rerun to insure the validity of the data.
6-14
-------
0.3
0.2
0.1
0
X
UCLR =
UWLR =
R =
0.20
0.15
0.06
X X
12345678
Order of Results
6 .4.4 Accuracy Control Charts
As in the above system, these charts are developed by collecting data for many samples, a
minimum of 15 to 20, but on spiked samples (preferably) or standards under assumed
controlled conditions. Again, these data should be generated over an extended period of
laboratory time, and be representative of normal operating conditions (7). The following
steps should be followed to construct accuracy control charts:
a. List the range (R) and the average (X)* of each subgroup of data.
Note: In the following example of nitrate data, subgroups of monthly data
involving four observations were used:
Month
Sept.
Nov.
Dec.
Jan.
Actual
1.1
1.1
1.1
1.1
1.1
1.1
1.2
1.2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
*X=
Found - Actual
Found
1.1
1.1
1.1
1.1
1.1
1.1
1.2
1.1
1.1
1.0
1.0
1.0
0.9
- 1.0
1.1
1.0
n
R
0
0.1
0.1
0.2
0
-0.025
+0.025
0
6-15
-------
b. Calculate the average range (R) by summing the list of R values and dividing by the
number of subgroups:
R = 0,4 = 0.10
c. Calculate the Upper Control Limit (UCL) on the range according to the formula:
UCLR = D4R,
where D4 is a constant dependent on the number of units in the subgroup. In this
, case, since four observations are in the subgroup, D4 = 2.28 (see Table 6-3).
UCLR = 2.28 R = 0.23
d. Calculate the Upper Warning Limit (UWL) on the range according to the formula:
UWLR = 2/3(D4R-R)+R
UWLR = 2/3 [2.28 (O.l)-0.1J+0.1 =0.19
e. Now graph R, UWLR, and UCLR in the following manner:
R
0.3
0.2
0.1
0
UCLR = 0.23
UWLR = 0.19
R=0.1
Order of Results
f. Turning now to the X values, calculate the UCL by the formula:
x = A2R
where A2 is a constant dependent on the number of units in the subgroup. In this
case, since four observations are in the subgroup, A2 = 0.73 (see Table 6-3).
= 0.73R = 0.07
6-16
-------
g. Calculate the UWLx by the formula:
= 2/3 A2(R)
= 2/3 [ 0.73 (0.1)] = 0.05
Note: Lower Warning Limit (LWLy) and Lower Control Limit (LCL^) are simply
the negative values of UWLx and UCL^, respectively.
h. Now graph the standard Nominal Value (set equal to zero),
x, and LCL^ in the following manner:
, and
.07
.05
.03
.01
-.01
-.03
-.05
-.07
= 0.05
Std Nominal Value
4 5
order of subgroups
= 0.05
= 0.07
In order to detail any trends forming within each subgroup, individual differences
may be plotted by preparing the following graph:
+.2
+.1
0
-.1
-.2
1
1
1 1
1 1
1
1
Nominal
subgroup
Individual value
j. Thus, as in the precision control charts, once all of the above accuracy control
charts have been constructed, all future data can be plotted on each set of
duplicate spiked samples or standards, to determine if the system is in control, out
of control, and/or to detect any trends developing within the system.
6-17
-------
6.5 References
1. Shewhart, W. A., Economic Control of Quality of Manufactured Product, 1931.
2. Anon., "Statistical Method-Evaluation and Quality Control for the Laboratory",
DHEW Training Course Manual in Computational Analysis, August 1968.
3. Anon., "Laboratory Quality Control Manual", Federal Water Pollution Control
Administration, Robert S. Kerr Water Research Center, 1969.
4. Griffin, D. F., "Systems Control by Cumulative Sum Method", Amer. J. Med. Tech.,
34,644(1968).
5. Duncan, A. J., Quality Control and Industrial Statistics, 3rd Ed., R. D. Irwin, Inc.,
Homewood, 111., Chap. 18 (1965).
6. ASTM Special Technical Publication No. 1'5-C, "Manual on Quality Control of
Materials", pp. 59-64, January 1951.
7. Grant, E. G., Statistical Quality Control, 3rd Ed., McGraw-Hill, New York (1964).
6-18
-------
CHAPTER 7
DATA HANDLING AND REPORTING
7.1 Introduction
To obtain meaningful data on water quality, the laboratory must first collect a
representative sample and deliver it unchanged for analysis. The analyst must then complete
the proper analysis in the prescribed fashion. Having accomplished these steps, one other
important step must be completed before the data are of use. This step includes the
permanent recording of the analytical data in meaningful, exact terms, and reporting it in-
proper form to some storage facility for future interpretation and use.
The brief sections that follow discuss the data value itself, recording and reporting the value
in the proper way, means of quality control of data, and storage and retrieval.
7.2 The Analytical Value
7.2.1 Significant Figures
The term significant figure is used rather loosely to describe some judgment of the number
of reportable digits in a result. Often the judgment is not soundly based and meaningful
digits are lost or meaningless digits are accepted.
Proper use of significant figures gives an indication of the reliability of the analytical
method used. The following definitions and rules are suggested for retention of significant
figures:
A number is an expression of quantity. A figure or digit is any of the characters 0, 1,2, 3, 4,
5, 6, 7, 8, 9, which, alone or in combination, serves to express a number. A significant figure
is a digit that denotes the amount of the quantity in the place in which it stands.
Reported values should contain only significant figures. A value is made up of significant
figures when it contains all digits known to be true and one last digit in doubt. For example,
if a value is reported as 18.8 mg/1, the "18" must be firm values while the "0.8" is
somewhat uncertain and may be "7" or "9".
The number zero may or may not be a significant figure:
a. Final zeros after a decimal point are always significant figures. For example, 9.8
grams to the nearest mg is reported as 9.800 grams.
b. Zeros before a decimal point with other preceding digits are significant, With no
other preceding digit, a zero before the decimal point is not significant.
c. If there are no digits preceding a decimal point, the zeros after the decimal point
but preceding other digits are not significant. These zeros only indicate the position
of the decimal point.
7-1
-------
d. Final zeros in a whole number may or may not be significant. In a conductivity
measurement of 1000 f/mhos/cm, there is no implication that the conductivity is
1000 ± 1 fimho. Rather, the zeros only indicate the magnitude of the number.
A good measure of the significance of one or more zeros before or after another digit is to
determine whether the zeros can be dropped by expressing the number in exponential form.
If they can, the zeros are not significant. For example, no zeros can be dropped when
expressing a weight of 100.08 grams in exponential form; therefore the zeros are significant.
However, a weight of 0.0008 grams can be expressed in exponential form as 8 x 10"4 grams,
and the zeros are not significant. Significant figures reflect the limits of the particular
method of analysis. It must be decided beforehand whether this number of significant digits
is sufficient for interpretation purposes. If not, there is little that can be done within the
limits of normal laboratory operations to improve these values. If more significant figures
are needed, a further improvement in method or selection of another method will be
required to produce an increase in significant figures.
Once the number of significant figures is established for a type of analysis, data resulting
from such analyses are reduced according to set rules for rounding off.
7.2.2 Rounding Off Numbers
Rounding off of numbers is a necessary operation in all analytical areas. It is automatically
applied by the limits of measurement of every instrument and all glassware. However, it is
often applied in chemical calculations incorrectly by blind rule or prematurely, and in these
instances, can seriously affect the final results. Rounding off should normally be applied
only as follows:
7.2.2.1 Rounding-Off Rules
a. If the figure following those to be retained is less than 5, the figure is dropped, and
the retained figures are kept unchanged. As an example: 11.443 is rounded off to
11.44.
b. If the figure following those to be retained is greater than 5, the figure is dropped,
and the last retained figure is raised by 1. As an example: 11.446 is rounded off to
11.45.
c. When the figure following those to be retained is 5, and there are no figures other
than zeros beyond the 5, the figure is dropped, and the last place figure retained is
increased by 1 if it is an odd number, or it is kept unchanged if an even number. As
an example: 11.435 is rounded off to 11.44, while 11.425 is rounded off to 11.42.
7.2.2.2 Rounding Off Single Arithmetic Operations
a. Addition: When adding a series of numbers, the sum should be rounded off to the
same numbers of decimal places as the addend with the smallest number of places.
However, the operation is completed with all decimal places intact and rounding off
is done afterward. As an example:
11.1
11.12
11.13
33.35 The sum is rounded off to 33.4.
7-2
-------
b. Subtraction: When subtracting one number from another, rounding off should be
completed before the subtraction operation, to avoid invalidation of the whole
operation.
c. Multiplication: When two numbers of unequal digits are to be multiplied, all digits
are carried through the operation, then the product is rounded off to the number of
significant digits of the less accurate number!
d. Division: When two numbers of unequal digits are to be divided, the division is
carried out on the two numbers using all digits. Then the quotient is rounded off to
the number of digits of the less accurate of the divisor or dividend.
e. Powers and Roots: When a number contains n significant digits, its root can be
relied on for n digits, but its power can rarely be relied on for n digits.
7.2.2.3 Rounding Off the Results of a Series of Arithmetic Operations
The rules for rounding off are reasonable for simple calculations, however, when dealing
with two nearly equal numbers, there is a danger of loss of all significance when applied to a
series of computations which rely on a relatively small difference in two values. Examples
are calculation of variance and standard deviation. The recommended procedure is to carry
several extra figures through the calculation and then to round off the final answer to the
proper number of significant figures.
7.2.3 Glossary of Terms
To clarify the meanings of reports and evaluations of data, the following terms are defined.
They are derived in part from American Chemical Society and American Society for Quality
Control usage (1, 2).
7.2.3.1 Accuracy Data
Measurements which relate to the difference between the average test results and the true
result when the latter is known or assumed. The following measures apply:
Bias is defined as error in a method which systematically distorts results. The term
is used interchangeably with accuracy in that bias is a measure of inaccuracy.
Relative error is the mean error of a series of test results as a percentage of the true
result.
7.2.3.2 Average
In ordinary usage, the arithmetic mean. The arithmetic mean of a set on ji^values is the sum
of the values divided by n.
7.2.3.3 Characteristic
A property that can serve to differentiate between items. The differentiation may be either
quantitative (by variables), or qualitative (by attributes).
7-3
-------
7.2.3.4 Error
The difference between an observed value and its true value.
7.2.3.5 Mean
The sum of a_series of test results divided by the number in the series. Arithmetic mean is
understood (X).
7.2.3.6 Population
Same as Universe. (See subparagraph 7.2.3.13).
7.2.3.7 Precision
Degree of mutual agreement among individual measurements. Relative to a method of test,
precision is the degree of mutual agreement among individual measurements made under
prescribed, like conditions.
7.2.3.8 Precision Data
*
Measurements which relate to the variation among the test results themselves, i.e., the
scatter or dispersion of a series of test results, without assumption of any prior information.
The following measures apply:
a. Standard Deviation (a). The square root of the variance.
n
a =
b. Standard Deviation, estimate of universe (s).
n- 1
c. Coefficient of Variance (V)._ The ratio of the standard deviation (s) of a set of
numbers, n, to their average, X, expressed as a percentage:
d. Range. The difference between the largest and smallest values in a set.
e. 95% Confidence Limits. The interval within which one estimates a given population
parameter to lie, 95% of the time.
7-4
-------
7.2.3.9 Sample
A group of units, or portion of material, taken from a larger collection of units, or quantity
of material, which serves to provide information that can be used as a basis for judging the
quality of the larger quantity as a basis for action on the larger quantity or on the
production process. Also used in the sense of a "sample of observations."
7.2.3.10 Series
A number of test results which possess common properties that identify them uniquely.
7,2,3.11 Skewness(k)
A measure of the lopsidedness or asymmetry of a frequency distribution defined by the
expression:
(Xj - X)3
na3
This measure is a pure signed number. If the data are perfectly symmetrical, the skewness is
zero. If k is negative, the long tail of the distribution is to the left. If k is positive, the long
tail extends to the right.
7.2.3.12 Unit
An object on which a measurement or observation may be made.
7.2.3.13 Universe
The totality of the set of items, units, measurements, etc., real or conceptual, that is under
consideration.
7.2.3.14 Variable -
A term used to designate a method of testing, whereby units are measured to determine, and
to record for each unit, the numerical magnitude of the characteristic under consideration.
This involves reading a scale of some kind.
7.3 Report Forms
The analytical information reported should include the parameter, the details of the analysis
such as burette readings, absorbance, wavelength, normalities of reagents, correction factors,
blanks, and finally, the reported value.
To reduce errors in manipulation of numbers, a good general rule is to keep data
transposition to an absolute minimum. If this were pursued, the ideal report form would
include all preliminary information of the analysis, yet it would be possible to use the same
form through to the final reporting of data into a computer or other storage device.
However, the ideal report form is not usually in use. Rather, a variety of methods are used to
record data. They are:
7-5
-------
7.3.1 Loose Sheets
Reporting of data onto loose or ring-binder forms is an older, but much used means of
recording data. It does allow easy addition of new sheets, removal of older data, or
collection of specific data segments. However, the easy facility for addition or removal also
permits easy loss or misplacement of sheets, mix-ups as to date sequence, and questionable
status in formal display, or for presentation as evidence.
7.3.2 Bound Books
An improvement in data recording is use of bound books which force the sequence of data
insertion. Modification beyond a simple lined book improves its effectiveness with little
additional effort. Numbering of pages encourages use in sequence and aids also in
referencing data, through a table of contents, according to time, type of analysis, kind of
sample, analyst, etc.
Validation can be easily accomplished by requiring the analyst to date and sign each analysis
on the day completed. This validation can be strengthened further by providing space for
the laboratory supervisor to sign off as to the date and acceptability of the analysis.
A further development of the bound notebook is the commercially available version
designed for research-type work. These note books are preprinted with book and page
numbers and spaces for title of project, project number, analyst signature, witness signature
and dates. Each report sheet has its detachable duplicate sheet which allows for up-to-date
review by management without disruption of the book in the laboratory. The cost is about
four times that of ordinary notebooks.
Use of bound notebooks is essentially limited to research and development work where an
analysis is part of a relatively long project, and where the recording in the notebook is the
prime disposition of the data until a status or final report is written.
7.3.3 Pre-Printed Report Forms
Most field laboratories or other installations doing repetitive analyses for many parameters
day in and day out, develop their own system of recording and tabulating laboratory data.
This may include bound notebooks; but a vehicle for forwarding data is also required. In
many instances, laboratory units tailor a form to fit a specific group of analyses, or to report
a single type of analysis for series of samples, with as much information as possible
preprinted to simplify use of the form. With loose-sheet multicopy forms (use of carbon or
NCR paper) information can be forwarded daily, weekly, or on whatever schedule is
necessary, while allowing retention of all data in the laboratory. Still, the most common
record is an internal bench sheet, or bound book, for recording of all data in rough form.
The bench sheet or book never leaves the laboratory but serves as the source of information
for all subsequent report forms (See Figure 7-1).
In most instances the supervisor and analyst wish to look at the data from a sample point in
relation to other sample points on the river or lake. This review of data by the supervisor,
prior to release, is a very important part of the laboratory's quality control program;
however, it is not easily accomplished with bench sheets. For this purpose, a summary sheet
can be prepared which compares a related group of analyses from a number of stations. An
example is shown in Figure 7-2. Since the form contains all of the information necessary for
7-6
-------
Figure 7-1. EXAMPLE OF BENCH SHEET
NL-C-88
(7-68)
Spectrographic Analyses Bench Data
Sample #__ Date Source.
ml. cone, to
Test Count
Sec
TDS.
ml. Factor
1.
2.
3.
Count
1. Zn
2. Cd
3. As
4: R
5. P
6. Fe
7 Mo
8. Mn
Q A1
10. Re,
11. Pii
12. Ag
n Mi
14. Co
15. Ph
16. Cr
17 V
18. Ba
19. Sr
Rerun Count
PPM (^g/l)PPB
Av. In Cone. Less Orig.
Count Sample Than Sample
|
1
1
r~
r~
i
i
rn
n
i
n
rn
n
n
i
*
l
i
L
i
1
I
, 1
^^^j
i
i
1
i
,
,
i
i
k
H
1
-------
Figure 7-2. EXAMPLE OF SUMMARY REVIEW SHEET
Table 2. MINERALS ANALYSES OF ZONE B, OHIO RIVER SAMPLES, CONC., mg/1.
STATION
Ohio at Ironton
Ohio at Greenup Dam
Ohio at Portsmouth
Scioto at Lucasville
Ohio at Maysville
Ohio at Meldahl Dam
Little Miami at Cincinnati
Ohio at Cincinnati
Licking at 12th Street
Ohio at Miami Fort
Ohio at Markland Dam
Kentucky at Dam I
Ohio at Madison
Great Miami at Eldean
Great Miami at Sellars Road
Great Miami at Liberty-
Fairfield Road
Great Miami at American
Materials Bridge
Whitewater at Suspension
Great Miami at Lawrenceburg
(Lost Bridge)
Storet
Number
200152
200001
200139
381710
200153
383070
380090
380037
200523
383072
200521
200522
1 74304
383047
383015
383007
383071
Date,
1969
Alkalinity
Hardness
Chloride
Sulfate
Fluoride
SOLIDS
Total
Diss.
Susp.
-------
reporting data it is used also to complete the data forms forwarded to the storage and
retrieval system.
The forms used to report data to data storage systems require a clear identification of the
sample point, the parameter code, the type of analysis used, and the reporting terminology.
Failure to provide the correct information can result in rejection of the data, or insertion in
an incorrect parameter. As a group of analyses is completed on one or more samples, the
values are reported in floating decimal form, along with the code numbers, for identifying
the parameter and the sampling point (station). Figure 7-3 shows an example of a preprinted
report form for forwarding data to keypunch.
7.3.4 Digital Read-out
Instrumental analyses, including automated, wet-chemistry instruments, such as Technicon
AutoAnalyzer, atomic absorption spectrophotometer, pH meter, selective electrode meter,
etc., now can provide direct digital readout of concentration, which can be recorded directly
onto report sheets without further calculation. Electronics manufacturers now produce
computer-calculators that will construct best-fit curves, integrate curves, and/or perform a
pre-set series of calculations required to obtain the final reported value for recording by the
analyst.
7.3.5 Key Punch Cards and Paper Tape
Since much of the analytical data generated in laboratories is recorded on bench sheets,
transferred to data report forms, key-punched, then manipulated on small terminal
computers, or manipulated and stored in a larger data storage system, there is a built-in
danger of transfer error. This increases with each transposition of data. It is suggested that
the analyst can reduce this error by recording data onto punch cards directly from bench
sheets. The cards can be retained, or forwarded immediately to the data storage system as
desired. IBM now offers a small hand-operated key-punch for this purpose.
It is anticipated that in future water quality systems, the intermediate report sheets will be
eliminated and the data will be punched automatically by the analytical instrument system
onto key-punch cards and/or paper tapes for direct use as computer input.
7.4 STORET-Computerized Storage and Retrieval of Water Quality Data
The use of computers with their almost unlimited ability to record, store, retrieve, and
manipulate huge amounts of data is a natural outgrowth of demands for meaningful
interpretation of the great masses of data generated in almost any technical activity.
In August 1961, an informal conference was held in the Basic Data Branch, Division of
Water Supply and Pollution Control, U.S. Public Health Service. A number of ideas were
brought together in the basic design of a system for storage and retrieval of data for water
pollution control, called STORET. In 1966, the STORET system was transferred, with the
Division, into the Federal Water Pollution Control Administration, U.S. Department of the
Interior. A refinement of this system is now operated by the Technical Data and
Information Branch, Division of Applied Technology, EPA.
If properly stored, the data can be retrieved according to the point of sampling, the date,
the specific parameters stored, etc., or all data at a sample point or series of points can be
7-9
-------
Figure 7-3. EXAMPLE OF STORE? REPORT FORM
WATER QUALITY DATA
LABORATORY BENCH DATA
STATION CI'<'GNATION
DATI O^ IAMPLB
YM. MO. DAY
P'TEOI COMPOSITE »AM»LC
ITEM
1
ITEM.
Fecal CoUform
UM1T MF/100
tit i ttoooooeo
. i 1 II
Fecal Streptococci
M ( 1 1
ITEM NH3-N + Org.N
ITEM
ITEM
II II
NHj-N
II II
NO2-N + NO3-N
II II
P, Total
ITEM
,
ITEM
ITEM
i
rr
ITEM.
II II
P, Soluble
i i i i i i
II II
TOC
II 111
Phenol
i i i « I i i
|ll II
Cyanide
II II
1 i 1
UM1T MF/100
Ml II
UNIT mg/1
III 1
uy.-, mg/1
III II
UNIT mg/1
III II
UNIT mx/1
III II
UNIT mg/1
o o e e « o e
M 1 1 1
UNIT mx/1
III II
UNIT UK/1
III III
UNIT mg/1
III II
COMPUTER CODED DATA
STATION CODE MKIAL YR. MO. 0»Y
ii ; i ;
: i :
PARAMETER CODE VALUE EXPONENT Pu*s
3 , 6 , UC m DDD
l»*a» 14-17 2* 2« I
3 1 6|7 9|| | || || I
0 0 6|3 5| || || || |
0 0 6|, 0 | | || |f 1
o 037311 IE DDDD
NEXT CARD . REPEAT COLUMNS 1-11 ABOVE
0 0 6|6 5| || || || I
IM» «»» at a> -c
0 0 6|6 6|| | | || || |
0 0|6|8 0|| | | || II 1
3 2|7|3 0|| | || || || |
COLUMN HO IBLAN-l
, .UT. DDDD
T-TI TI-T» T« . 77 ?* ?»
7-10
-------
extracted as a unit.
There is a State/Federal cooperative activity which provides State water pollution control
agencies with direct, rapid access into a central computer system for the storage, retrieval,
and analysis of water quality control information.
Full details on use of the STORET system are given in the STORET handbook recently
revised (3).
7.4 SHAVES-A Consolidated Data Reporting and Evaluation System
Information systems have been developed to bridge the gap between the analyst and his raw
data, and a complex data storage and control system. These systems include preprinted
report forms, computerized verification, and evaluation of data and data storage. An
example is the SHAVES system.
The term, SHAVES, is an acronym for "Sample Handling and Verification System," which
originated at the Great Lakes-Illinois River Basin Comprehensive Project Laboratory at
Grosse Isle, Michigan. Although the system's original purpose was verification of the
calculations following laboratory analyses, it now includes data storage, checks for
completeness and consistency of data, procedures for submitting analytical requests, a set of
forms for recording sampling and analytical information, and a clerical procedure to account
for analyses completed and pending. The primary purposes of SHAVES are the
standardization, automation and control of reporting analyses. All samples received at the
Pacific Northwest Water Laboratory for routine analysis are processed through the system.
Although SHAVES uses a computer to perform its operations, it is not primarily a computer
program. It is intended for use as an intra-laboratory quality control tool, and as such
compliments the STORET system. It is described in detail-elsewhere (4).
7.6 References
1. "Guide for Measure of Precision and Accuracy," Anal. Chem., Vol. 33, p. 480, (1961).
p. 480.
2. "Glossary of General Terms Used in Quality Control," Quality Progress, Standard
Group of the Standards Committee, ASQC, II, (7), pp. 21-2, (1969).
3. Water Quality Control Information System (STORET), EPA, Washington, D.C. 20460,
Nov. 15, 1971.
4. Byram, K. V. and Krawczyk, D. F., "An Evaluation of SHAVES: A Water Quality
Sample Handling System," Environmental Protection Agency, Pacific Northwest Water
Laboratory, 1969.
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CHAPTER 8
SPECIAL REQUIREMENTS FOR TRACE ORGANIC ANALYSIS
8.1 Introduction
The high sensitivity of the instrumentation used in trace organic chemical analysis, and the
low concentration of compounds being investigated, dictate that special attention be given
this field of analytical endeavor. Contamination of the sample from any possible source
must be diligently guarded against, and interferences in the sample must be carefully
controlled. Finally, strict attention to method and highly refined technique are required to
produce valid quantitative results.
8.2 Discrete Bottled Samples
Sample collection should be done with wide-mouth glass bottles, equipped with screw caps
fitted with Teflon liners. The use of a screw cap without a Teflon liner may cause
contamination of the sample by the liner or adhesive used in sealing the liner to the cap.
Plastic bottles (polyethylene) are not used because traces of plasticizer may be leached from
the plastic by the water, and can be a source of analytical interference. Moreover, organics
from the sample may be adsorbed on the plastic. It has been suggested that high grade
Teflon bottles may be satisfactory for this use; however, the cost is prohibitive at present.
Many investigators avoid the use of glass sample bottles, because breakage in shipment
frequently causes loss of sample. This is overcome by the use of relatively inexpensive,
expanded polystyrene foam shipping containers molded to fit the bottle. These shipping
containers can be purchased from Preferred Plastics Corp., North Grosvenordale,
Connecticut.
To insure freedom from organic contaminants, bottles are rinsed successively with chromate
cleaning solution, running tap water, distilled water, and finally several times with redistilled
solvent (e.g., acetone, hexane, petroleum ether, chloroform). Caps are washed with
detergent, rinsed with tap water, distilled water, and solvent. Liners are treated in the same
way as the bottles and are stored in a sealed container.
Each method designates a recommended sample size for surface water analysis. Duplicate
samples are recommended. If analysis by more than one method is to be requested on the
same sample, sufficient sample must be simultaneously obtained to supply the needs of each
analysis.
It is also recommended that when requesting a non-specific analysis, any information that
could help direct the analytical approach, or aid in interpretation of results, be supplied.
Such information could include industrial or agricultural activities in the area from which
the sample was obtained, spills, or other accidents that may have occurred in the area. Also
mention of similar upstream activity could provide valuable assistance.
Samples should be stored in a cool, dark place, and analyzed as soon as possible. If the
sample cannot be analyzed immediately, reporting the holding time can help in interpreting
results where die-off rates are known.
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8.3 Carbon Adsorption Samples
The affinity of carbon for organic substances requires that supplies of carbon be protected
from extraneous sources of contamination. For example, carbon can adsorb organic
substances such as paint vehicles and insecticides from the air. Therefore, the carbon is
stored and processed in an area adequately protected from such sources of contamination.
As an additional precaution, the ventilating, heating, and air-conditioning systems for the
laboratories in which carbon adsorption samples are processed are completely isolated from
all other laboratories. All carbon is obtained from the manufacturer in sealed metal drums.
Obviously, spraying with pest-control chemicals is not permitted in these areas. Carbon
blank determinations supplement these precautions.
8.4 Glassware
Proper calibration of volumetric glassware is essential to valid analytical results, because
quantitation is performed by comparison to measured amounts of standard compounds, and
by accurate measurement of sample volume.
Individual concentrator tubes, used to measure final concentrate volumes, must first be
calibrated at the working volume. This is particularly important for volumes less than 1 ml.
Calibration should be made by noting the number of microliters of solvent required to bring
the liquid level (lower miniscus) up to a particular graduation mark. A precision 100 /zl
syringe should be used to measure calibration volume.
It is also very important in trace organic analysis that glassware be as free as possible of any
organic contaminants. A chromic acid cleaning solution is required for removing all traces of
organic material from glassware.
8.5 Reagents and Chemicals
The minimum purity of reagents and chemicals should be analytical reagent grade.
Analytical standards should be reference grade, when available. The analyst should take
special note of the assay of less pure materials (most often pesticides). All reagents and
chemicals should be stored according to manufacturer's instructions to prevent degradation.
Proper storage is especially important if the chemical is to be used in preparing an analytical
standard. Refrigerated chemicals should be allowed to come to room temperature before
exposing them to the atmosphere.
When preparing stock solutions, it is recommended that at least 0.100 gram of material be
used for greater accuracy in weighing. Solutions should be carefully stored so as to preserve
their concentration, and to protect them from ultraviolet radiation. Usually storage in
ground-glass stoppered bottles, either amber-colored, or out of the line of direct lighting, is
sufficient.
Standard solutions should be prepared using precision syringes, preferably equipped with a
Cheney adapter, to measure the volume of stock solution to be diluted. The syringe barrel
should be pre-wetted with solvent and air bubbles expelled. Dilution should be done in a
Class A volumetric flask to insure accurate measurement. If these solutions are to be used
frequently, they are best stored in a screw-cap, septum, sealed vial. These vials allow instant
access to the solution and offer good protection against concentration changes of the
standard solution. Evaporation of the solvent caused by repeated removal of the cap is a
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serious problem with other containers. If septum vials are not available it is advisable to
prepare standard solutions in a volumetric container of 100 ml or more and transfer a small
portion to a separate container for daily use, then discard that portion at the end of the day.
All stock or standard solutions should be carefully watched for signs of changes in
concentration or deterioration. As an aid to monitoring these solutions, it is wise to label
them as to compound, concentration, solvent used, date, and preparer. Also, in the case of
GC solutions, it is necessary to retain some evidence of its chromatographic behavior as a
fresh solution for comparison at a later date.
Distilled water used as dosed, control samples must be free of organic interferences. A very
effective way of removing organic interferences from distilled water is to pre-extract the
water with the solvent that is to be used in the analysis, then boil the water to remove the
residual solvent.
Organic solvents used in pesticide analysis should be pesticide quality, and demonstrated to
be free of interferences in a manner compatible with whatever analytical operation is to be
performed. Solvents can be checked by analyzing a volume equivalent to that used in the
analysis and concentrated to the minimum final volume. Possible interferences are noted in
terms of factors such as relative retention times, peak geometry, peak intensity, and width
of solvent response. Interferences noted under these conditions can be considered
maximum. If necessary, a solvent must be redistilled in glass using a 60-cm column packed
with 1/8" glass helices, or an equivalent system.
Hexane - ethyl ether and benzene are commonly used in the extraction of water and
wastewater in conjunction with analysis by electron-capture gas-liquid chromatography.
Because electron-capture detection methods are extremely sensitive to interferences
normally found in these solvents, the cleanest possible reagent grade or pesticide-quality
solvents must be used. Redistillation in the lab in an all-glass system is usually necessary.
Experience in the Analytical Quality Control Laboratory has shown Burdick-Jackson
hexane and Baker Chemical Co. ethyl ether to be satisfactory. All solvents vary from lot to
lot. Therefore, when a good lot is found, subsequent order should specify that lot number.
Solvents in the same lot may also vary and therefore each container should be checked.
8.6 Common Analytical Operations
Adequate steps must be taken to eliminate or minimize interferences from solvents and
other materials. A blank should be run simultaneously under the same analytical conditions
as any block of samples analyzed. A block of samples is defined as any group of one or more
samples analyzed using a common batch of analytical supplies. Should any one of the
supplies be changed (i.e. solvent, silica gel, Florisil, etc.) a new blank is required.
Quantitation of micro amounts of organic materials requires extremely careful technique to
avoid loss of sample. Quantitative transfers are essential to obtain accurate and precise
results. Practice in these manipulations is recommended for the inexperienced analyst.
Concentration of sample extracts to very small volumes for micro analysis requires great
care to avoid loss of constituents. A Kuderna-Danish evaporator is a very useful apparatus to
accomplish this operation. Instructions in the use of this evaporator must be strictly
followed to avoid loss of desired sample (1). Final concentration in an ampule or calibrated
tube is accomplished in a warm water bath with a gentle stream of clean, dry air, if air
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oxidation is not a problem; otherwise, nitrogen should be used. During the final
concentration, the inside walls should be rinsed repeatedly with the working solvent to
insure the total sample is contained in the bottom of the tube. Complete evaporation of
solvent must be avoided to prevent loss of sample constituents. The step should be
accomplished within 10 or 12 minutes for best results.
8.7 Gas-Liquid Chromatography
To obtain reproducible results, it is necessary to have very accurate control of the column
oven temperature. The temperature should be reproducible within ±1°C, and have minimum
gradients of 2°C throughout the oven. For temperature-programmed operation, a low mass
oven is required to allow rapid heating and cooling of the column. Most manufacturers
produce gas chromatographs which meet these requirements. Many organic compounds
decompose when they come in contact with hot stainless steel. For this reason the injection
block should be capable of accepting a quartz or glass tube to prevent the compounds from
contacting hot metal. Memory peaks observed using direct, aqueous-injection, gas
chromatography can be eliminated or greatly reduced by employing direct on-column
injection. A Bio-med injection kit manufactured by Tracer Instrument Co. is very suitable
for this purpose.
The septum should be changed at the end of each day's use. Changing at the end of each day
allows overnight purging of the system of any bleed-off of contaminants from the septum.
To avoid most of this bleed-off the septa can be preconditioned by heating at 250°C in a
vacuum oven for two hours.
The gas chromatograph should be equipped with accurate needle-valve, gas-flow controls. If
these flow controls are not previously calibrated this can easily be done using a soap-bubble
flow meter and a stopwatch.
The nature of constituents to be measured dictates the type of detector to be employed.
The electron capture detector is extremely sensitive to electronegative functional groups and
substituents, such as: halogens, conjugated carbonyls, nitrites, nitrates, and organometals. It
is virtually insensitive to hydrocarbons, alcohols, and ketones. The selective sensitivity to
halides makes this detector particularly valuable for the analysis of many pesticides.
Electron capture detectors employing two sources of ionization are available: tritium (H3)
and radioactive nickel (Ni6 3). Each has its advantages. Most H3 detectors possess greater
sensitivity than Ni63 detectors. However, the H3 detector is limited to 225°C because of
radioactive leakage. This temperature limitation makes the H3 detector susceptible to a
buildup of high-boiling contaminants which reduce its sensitivity. The Ni6 3 detector can be
operated up to 400°C to prevent this buildup of contamination.
The microcoulometric detector is specific for halogen-, sulfur-, or nitrogen-containing
compounds depending on the conditions used. Although the sensitivity of this detection
system is not as great as others available, the extreme specificity makes it a very valuable
device in indentification, minimizing the need for cleanup of the extract.
Another selective detector, with about the same sensitivity for chlorinated compounds as
microcoulometric titration, is the Coulson electrolytic conductivity detector. The specificity
of this detector makes it very useful in the identification of pesticides, and sample cleanup
and pre-treatment are less critical. However, if chlorinated compounds are being detected, a
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scrubber tube must be used with this instrument to remove any SO2 produced by
sulfur-containing compounds in the sample.
The flame ionization detector (FID) responds to virtually all compounds. Some notable
exceptions are air, water, carbon disulfide, and the fixed gases. This makes the FID very
useful for direct- aqueous-injection gas chromatography. However, since the detector is
sensitive to such a wide range of compounds, it is also subject to interference from
extraneous material. This means that the extract must be cleaned up considerably before
analysis. The flame photometric detector (FPD) is used to analyze residues of phosphorus-
and sulfur-containing pesticides and their metabolities. Little or no cleanup of the sample
extract is required, and extraneous material causes no appreciable interference.
The alkali flame detector (Thermionic) can also be used in the analysis of organophosphate
pesticides. Even though this detector gives an enhanced response to phosphorus, a large
amount of extraneous material demands that the extract be cleaned up before the
phosphorus-containing constituents are quantitated. An enhanced response to sulfur is not
obtained with the alkali flame.
A one-millivolt, one-second, full-scale-response, strip-chart recorder should be used to
maintain a permanent record of the results.
The type of detector employed dictates the type of carrier gas that must be used. Nitrogen
can be used for the FID, electron-capture, flame-photometric, and microcoulometric
titration detectors. The Analytical Quality Control Laboratory has used nitrogen from
various suppliers, and has found the J. T. Baker Chemical Company's extra dry grade to be
satisfactory. However, to avoid the risk of system or detector contamination from materials
possibly present in the gas cylinder, the cylinder should be replaced when the tank pressure
reaches 200 psi. It is also recommended that some type of gas purifier which is composed of
a molecular sieve, desiccani, and filter be used on ail combustion gases and carrier gases used
with electron capture detectors.
A precision, gas-tight, microliter syringe, which can be accurately filled, will deliver
reproducible injections, and can be easily and thoroughly cleaned, is recommended for gas
chromatographic use. A Teflon plunger seal to prevent backwash, and no dead volume are
desirable features. These types of syringes are available from various suppliers, but the
Glencoe syringes have additional design features to prevent bending plungers.
It is not possible to enumerate here the many types and applications of liquid phases and
solid supports. The literature (2, 3) will provide helpful guidance in the selection of these
materials.
The type of column tubing required to perform a specific GC analysis is prescribed by the
method for that analysis. Aluminum has been found suitable for chlorinated organic
pesticides and stainless steel for phenols. Generally 1/8" or 1/4" OD tubing is used. The
1/4" column is capable of accepting larger injections but the 1/8" column is more efficient
at chromatographing small injections. It should be noted that the use of glass columns will
prevent the degradation of material often associated with metal columns.
Silanized glass wool should be used to minimize degradation or absorption of organic
compounds by the glass-wool plugs. Glass wool can be silanized by treating with 10%
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dimethyldichlorosilane in toluene for 10 minutes, or the prepared material can be purchased
from Applied Science Laboratories.
Column packings can be prepared by the analyst, or purchased already prepared and .
conditioned. If prepared in the laboratory a highly refined technique should be developed
by the analyst. A well prepared packing is essential to an acceptable chromatographic
analysis. Particular care should be taken to accurately measure loadings, uniformly
distribute the liquid phase, and preserve the structure of the fragile solid support. One
method used successfully is the beaker slurry method in which the proper amounts of
stationary phase and solid support are mixed together in a solvent in a beaker. The solvent
can be evaporated by immersing the beaker in a warm water bath or filtering it through a
Buchner funnel. The mixture is then dried at 110° C to remove residual solvent. A fluidized
dryer (4) may also be used to remove the remaining solvent. Details on techniques for
preparation of Teflon packings may be obtained from Applied Science Laboratories
(Bulletin FF124).
An important consideration in packing a column is obtaining a uniform density, not so
compact as to restrict gas flow, and not so loose as to create voids during use. Also
important is the need to exercise care not to crush particles during packing.
It cannot be overemphasized that column conditioning is essential to obtain acceptable GC
analysis. Proper conditioning further distributes the liquid phase over available active sites,
and removes excess liquid phase that may bleed off the column and impair GC performance.
Unstable performance in the form of baseline drift, varying sensitivity, and wide solvent
peaks are usually the result of column bleed due to improper conditioning. This also
contaminates the detector and makes frequent cleaning necessary.
A good general procedure for conditioning columns is as follows: install the column, leaving
it disconnected from the detector; heat the column in the GC oven to just above maximum
recommended operating temperature for the liquid phase, without gas flow, for 2 hours;
follow with a half-hour equilibration period at a temperature at least 40 degrees below the
maximum recommended temperature, still without flow; raise the temperature 20 degrees
above operating conditions (do not exceed the maximum recommended operating
temperature for the liquid phase), adjust gas flow to 50 ml/min, and maintain these
conditions 24 to 48 hours.
There are many factors that must be considered in any attempt to obtain optimum
operating conditions. As indicated, optimum conditions begin with the proper selection of
column materials including tubing, glass wool, liquid phase, and solid support. Each method
dictates the selection of these items including the percent liquid phase. Liquid load and
operating temperature are critical to optimum resolution and elution rate. Uniformly
coating the solid support, properly packing the column, and properly conditioning the
column all contribute greatly to attaining optimum conditions. Improper attention to these
factors contributes to either column bleed, which fouls the detector, or tailing peaks, which
prevent resolution of closely eluting compounds.
The system must be free of gas leaks since these affect sensitivity and reproducibility. The
proper selection of a carrier gas is important. InjectionA'blocks should be of proper flow
design, and should be kept clean. Temperature control of injection port, column oven, and
detector must be accurate and constant, and must be at equilibrium to obtain reproducible
results.
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Considerable attention must be given to those factors affecting detector response, such as
gas flows and regulated current. Also, standard solutions and condition of electrodes for
MCT detectors, condition of radioactive source for EC detectors, and ratio of combustion
gases in flame ionization detectors must be carefully attended to.
The gas chromatograph must either be operated within the linear range of the detector, or a
suitable calibration curve must be employed. The linear range can be determined by
chromatographing different amounts of a compound and observing the response. The peak
area of the responses should be proportional to the amount of compound injected
throughout the full range of the recorder. If these conditions are not fulfilled at the
attenuation investigated, a more sensitive attenuation should be selected until the linear
range is found.
Once having obtained optimum operating conditions, the analyst must assure sustained
optimum performance through a routine maintenance program, as prescribed in the
instrument manual. Such common practices as inspecting for gas leaks, changing the septum
after daily use, replacing gas tanks before they run too low, and keeping a close check on
injection block, oven, and detector temperatures must be performed frequently.
Column performance can be monitored by observing daily response to a selected group
standard, and comparing it to response to that same standard under previously determined
optimum conditions. Changes in elution pattern, relative proportions of peaks, and peak
geometry are signs of a deteriorating column if the rest of the system has been properly
maintained. A column should be replaced as soon as deterioration is observed.
A good syringe-handling technique is important when doing GC analysis. Before measuring
the volume to be injected into the gas chromatograph, first wet the barrel of the syringe and
expel all air bubbles. The volume injected is determined by drawing a selected portion of the
extract entirely into the glass, calibrated, syringe barrel, noting the volume, injecting the
sample, partially withdrawing the plunger, and measuring the liquid remaining in the syringe
barrel. Injections are made rapidly after the needle is in the gas chromatographic injection
port. The needle is immediately removed from the system to prevent volatilization of any
sample in the needle. This method improves the analytical accuracy since any absolute
volume is injected and reproducible technique is not required. It also eliminates the built-in
bias of injecting slightly more than expected each time, when direct barrel readings are used
exclusively.
The standard solutions can be more accurately used if prepared at a concentration that
allows injection volumes similar to those of the sample.
8.8 Qualitative Analysis
The retention time (Rt) of a component on a given column, under given conditions, is
characteristic of that particular component, and is used for qualitative identification. The
standard way of reporting retention data is to give the relative retention (RRt) defined as Rt
(component) = Rt (reference compound). The retention time may be measured as the time
elapsed from an unrestrained solute peak elution, to the apogee of the peak of interest. This
works well when a flame ionization or electron-capture detector are used. However, when
the microcoulometric titration or the flame photometric detector are used (the flame is
extinguished by the injection), no unrestrained solute peak is observed. In such cases, the
injection point is manually or electrically marked, and used as the point of reference for
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retention times. Caution must be exercised when manual marking is practiced, so that an
accurate reference point is provided.-
Tentative identification is made by matching the relative retention time of the unknown
component with that obtained from a known compound analyzed under identical
conditions, provided that peak geometry is also similar. It must be pointed out that
similarity of retention time and peak geometry to a known compound does not
unequivocally identify an unknown. Additional gas chromatographic columns of different
polarity and other detectors may be used for confirmation. If retention time and peak
geometry match on two or more columns, the identity is corroborated. However, further
corroboration using analytical tools such as infrared spectroscopy, mass spectrometry, or
thin-layer chromatography should be used whenever possible.
Proof of identity of compounds which produce a multi-peak response should be evidenced,
not only by relative retention and peak geometry, but also by the correct number and
relative proportion of each peak in the total chromatogram. This is called a "fingerprint"
comparison of the known standard chromatogram with that of the unknown constituent.
8.9 Quantitative Analysis
The quantitative interpretation of a gas chromatogram is based either on the peak height or
the peak area. The area measurement is generally preferred because peak height is extremely
sensitive to small changes in the operating conditions, particularly in the column
temperature. However, in chromatograms where the peaks are extremely sharp and narrow,
the error involved in the area measurement makes height measurements more reliable.
Peak area measurement should be carried out using height x width at half height, disc
integrator, or electronic digital integrator. These techniques are rapid and simple, and give
good results with symmetric peaks of reasonable width. The use of a planimeter, although
less precise than other methods, is presently found to be the best method for measuring the
area of unsymmetric peaks which do not originate at the baseline. Precision is improved by
tracing each peak several times and taking an average value. The electronic integrator is also
recommended for those unsymmetric peaks which originate at the original baseline.
Concentrations of constituents are determined from standard calibration curves obtained
under identical conditions. An absolute calibration curve is obtained from peak areas or
peak heights plotted against known weights of a compound chromatographed under
identical conditions. These standard injections must be made during the sample run to
detect any change in instrument conditions or response which would invalidate the
calibration.
The use of an internal standard is the most accurate method of quantitating constituents in
a sample. A calibration curve can be obtained by simultaneously chromatographing the
previously identified sample constituent and a standard, in known weight ratios, and
plotting, the weight ratios versus area ratios. An accurately known amount of the standard is
then added to the unknown sample and the mixture chromatographed. The area ratios are
calculated, and the weight ratio of the sample constituent to the standard is read from the
curve. Since the amount of standard added is known, the amount of the sample constituent
can be calculated. Using this method, injection volumes need not be accurately measured
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and detector response need not remain constant since any change in response will not alter
the ratio.
If the following requirements can be met, the internal standard is the preferred method of
calibration. The internal standard must be well resolved from other peaks, must elute close
to peaks of interest, should approximate the concentration of unknown, and should have
structural similarity to unknown.
8.10 Thin-Layer Chromatography
Special equipment for thin-layer chromatography as supplied by the Brinkman Instrument
Company has been found by the Analytical Quality Control Laboratory to be particularly
convenient to use. Adsorbents supplied by Warner-Chilcott Laboratories, particularly Silica
Gel G and Alumina G, are' found to be free of impurities. In any case, it is best to check the
adsorbent for impurities, and discard it if necessary. Plates precoated by the manufacturer
can often be used when quantitation is visual; however, trials of plastic-backed plates in the
Analytical Quality Control Laboratory have shown that they often develop unevenly and
very slowly. The use of precoated glass-backed plates is therefore recommended.
Prepared plates should be carefully inspected for flaws that might interfere with proper
chromatographic development. Plates should be marked in such a way that any grain lines
would be perpendicular to the direction of development when used. Prepared plates are best
activated by heating at 110° C in a convection oven for 30 minutes. Phosphorous pentoxide
is recommended as the desiccant for use when storing plates.
Two important points to remember before using the developing chambers are to give the
chamber atmosphere time to become saturated with solvent vapor, and to keep the chamber
in constant temperature surroundings shielded from drafts. Failure to observe these points
could result in an erratic chromatographic development, generally off line.
When spotting a plate, care must be taken not to overload the spot. Overloading results in
poor chromatographic efficiency. Also, when applying a gentle stream of air or other gas to
evaporate the solvent, care must be taken not to blow away any of the layer or sample
droplets. The analyst should also be aware of whether or not the use of air to evaporate the
solvent could cause any oxidation of the sample material, thereby causing erroneous results.
If oxidation is a problem an inert gas should be substituted.
When spraying a plate with aqueous or other non-volatile sprays, care should be taken not to
soak the plate. Soaking with such sprays may cause spots to run as the plate stands
vertically. When using any spray it is better to use a stronger solution, then apply increased
amounts to develop the spots. Overspraying can produce a background color which impairs
the visibility of the developed spot.
Quantitation by visual comparison of sample response to a series of standards is
semi-quantitative at best. However, by making the spot size the same for samples and
standards,by using the same solvent for both, and by using careful development techniques,
a fair degree of reliability can be obtained, when comparing the factors of spot size and
color intensity.
When zone scraping and collecting the layer material with the aid of a vacuum, the
collection apparatus is usually plugged with glass wool to trap the adsorbent. Since poorly
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packed glass wool can cause high loss of material by failure to trap the dust, the scraping of
the periphery of the zone should be done first. A clear tygon vacuum line, or a glass section
near the end of the tube makes it easy to monitor for untrapped adsorbent.
8.11 Column Chromatography
The features of a chromatographic column which define its utility are shape, liquid capacity,
and elution rate. The liquid capacity is principally a matter of convenience and should be
weighed against the increased labor required for cleaning equipment. A flow-control device
is critical to an efficient separation because increased flow decreases resolution between
emerging components. A Teflon, flow-control stopcock is required because lubricant will
produce an analytical interference.
Most adsorbents used in column chromatography are preactivated by the manufacturer and
shipped in air-tight containers. Storage in the laboratory should also be in an air-tight
container because moisture will greatly affect the activation state of an adsorbent. If a
second activation step is required, the material should be heated at 130°C for at least five
hours. The activity of the adsorbent can be monitored by eluting a mixture of
chromatographically pure dyes (5). The elution rate and degree of separation of the
individual dyes is a function of the activation state of the adsorbent. This enables the
analyst to accurately attain the same activation for different batches of adsorbent.
When the adsorbent is added to the column, gentle tapping or a vibrator should be used to
settle the material. This minimizes the space between particles and prevents channeling of
the eluting solvent through the adsorbent which reduces separation efficiency.
Liquids should be added slowly, down the inside wall of the column, to avoid disturbing the
packing surface. Mixing of the solution above the adsorbent with the fresh eluting solvent
can be minimized by introducing the new solvent just as the last of the solution reaches the
packing surface. The column top must not go dry or air may be introduced which will lower
the separation efficiency of the system.
Before addition of the sample, columns should be pre-eluted with 50-75 ml of the solvent
prescribed by the procedure. This is done to remove trapped air and to clean the column
material of trace contaminants. During this pre-elution it is often necessary to tap the
column to free all trapped air, especially if a volatile solvent is used in the pre-elution.
8.12 References
1. Gunther, F. A., et al, Anal. Chem.,.23, No. 2, p. 1835 (1951).
2. Lynn, T. R., et al, Guide to Stationary Phases for Gas Chromatography. Analabs, Inc.,
1968.
3. McNair, H. M. and Bonelli, E. J., Basic Gas Chromatography. Varian Aerograph, 1969.
4. Kruppa, R. F., et al, Anal. Chem.,39, 851 (June 1967).
5. Brockman, H. and Schodder, H., Chem. Ber., 74, p. 73 (1941).
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CHAPTER 9
SKILLS AND TRAINING
9.1 General
Analytical operations in the laboratory can be graded according to the degree of
complexity. Some analyses require no sample treatment, with the measurement performed
in minutes on a simple instrument. Other determinations require extensive sample
preparation prior to complex instrumental examination. Consequently, work assignments in
the laboratory should be clearly defined. Each analyst should be completely trained and
fully understand all the assignments of his job before being given new responsibilities. In this
regard, all analysts, sub-professional or professional, should be thoroughly instructed in
basic laboratory operations, according to the degree of professional maturity. Some of the
basic operations that should be reviewed periodically with laboratory personnel follow:
a. Sample Logging
Emphasize the routine procedure for recording of samples entering the laboratory,
and assign primary responsibility. Establish what information is required, and how
sample is routed to analyst. Discuss stability of samples, and how they should be
stored prior to analysis.
b. Sample Handling
The analyst should understand thoroughly when the sample is to be settled,
agitated, poured, pipetted, etc., before removal from the container.
c. Measuring
The analysts, especially new employees and sub-professionals, should be instructed
in the use of volumetric glassware. The correct use of pipettes and graduates should
be emphasized as discussed in Chapter 4.
d. Weighing
Because almost every measuring operation in the analytical laboratory is ultimately
related to a weighing operation, the proper use of the analytical balance should be
strongly emphasized. Maintenance of the balance, including periodic
standardization, should be reiterated to all personnel.
e. Glassware
All glassware should be washed and rinsed following the requirements of the
analysis to be performed. Not only must the personnel assigned to this task be
instructed, but all lab personnel should know the routine for washing glassware, and
also special requirements for particular uses. In addition, the precision tools of the
laboratory such as pipets, burets, graduates, Nessler tubes, etc., should be inspected
before use for cleanliness, broken delivery tips, and clarity of marking. Defective
glassware should be discarded or segregated.
9-1
-------
In summary, quality control begins with basic laboratory techniques. Individual operator
error and laboratory error can be minimized if approved techniques are consistently
practiced. To insure the continued use of good technique, laboratory supervisors should
periodically review the basic techniques with each analyst and point out, when necessary,
areas of needed improvement.
Continuing improvement of technical competence for all laboratory personnel is, of course,
the final responsibility of the laboratory supervisor. In a well organized laboratory, however,
a big brother attitude of higher ranking to lower grade personnel should be encouraged; each
person should be eager to share experience, tricks-of-the-trade, special skills, and special
knowledge with subordinates. Obviously, improved efficiency and improved data quality
will result.
9.2 Skills
The cost of data production in the analytical laboratory is based largely upon two
factorsthe pay scale of the analyst, and the number of data units produced per unit of
time. However, estimates of the number of measurements that can be made per unit of time
are difficult, because of the variety of factors involved. If the analyst is pushed to produce
data at a rate beyond his capabilities, unreliable results may be produced. On the other
hand, the analyst should be under some compulsion to produce a minimum number of
measurements per unit of time, lest the cost of data production become prohibitive. In the
following table, estimates are given for the number of determinations that an analyst
should be expected to perform on a routine basis. The degree of skill required for reliable
performance is also indicated. The arbitrary rating numbers for the degree of skill required
are footnoted in the tables, but are explained more fully below:
a. Rating 1indicates an operation that can be performed by a semi-skilled
sub-professional with limited background; comparable to GS-3 through GS-5.
;
b. Rating 2operation requires an experienced aide (sub-professional) with
background in general laboratory technique and some knowledge of chemistry, or a
professional with modest training and experience; comparable to GS-4 through
GS-7.
c. Rating 3indicates a complex procedure requiring a good background in analytical
techniques; comparable to GS-7 through GS-11.
d. Rating 4a highly involved procedure requiring experience on complex
instruments; determination requires specialization by analyst who interprets results;
comparable to GS-9 through GS-13.
The time limits presented in the table are based on use of EPA methods.
A tacit assumption has been made that multiple analytical units are available for
measurements requiring special equipment, as for cyanides, phenols, ammonia, nitrogen and
COD. For some of the simple instrumental or simple volumetric measurements, it is assumed
that other operations such as filtration, dilution or duplicate readings are required; in such
cases the number of measurements performed per day may appear to be fewer than one
would normally anticipate.
9-2
-------
Table 9-1
SKILL-TIME RATING OF STANDARD ANALYTICAL OPERATIONS
Measurement
pH
Conductivity
Turbidity (HACK 2100)
Color
DO (Probe)
Fluoride (Probe)
Skill Required (Rating No.)
(Simple Instrumental)
1
1
1
1
1,2
1,2
No./Day
100-125
100-125
75-100
60-75
100-125
100-125
(Simple Volumetric)
Alkalinity (Potentiometric) 1
Acidity (Potentiometric) 1
Chloride 1
Hardness 1
DO(Winkler) 1,2
50-75
50-75
100-125
100-125
75-100
Solids, Suspended
Solids, Dissolved
Solids, Total
Solids, Volatile
Nitrite N (Manual)
Nitrate N (Manual)
Sulfate (Turbidimetric)
Silica
Arsenic
(Simple Gravimetric)
1,2
1,2
1,2
1,2
(Simple Colorimetric)
2
2
2
2
2,3
20-25
20-25
25-30
25-30
75-100
40-50
100-125
100-125
20-30
SKILL REQUIRED
1 - aide with minimum training, comparable to GS-3 through GS-5
2 - aide with special training or professional with minimum training,
comparable to GS-5 through GS-7.
3 - experienced analyst, professional, comparable to GS-9 through GS-12.
9-3
-------
Table 9-1 (continued)
SKILL-TIME RATING OF STANDARD ANALYTICAL OPERATIONS
Skill Required (Rating No.)
No./Day
(Complex, Volumetric or Colorimetric)
2,3
2,3
2,3
2,3
) 2,3
et) 2,3
2,3
2,3
(Special Instrumental)
2,3
atment)-
2,3
reatment)
3,4
3,4
30-40*
25-30
25-30
50-60
20-30
15-20
25-30
10-15
150
60-80
3-5
2-4
Measurement
BOD
COD
TKN
Phosphorus, Total
Phenol (Dist'n only)
Oil & Grease (Soxhlet)
Fluoride (Dist'n)
Cyanide
Metals by AA
(No preliminary tn
Metals by AA
(With preliminary 1
Pesticides by GC
(Without cleanup)
Pesticides by GC
(With cleanup)
SKILL REQUIRED
2 - aide with special training or professional with minimum training,
comparable to GS-5 through GS-7.
3 - experienced analyst, professional, comparable to GS-9 through GS-12.
4 - experienced analyst, professional, comparable to GS-11 through GS-13.
* - depends on type of sample.
9.3 Training
For more experienced, higher grade personnel, formal training in special fields, possibly
leading to specialization, should be almost mandatory. Such training can be fostered
through local institutions and through the training courses provided by the Environmental
Protection Agency. Regional policies on after-hours, government-supported training should
be properly publicized.
Formalized training for lower grade personnel, comparable to GS-3 to GS-5, is relatively
scarce. However, skills can be most efficiently improved at the bench level on a personal,
informal basis by more experienced analysts working in the same area. Exposure to
pertinent literature should also be a definite program policy.
9-4
if U. S. GOVERNMENT PRINTING OFFICE: 1977 0 - 758-469
-------
APPENDIX H
ANALYTICAL CHEMISTRY. VOL. 50, NO. 1. JANUARY 1978 91
Automated Determination of Mercury in Sediments
l
idrea M. Jirka* and Mark J. Carter
l:ed States Environmental Protection Agency, Cen'.rat Regional Laboratory, 536 South Clark Street, Chicago, Illinois COS05
An automated method for the determination of total mercury
In sediment samples is reported. Aqueous suspensions of
sediment samples are automatically analyzed using the
co!d-vapor detection method following a persulfate oxidation
and stannous chloride reduction. The method completely
recovers mercuric sulfide and produces data that are com-
parable to those obtained by the standard method. The
method is safer, faster, and easier to perform than the standard
method. Samples are analyzed at the rate of 30 per hour, with
a routine detection limit of 0.1 mg Hg/kg of sample, and an
average relative standard deviation of 6% at the level of 20-30
mg/kg.
In recent years, mercury has been recognized as a toxic
contaminant to the environment. Reliable and efficient.
methods are required for determining mercury in all types
of environmental samples. Most of the reported methods are
for the determination of mercury in water samples. Nearly
all of these methods involve the cold-vapor detection method
Kscribed by Hatch and Ott (1). The measurement of total
rcury involves a rigorous digestion procedure.
n lakes and streams, mercury can collect in the bottom
sediments, where it may remain for long periods of time. It
is difficult to release the mercury from these matrices for
analysis. Several investigators have liberated mercury from
soil and sediment samples by application of heat to the
samples and collection of the released mercury on gold
surfaces. The mercury was then released from the gold by
application of heat or by absorption in a solution containing
oxidizing agents (2,3).
Bretthaur, Mognissi, Snyder, and Mathews 'described a
method in which samples were ignited in a high-pressure
oxygen-filled bomb (4). After ignition, the mercury was
absorbed in a nitric acid solution. Pillay, Thomas, Sondel,
and Hyche used a wet-ashing procedure with sulfuric acid and
perchloric acid to digest samples (5). The released mercury
was precipitated as the sulfide. The precipitate was then
redigested using aqua regia.
Feldman digested solid samples with potassium dichromate,
nitric acid, perchloric acid, and sulfuric'acid (6). Bishop,
Taylor, and Neary used aqua regia and potassium per-
manganate for digestion (7). Jacobs and Keeney oxidized
sediment samples using aqua regia, potassium permanganate,
and potassium persulfate (8). The approved U.S. Environ-
mental Protection Agency (EPA) digestion procedure requires
aqua regia and potassium permanganate as oxidants (9).
These digestion procedures are slow and often hazardous
because of the combination of strong oxidizing agents and high
P*' .peratures. In some of the methods, mercuric sulfide is
adequately recovered. The oxidizing reagents, especially
potassium permanganate, are commonly contaminated
with mercury, which prevents accurate results at low con-
centrations.
?..!-.-' V.-;H!.V: Miller, and Carter have described > automated
:.".-.;....! :'..- iht dcttrrninKUon of total mercury in vvjiier-, and
'*;.;'.:; v.v:?vi t (ifi]. In that method, potassium ptrbulfate and
K.,:f.-.-;',: ;:(!:! v.t-re used to digest sample* for analysis by the
cold-vapor technique. The use of potassium permanganate
as an additional oxidizing agent was proved unnecessary,
which reduced the level of contamination in tKe system, and
allowed a routine detection limit of 0.1 pg Hg/Ltobe attained.
1 Use of the described method resulted in significant savings -
in time, reagent costs, and laboratory space when analyzing
water and wastewater samples.
The same advantages can be realized when analyzing sed-
iment samples by use of the method described here.
EXPERIMENTAL
Apparatus. A Tekmar model SDT homogenoer was used to
blend samples prior to analysis. All other apparatus used was
described by El-Awady (JO). Additional air lines were added lo
the analytical system, and an air-bar was used for all air lines.
The GO fitting on the sample line was changed to G3. The
analytical manifold is shown in Figure 1.'
Reagents. A preservative solution was prepared by addition
of 250 mL of coned HN03 and 25 g of K2Cr2O7 to 500 ml, of
distilled water and dilution to 1000 mL. All other reagents -were
those described by El-Awady (JO).
Procedure. Sediment samples were passed through a Nov 10
polypropylene sieve to remove large debris. If necessary, the
samples were blended using a Waring blender. Approximately
1 g of wet sediment was accurately weighed into a 36O-mL
polyethylene bottle. Five mL of preservative solution was added
to drive offer oxidize any free sulfides as well as to preserve the
sample (10,11). If the J^CrjO; was entirely reduced, as indicated
by a green color, additional preservative solution was added. Then
245 mL of distilled water was added, and the aqueous samples
were blended using a Teckmar blender. The samples were allowed
to stand overnight. Additional preservative solution was added
if the dichromate was entirely reduced after standing. The
aqueous samples were then analyzed, using the modified auto-
mated analytical system in the manner described by El-Awady
(10).
To convert the mercury concentrations in the aqueous samples
to the concentrations in the original sediments, separate de-
terminations of percent solids were made, and the following
formula was applied:
mg Hg/kg (dry sample) =
jig Hg/L (aqueous) X 25
(g sediment) X (% solids)
RESULTS AND DISCUSSION
Recovery of Mercuric Sulfide. Jacobs and Keeney
observed that mercuric sulfide required digestion prior to
analysis, using aqua regia and KMnO4 plus K2S2O» for
complete recovery of mercury (8). El-Awady observed variable
results when analyzing HgCl2 solutions which contained more
than 20 mg S2"/L, but he reported no interference for HgCl2
solutions containing less than 20 mg S2~/L (10).
To determine the recovery of HgS when analyzed by the
automated method, organic and inorganic mercury standards
were analyzed after they were spiked with Na^. Inorganic
mercury reacts with sulfide:
S»- + Hg'*- HgS
To ensure that the- reaction was complete under the
conditions sui'jj&o. 3 stsrdsrci containn-^ ;UvU ^2 Kj;i"~/i, was'
reacted with Ma2S and filtered to rernc-ve 'HgS. The filtsate.
\vns diluted 1/1.0, and dichromate preservative solution was
-------
92 . ANALYTICAL CHEMISTRY, VOL. 50. NO. 1, JANUARY 1978
Sampler IV
Table I. Recovery of Mercury Standards
Spiked with Sulfide
Recorder
Figure 1. Modified automated total mercury manifold. Numbers in
parentheses correspond to the flow rate of the pump tubes in mL/min.
Numbers adjacent to glass coils and fittings are Technicon Corp. part
numbers
added. Less than 0.1 ng Hg/L was recovered from analysis
of the filtrate, which indicated that the formation of the
insoluble HgS was complete.
When the same experiment was performed using organic
mercury, no precipitate was observed, and 80% of the mercury
was recovered in the filtrate. The loss of 20% was probably
due to mercury which adhered to the glassware during fil-
tration, since oxidizing conditions were not maintained.
Table I contains the results of analyses for organic and
inorganic mercury standards which were spiked with sulfide.
There was no significant interference due to S2" in the so-
lutions containing 10 mg S2~/L- However^ a negative in-
terference was observed for both organic and inorganic
standards containing 100 mg S2'/L which is equivalent to
25000 mg S2~/kg in the sediment. The spiked blank also
resulted in a small negative interference.
It is interesting to note that exactly the same interference
occurred for both organic and inorganic mercury standards,
since methyl mercuric chloride does not react directly with
sodium sulfide to form mercuric sulfide. Therefore, the
interference could not be the result of incomplete digestion
of HgS or CH3Hg+.
Sulfur, ozone and H2S were investigated as possible causes
for the interference. Sulfur added to a standard did not cause
an interference. Ozone and H2S were introduced directly into
the mercury detector. No interferer.ee was observed.-'
When the automated method was used, the interferences
which were- observed for the standards and blank spiked with
100 mg S2"/L. occurred when an excess of dichromate did not
exist in the solutions. As excess of dichromate exists when
the sulfide concentration is less than 36 mg/L. Above that
concentration, there is no dichromate. When no additional
dichromate preservative solution v.-as added, a negative in-
U-:/".: - .. . .-:: ohstrvcd betv.'f.-&n ".- .r.-.-j 50 nig S-'/L for bo'n
or;-.;.':':: - ' :;-;organic mercury s^j'durds. However, no in-
ier'V-i't - .-..;.; observed for su'fu'f- '~i-.:icentrattons as hi^h a.s
Standard solution
Blank
1 jig Hg/L (CHjHgCl)
lMgHg/L(HgCl,)
1 ng Hg/L (CHjHgCl)
Blank
1 /.g Hg/L (HgCl,)
1 Mg Hg/L (CHjHgCl)
Blank
1 ^g Hg/L (HgCl,) +4
times normal preservative
1 pg Hg/L (CHjHgCl) +4
times normal preservative
Blank +4 '
times normal preservative
Odd shaped peak.
spike,
mg/L
10
JO
10
100
100
100
100
100
100
Observ:
Hg
i
O.O
3.0
5..0
l-.O
1.0
O.O
0.4°
0.4°
-O.I
1.2
1.1
O.O
100 mg/L if additional dichromale preservative solution was
added and oxidizing conditions were maintained.
Therefore, it is our conclusion that the negative interference
which was observed for the standards when oxidizing con-
ditions were not maintained was caused by the adsorption of
mercury on the inside surfaces of the culture tubes and the
analytical system prior to the introduction ofKySyOg. The
small negative interference in the blank can be explained l>y
the fact that a small amount of mercury was present ij
arialytical reagents. When the blank, which contained.'
pumped through the system, oxidizing conditions
maintained and a slight negative signal was observed.
The mercury which was adsorbed on the surfaces of the
analytical system was released when oxidizing conditions were
again present. This was observed as a peak which occurred
when sulfide was removed from the system, or by a positive
baseline drift.
Organic Interferences. Aromatic organic compounds
such as benzene, which are not oxidized in the digestion,
absorb at the same wavelength as mercury. This represents
a positive interference in all cold-vapor methods for the
determination of mercury. For samples containing aromatics,
i.e., those contaminated by some industrial wastes, a blank
analysis must be performed, and the blank results must be
subtracted from the sample results. The blank analysis is
accomplished by replacing the KjjS2Os reagent and the SnCl^
reagent with distilled water, and re-analyzing the sample.
Comparison of Methods. A diverse group of 25 sediments
were analyzed by the automated rnt-lhod, and also after di-
gestion by the standard EPA method. The standard EPA
method was a micro modification of an aqua regia/KMnQj
digestion (9). The results are shown in Table II. For those
samples containing detectable concentrations of mercury, the
automated method results averaged 94% of the standard EPA
method results. The mean results for. the first sample listed
under "high org<-unics" are quite different, with the automated
method producing low results. However, when these results
are compared to the standard deviations, 41 ± 11 nig/kg;
23 ± 5 mg/kg, the bias is not significant when compared
the data scatter. The variation in detection limits report!
for the standard method is the result of the different weights
of the samples taken for analysis.
Table III contains the results of inter-laboratory comparison
testing for five s^dirnvnt samples contain!:!;; iov/ mercury
The results which were obtained usins the
:-rrt os described
suits
;aj«
1
the analytica
-------
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1. JANUARY 1978 . 93
Tab/e II. Comparison of Methods for Mercury
Determination in Sediments
Standard Automated
method (7) method
Sample type
Sand
Industry
Industry
Harbor
Harbor
Harbor
Harbor
Harbor
Harbor :
Harbor
Clay
Creek
Harbor
Harbor
Harbor
Island
Island
Island
Harbor
High organics
Industrial
Creek
Sludge
Sludge
Sludge
Industrial
Harbor
Island
Mean
«
mg
Hg/kg
(S)
1.2
2.6
0.5
<0.2
<0.2
<0.1
<0.1
<0.2
<0.1
<0.1
1
<0.2
<0.5
<0.2
<0.6
<0.5
<0.2
41
<0.2
28
16
30
0.5-
0.3
<0.9
No.
of
dctns
4
2
2
2
2
2
2
2
2
2
4
2
4
2
2
2
2
4
2
2
2
2
2
2
2
mg
Hg/kg
(A)
1.4
2
0.4
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.1
0.8
<0.1
0.2
<0.1
<0.1
<0.1
<0.1
23
0.1
24
18
27
0.7
0.3
<0.1
No.
of
detns
8
2
2
2
2
2
2
2
2
2.
6
2
6
2
2
2
2
8
2
2
2
2
2
2
2
% Re-
covery
(A/S
X
100)
117
77
80
*
-- . »
""'"""' ' *
' »
80
*
*
. » *
56
,
86
112
90
140
100
94
Table III. Comparison of Methods for Mercury
Determination in Sediments
Results of interlaboratory
study (28 labs)0
Sample
1
2
3
4
5
Mean, Acceptable
mg range,
Hg/kg mg Hg/kg
0.0873 0.063-0.111
0.179 0.126-0.232
0.229 0.166-0.292
0.667 0.515-0.820
0.625 0.495-0.754
Automated
method
Without
modifi-
cation,
mg
Hg/kg
0.072
0.14
0.12
0.42
0.45
With
modifi-
cation,
mg
Hg/kg
0.1
0.2
6.60
0.61
" Unpublished results obtained from Aspila and
Carron (/2),
did not flow uniformly through the system. Some of the
sediment settled in the GO fitting before the pump. However,
when additional air lines were added, an air-bar was used for
all air lines, and the GO fitting was changed to a G3 fitting,
the sediments were easily pumped through the system, and
comparable results were obtained. '
There was a limit to the amount of sediment that could be
pumped through the manifold. If precipitation of sediment
v.-as observed in the sample lines, a smaller amount of sample
v.-a5 taken for analysis. If this precaution was not taken, results
re biased low.
Precision, Accuracy, and Spike Recovery. The pre-
ion of the automated method at low levels was evaluated
by analyzing four sand samples (which contained less than
1 Tn% H>'/V;--> five times. The mean vnkies ninycd from 0,13
; 0.2: in::: liV/ki/. The-standard fiovinJioris raiijjsd !>o:n O.Oil
; ; 0.020 mjc i 'g/kg, with relative standard deviations ranging
Table IV. Precision at Low Levels .
Sample
'Hg, mg/kg
Mean
Std dev
Rel std dev, %
1
0.22
0.19
0.22
0.22
0.18
0.21
0.020
9.5
2
0.11
0.12
0.14
0.15
0.13
0.13
0.016
12
3
0.13
0.15
0.13
0.12
0,13
0.13
0.011
8.4
4
0.15
0.18
0.14
0.16
0.14
0.15
0.017
11
Table V. Precision and Spike Recovery
for Sludge Samples - , -
Sample
Hg, mg/kg
Mean
Std dev
Rel std
dev
5
18
19
18
20
16
18.2
1.48
8.1%
6
16
17 .
18
18
17
17.2
0.84
4.9%
7
35
34
30
30
32
32.2
2.28
7.1%
mg Hg/kg
Sample
5
5
6
6
7
7
8
8
Mean
18.2
18.2
17.2
17.2
32.2
32.2
31.2
31.2
Spike
6.02
5.43
5.18
4.93
22.5
23.6
9.52
16.0
Sam-
ple +
spike
26
24
22
21
61
61
36
53
Spike
re-
covery
8
e
5
4
29
29
5
22
8
30
32
33
30
31
31.2
1.30
4.2%
Spike
re-
covery.
%
130
110
93
77
130
12Q
50
140
from 8.4% to 12%. (See Table IV).
The precision of the automated method at high levels was
evaluated by analyzing four sludge samples five times. The
mean values ranged from 17.2 to 32.3 mg Hg/kg (Table V).
These data indicate that results should be reported to no more
than two significant figures. The same sludge samples were
spiked in duplicate with methyl mercuric chloride, and then
analyzed by the automated method. The average spike re-
coveries were 85%, 95%, 120%, and 125% (Table V).
Detection Limit. The detection limit for the automated
method is dependent upon the weight of sample taken for
analysis. It is 0.1 fig Hg/L in the aqueous samples- The results
for the automated method are routinely reported to a lower
limit of 0.1 mg/kg which corresponds to a dry sample weight
of 0.25 g.
If a lower detection limit is necessary, the samples may be
pulverized and a larger weight may be used. However, if any
material settles in the manifold, the results wflLbe biased low.
ACKNOWLEDGMENT
The authors thank K. I. Aspila and J. M. Carron, Inland
Waters Directorate, Water Quality Branch, Special Services
Section, Department of Fisheries and Environment, Bur-
lington, Ontario, Canada, for providing comparison data used
in this work.
LITERATURE CITED
(1) W. R. Hatch and W. L. Oil. Anal. Chem.. 40. 2065(1968).
(2) P. C. Leorg. and H. P. Gng, Ane.l. Chem., 43, SJS (1971).
Hi O. H. ArvV,T-..",n. J. H £.--: J. J. M.jrphv, d.nci Vv. ','.'. ,.:-':*, /..*-./. Cf--em..
33, 15n (-S71).
U, c. W. BK::^SU-. A. A ?.';^hisji. S. S. Sr.yc'er. sr.d ,'i. "»V. f.'^.^e-*j. An.ji.
Chum., 46, «5 (107';
-------
94 « ANALYTICAL CHEMISTRY. VOL. 50. NO. 1. JANUARY 1978
(5) K. K. S. Pitoy, C. C. Thomas. J. A. Sondel. and C. M. Hyche, Anal. Ctem..
43. 1419(1971).
(6) C. FelfJman. Anal. Chem.. 46, 1606 (1974).
(7) J. N. Bishop, L. A. Taylor, and B. P. Nsary, "The Determination of Mercury
in Environment Samples". Ministry of the Environment. Ontario. Canada.
1973.
(8) L. W. Jacobs and D. R. Keeney, Environ.. Sci. Techno!.. 8. 267 (1976).
(9) "Methods for Chemical Analysis of Water and Wastes". U.S. Environmental
Protection Agency. Cincinnati. Ohio, 1974, p 134-138.
(10) A. A. Et-Awady. R. B. Miller, and M. J. Carter. Anal. Chem., 48, 110
(1976).
(11) K. I. Aspila and J. M. Carron. "Jnier-Laboratory Quality Control Study
No. 18-Total Mercury In Sediments". Report Series. Inland Waters
Directorate Water Quality Branch. Special Servfces Section D»partm»nt
of Fisheries and "Environment. Burlington. Ontario. Canada
RECEIVED for review June 28, 1977. Accepted OcloIL
1977. Mention of a product nams dors not imply cndorsS*
by the Central Regional Laboratory, U.S. Environmental
Protection Agency, Region V.
* ...''
Enzymic Substrate Determination in Closed Flow-Through
Systems by Sample Injection and Amperometric Monitoring of
Dissolved Oxygen Levels
Ch-Michel Wolff1 and Horacio A. Moltola*
Department of Chemistry, Oklahoma Stats University, Stillwater, Oklahoma 74074
Repetitive determinations using Injection of the sample
containing the sought-for species (substrate, S) into a con-
tinuously circulated reagent mixture (enzyme + buffer), is
described. The glucose oxidase-catalyzed oxidation of /?-
o-glucose has been chosen to illustrate methods of substrate
determination based on: S + H2O + O2 * P -f- H2O2, with P
= oxidation product(s), E = enzyme. Oxygen depletion is
monitored by a three-electrode amperomelric system allowing
determination rates as high as 700 determinations/h, and with
due precautions even about 1700 determinations/h, and
relative standard deviations (population) of less than 2%. The
method was compared with the "Beckman Oxygen Rate
Analyzer" technique, which requires about 1 min per deter-
mination. The correlation factor between the two methods
was found to be r = 0.97 (for 44 samples of human blood
serum). The proposed approach allows continuous use of the
enzyme and more than 10000 serum samples have been
Injected into the same reservoir solution without any observable
interference.
Reagent recirculation in closed flow-through systems has
been shown to be a useful ancillary device in repetitive de-
terminations by sample injection .(1-3). Enzymes, because
of their catalytic nature, suggest themselves as main reagents
to be recirculated in the implementation of such procedures.
Several enzymic methods are based on the general scheme
illustrated by the equation below:
S (substrate) + H2O + O2 5. Product(s) + H2O2 (1)
in which E is the appropriate enzyme. Determination of
glucose, uric acid, galactose, and D- and L-amino acids can be
citod as examples of substrate determinations currently done
with methods based on Equation 1. Both equilibrium and
kinetic methods are available in which the H2O2 produced is
measured by coupling reaction 1 with a second reaction in
which the H2O2 oxidizes an organic compound whose color
change(s)'or fluorescence is monitored. Methods based on
the measurement of oxygen decrease (as a result of reaction
1), mainly amperometrically, with membrane-Clark type-
electrodes or a platinum disk covered with an immobilized
enzyme layer acting as a thin reaction zone (4, 5), can also be
found in the literature, and are both useful and popi
Details of these and similar procedures can be foi:
monographs dedicated to enzymic determinations {6, _
recent reviews on the subject (8). Specificity and regeneraTOn
(through the catalytic cycle) are, perhaps, the most significant
properties of an enzyme as an analytical reagent. The first
of these properties is widely recognized and used; the second,
however, has not yet been thoroughly exploited. To our
knowledge only a few papers report the re-use of the enzyme
and attention has been mainly focused on immobilized en-
zymes (1, 9). An interesting exception to the use of immo-
bilized enzymes is a Letter to the Editor recently published
by Case and Phillips (JO), when the work reported here was
well under way, reporting recycling of the enzyme solution
in the Beckman Glucose Analyzer.
The major objective of the work reported here was to effect
recycling of the enzyme solution with sample injection
techniques in continuously flowing streams with amperometric
monitoring of changes in oxygen levels as a result of a reaction
of the type illustrated in Equation 1. The advantages of
enzyme recirculation are obvious and have been briefly
discussed above; sample injection affords the use of small
sample volumes and in conjunction with flow-through systems
allows processing of a large number of samples per unit time.
To take advantage of the latter, fast detection is needed, and
in this paper we describe the application of the three-electrode
nonmembrane system reported previously (11). Because of
the relatively low price of glucose oxidase, the determination
of glucose in:
/3-D-Glucose + O2 + H2O
c-Gluconic acid + H2O2
in which GOD = glucose oxidase, was chosen as a modeTTb
illustrate the application that is the subject of this paper. The
method illustrated here is another example of the analytical
u-e of ti an^ivnt signals generated by senes reactions and/or
processi-s (2, ,';, /:?;. As =urh it involves signal incnsMrenients
rnsd« urcltr dynamic conditions in a x.siern aporoach'.nj
-------
APPENDIX I
Interim Methods for the Sampling ?.ncl Analysis of
Priority Pollutants ir. Sedinents
and Tish Tissue
U..S. Environnental Protection Agency
Environncntal '?onitorin5 and Support Laboratory
Cincinnati, Ohio 45263
-------
APPENDIX I
CONTENTS
Sample Handling
Analysis of Sediment for Chlorinated Pesticides, Polychlorinated Biphenyls
and Non-polar Neutrals - " ;.--- . .. : -':-- -
Analysis of Fish for Chlorinated Pesticides and Polychlorinated Biphenyls
Analysis of Sediment for General Organics by Mechanical Dispersion Extraction
Analysis of Fish for General Organics by Solvent Extraction
Analysis of Sediment for Volatile Organics by Head Space Analyses
Analysis of Sediment for Cyanide
Analysis of Fish for Cyanide
.Analysis of Sediment for Phenols
Analysis of Fish for Phenols
Analysis of Sediment for Mercury
Analysis of Fish for Mercury
Analysis of Sediment for Metals (Sb, Be, Cd, Cr, Cu, Pb, Ni, Ag, Tl, Zn)
Analysis of Fish for Metals
Analysis of Sediment for Arsenic and Selenium
References
Table 1. Priority Pollutants Analyzed by Soxhlet Extraction
Table II. Base-neutral Extractables
Table III. Acid Extractables
Table IV. Characteristic Ions of Volatile Organics
-------
APPENDIX I
Interim Methods, for the Sampling and Analysis of
Priority Pollutants in Sediments
and "Fish Tissue '.
U..5. Environmental Protection Agency
Invironncntal Monitoring and Support Laboratory
Cincinnati, Ohio 45263
-------
The ac cbrip lishnent of our objective in protecting the environment
requires a reliable assessment of the present condition and a
deternination of the effectiveness of corrective measures. Decisions
which nust be nade on the need for pollution abatement and the nost
efficient neans of achieving environmental quality depend upon the
availability of sound data. Test procedures for measurement of the
presence and concentration .of substances hazardous to hunan health as
'rail as an evaluation of the quality of the environment are essential
to satisfactory decision-making.
These guidelines for sample preparation and analysis of sediment
and fish have been prepared by the staff of the Environmental
Monitoring and Support Laboratory - Cincinnati, at the request of the
Ronitnring and Data Support Division, Office of t'ate'r- and Hazardous
Bastes, with the cooperation of nany US KPA Regional Laboratories, thr>
Food and T^rug Administration, the Southeast *7ater Research Laboratory,
the Environmental Research Laboratory - Duluth, and the National
Institute for Occupational Safety and Health.
The procedures represent the current state of the art, but
improvements are anticipated as more experience is obtained. Users of
these methods are encouraged to identify problems to assist in
updating the test procedures by contacting the Environmental
Monitoring and Support Laboratory, U.S. Environmental Protection
Agency, Cincinnati, Ohio 45263.
The Manual is published and distributed by the Environmental
Research Information Center (ERIC), as one of a series designed to
|nr,ure that the latest technologies developed by. SPA and private
-------
industry are disseminated to states, municipal ities, and industries
who are responsible for environmental pollution control.
The other mar.unls in this series are:
Methods for Chenical Analysis of Hater and '..'astes
Handbook for Analytical Duality Control in Water and Uastewater
*
Laboratories *
These are available froa EP.IC, Environnen tal Research Center,
Cincinnati, Ohio 45263.
Robert C. Croue, Director
Environmental Research
Info mat ion Center
Cincinnati, Ohio 45263
DT7isht G. Ballinger, director
Environmental Monitoring and Support
Laboratory
Cincinnati, Ohio 45263
-------
SAMPLE HANDLING
Collection
1.1 'Samples shall be collected according to recognized
procedures. Preferably, all analyses should be performed
on the same sample. A minimum of 250-<>rans are required
for the total protocol.
1.2 The recotnnended container for the sediment sample is a
standard one-quart, wide-mouth, screw-cap, glass bottle
with a Teflon lid liner. It is particularly important that
glassware used in organic residue analyses be scrupulously
cleaned before initial use. At. the tine o? collection the
bottle should be filled nearly to the top with the sediment
sanple. If the sample is collected below a water coluan,
the threads and sealing surfaces should be washed off with
sanple water. "Top off" the collected sediment sample with
sample water and seal with the Teflon-lined screw cap.
Maximum effort must be nade to seal the sample with a
ninimun of gaseous head space. The sanple must remain
sealed until the aliquots for volatile organics are taken
for analyses.
1.3 In the case of small fish, a sufficient number should be
combined bv sampling site location and species to ottain
the minimum weight. The collected samples are wrappe-i in
aluminum foil , labeled 'wi t!i freezer tape, and placed in the
freezer chest with-dry ice.
Preservation
-------
2.1 The sediment sample should be l^hel.ed with freezer tape and
transferee to the laboratory in an ice chest maintained ?.t
o
or near 4 C. The sanples should be processed as soon as
possible.
2.2 Fish sanples are to be frozen at the tine of collection and
nust remain frozen until the subsannles are taken for
purgeable orsanics.
3. Processing
3.1 Sedinent . .
3.1.1 Decant the water from the top of the sediment.
Transfer the sediment into a Pyrex tray and nix
thoroughly with a Teflon spatula. Discard sticks,
stones and other foreign objects if present. 'Jei»h
five 19.0-gran portions of the sample into separate
125-ml vials. Using a crimper, tightly secure a
septum to each bottle with an aluminum seal. Store
these sample aliquots in a freezer until ready for
volatile opanics analysis.
3.1.2 Determine the percent solids in the sediment by
drying a 10-25-g portion in a tared evaporating dish,
overnight, at 103 C.
Calculate the solids using the equation:
T, solids = A x 1.00
D
where: A = weight of dry residue in grans 3 ~
weight of wet sample in jjrar.s
3.1-3 Transfer half of the remaining sediment sample back
to the original sample bottle and store at 4°C. This
-------
portion will be used for those analyses requiring a
wet sample. Spread the other half of the sample
uniformly in the tray and allow to dry at roon
tenperature'for four or five days in a contaninant-
free environment. TJhen dry less than 10% waters-
grind the sanple with a large nortar and pestle to a
uniform particle size. discard any foreign objects
found during grinding and transfer the powdered
sedinent into a wide-nouthed glass jar and seal with
a Teflon lined lid. This air dried sample will be
used for those analyses requiring an air dried
sample
3.2 Fish
3.2.1 To prepare the fish sample for analytical
pr e treatment, unwrap and weigh each. fish. Combine
snail fish by site and species until .a minimum
conbined weight of 250-g is obtained Chop the sanple
into 1 in chunks using a sharp knife and mallet.
3.2.2 Grind the sample using a large commercial meat
grinder that has been precooled by grinding dry ice.
Thoroughly mix the ground material. Regrind and mix
material two additional times. Clean out any
material remaining in the grinder; add this to the
sample and nix well.
3-2.3 Heigh five 10.0-g portions of the sample into
separate 125-nl vials. Using a crimper, tightly
secure a septum to each bottle with a seal. Store
-------
these s.triple aliquots in a freezer until ready for
volatile organics analysis.
3.2.4 Transfer the renaining fish sample to a glass
container and stor-e in a freezer for later
subsanplin?; and analysis.
4. Special Equipnent and Materials
4.1 Ice chest .
4.2 wide-nouth quart bottles x/i'th Teflon lid liners.
4.3 Teflon coated or porcelain spatula.
4.4 Pyre:: glass tray, 8 :c 12 :c 2 inch.
4.5 Tlortar and pestle (large).
4.6 Knife, heavy blade (or neat cleaver).
4.7 Mallet, plastic faces, 2 to 3-lb.
4»3 Electric neat grinder, 1/2 HT>.
4.9' Dry ice.
4.10 Aluninura foil.
4.11 Freezertape,forlabels.
4.12 Freezer.
4.13 Vials, 125-ml Hypo-Vials (Pierce Chemical Co., #12995),
or equivalent .
4.14 Septa, Tuf-Sond (pierce "12720), or equivalent.
4.15 Seals, aluninuri (Pierce r'13214), or equivalent.
4.16 Crimper, hand (?ierce "13212), or equivalent.
-------
Analysis of Sedinents for Chlorinated Pesticides,
Polychlorinated Biphenyls and non-polar '"eutrals
1 . Scope
1.1 The conpounds listed in Table I are extracted fron air-
dried sedinent by the soxhlet extraction technique. The
extract is subsequently analyzed for pesticides and ?CBs
using approved nethodsSu 1,2Sp as cited in the Federal
Resistsr'Su 3$p . ' The renaining conpounds are deterniaed
using the nethods described in "Sanpling and Analysis
Procedures for Screening of Industrial Affluents for
Priority Pollutants" ('»)... While the above "referenced
nethods have been proven for pesticides and °C3s, they have
not been sufficiently tested through extensive
experinentation for the non-polar neutral conpounds in
Table 1.
2. Special Apparatus and Materials »
2.1 Soxhlet extractor, 40-nsi 10, :/i tji 500-nl round botton
flask.
2.2 Kuderna-Oanish, 500-nl, with 1 Q-rnl graduated receiver and
3-ball Snyder colunn.
2.3 Chrornato^-raphic colunn - Pyrex, 20-nn ID :-. approximately
AOO-mn long, with coarse fritted plate on botton.
3 Procedure
3*1 Extraction
3.1.1 T'ei£h 30.0-grans of -the previously air-dried sanple
into a tared 200-r.l beaker. Add 3-r.l distilled vater
-------
(10"' of sanple weight), ni" well and allow to stand
for 2 hours while nixing occasionally.
3.1-2 Place about 1/2" of pr e-ext rac tec! g.lass wool in the
botton of the soxhlet extractor chanber and
quantitatively transfer the contents of the beaker
into the chanber. Place a second glass wool plug on
top of the sanplc. Wash the 200-al beaker and all
nixing tools several tines x/ith a 1:1 hexane acetone
mixture. Cycle the wash nixture through the
extractor using a total of 300-nl of the nixed
'solvent.
3.1.3 Attach the extractor to a 500-nl round botton flask
containing a boiling stone and extract the solids for
16 hours*
3.1*4 After extraction is connlete, dry and filter the
extract by passing it through a 4" colunn of hexane-
»
washed sodiun sulfate. Hash the 500-cl flash and the
sodiun sulfate with liberal anounts of hexane.
Collect the eluate in a 500-nl K-D evaporative flask
with a 10-nl anpul. Concentrate the sanple extract
to 6-10-nl.
3.2 Clean u? and set>oration.
3*2.1 Adjust the sanple extract volune to 10-nl" and cleanup
the extract by Florisil colunn chronatography
according to the 304 (g) nethodoic^y for ?CBs, part
10.3. For sulfur renoval continue with part IP.5.3.4-
of that method. "OTE: If sulfur crystals are
-------
present in the extract seperate the crystals fron the
sanple by decantation.
3.2.2 Analyze the Florisil cluates for the pest.icides
appearing in Table I, according to the approved
method for pesticides. The PC3s appearing in Table
I, should be determined as d-escribed in Reference 2.
3.2.3 Analyze regaining compounds of Table I, column C, by
"Sampling and Analysis Procedures for Screening of
Industrial Effluents for Priority Pollutants" A- )
3.3 Standard quality assurance protocols should be employed,
including blanks, duplicates and dosed samples as described
in the "Analytical Quality Control Handbook". Dosing, can
be accomplished by injecting 1-20-ul of a standard into the.
homogenized sediment contained in the soxhlet extractor
chamber.
4. Reporting of Data
»
4.1 Report results in ug/kg on a dry weight basis using the
percent moisture values determined earlier. Report all
quality control data with the analytical results for the
samples.
-------
Hlvsis'.of Fish for Chlorinated Pesticides and Pplychlor in a ted Diphenyls
1. Scope
1.1 The chlorinated pesticides and polychlorinated biphenyls
(PCBs) listed in Table I are extracted fron fish using
either nethod A or B as described below. Method A enploys
a blender, whereas a Tissumizer or the equivalent is
required for Method B. Either procedure results in an
extract that can he incorporated directly into approved US
EPA test procedures for pesticides or PC3s as cited in
the Federal Register.(3) .
2. Special Apparatus and Materials . >
2.1 Method A Only
' . '
2.1.1 Blender, hij»h-speed - Waring Blender, Gourdes, Qnni
''
Mixer, or equivalent. Explosion proof nodel
reconnended. Quart container is suitable size for
.
routineuse.
. ' .
2.1.2 Suchner funnel - porcelain, 12-cn.
*
2.1.3 Filter paper - 1 10-nn sharkskin circles.
2.1. A Flask, vacuua. filtration - 500-nl. '
2.2 Method B Only .
2.2.1 tissunizcr SDT-1S2EN'(available fron Teknar Conpany,
P.O.' Box 37202, Cincinnati, Ohio 45222) or
equivalent.
2.2.2 Centrifuge - capable of handling 100-nl centrifuge
tubes. ,'
2.3 Method A & B
-------
2.3.1 ^.uderna-Danish concentrator - 5vO-n.l, with IH-nl
graduated receiver and 3-ball Snyder coluan.
2.3.2 Chroriatographic column - pyre::, 20-nn id x
approximately 400-nn Ion?,, with coarse fritted plate
on botton.
3. Proc edures
3.1 ' Method A . -
3.1.1 T7eigh a 25 to 50-3 portion of frozen, ground fish and
add to a high speed blender. Add 100-g anhydrous
Na^SO^ . to combine with the water present and
to disintegrate the sample. Alternately, blend and .
nix with a spatula until the sample and sodiun
sulfate are well nixed. Scrape dovn the sides of the
blender jar and break up the caked material with the
spa'tula. Add 150-nl of hexane and blend at high
speed for 2 tain.
3.1.2 Decant the hexane supernatant through a 12-cn Buchner
filter with two sharkskin papers into a SOQ-nl
suction flask. Scrape down the sides of the blender
jar and break up the caked material with the spatula.
Re-e?
-------
blending, transfer the residue from the blender jar
to the P.uchner, rinsing the hlendcr jar and material
in the Buchner with three 25 to 50-nl portions of
hexane. Iranediately after the last rinse, press the
residue in the Buchner v/ith the hot ton of & clean
beaker to force out the regaining hexane.
3,1,4 Pour the combined extracts and rinses through a
colunn of anhydrous *TA. ^'S'O,*' . -, 2'0-na x 100-an,
and collect the eluate in a 500-m'l Kuderna-Danish
concentrator. T7ash the flash and then the-column
with snail portions of hexane and concentrate the
extract below 10-r:l.
3.2 Method 3 .
3.2.1 "eish 20.0-g of frozen, ground fish and add to a 100-
ral centrifuse tube. Add 20-nl of hexane and insert
the Tissuriizer into the sanple. Turn on the
Tissunizer and disperse the fish in the solvent for
1 nin. Centrifuge and decant the solvent through a
colunn of anhydrous Na.^SOj-i » 20-nn x lOO-nm,
and collect the eluate in a 590-i?.l Kuderna-Ranish
concentrator. ,
3.2.2 Repeat the dispersion twice usins a 20-nl aliquot
: each tine, combining all dried portions of solvent in
the concentrator. Rinse the Tissunizer and the
colunn with snail portions of hexane and concentrate
the extract below 10-nl.
3 Cleanup and Analysis
-------
3.3-1 Unless prior experience would indicate the fish
species fat content is low (less than 3 g/per
extract), thehexaneacetonitrile clean-up procedures
described in the reference methods should be
followed. In all cases, Florisil column
chrornatog raohy should be used to clean up the
extracts before gas chroria togr aphy f g^j . An
electron capture detector is used for "final
measurement, and results are calculated in u.°/kg.
Identifications can be confirmed by GC/MS techniques
as described in the analytical protocol for
was t ewa t ers.. f.4 I . .
3.4 Quality Control
3.4.1 Standard quality assurance protocols should be
employed, including blanks, duplicates, and dosed
samples as described in the "Analytical Quality
Control Handbook"./ 7\ ..
3.4.2 Dose fish sample aliquots by injecting raininun
amounts (^20-ul total), of concentrated pesticide or
PCB solutions into the solid subsanple 10 to 15
minutes before extraction.
Reporting of Data .
4.1 Report results in ug/k» on a wet tissue basis. Report all
quality control data with the analytical results for the
samples.
-------
Analysis of Sediment for General Organics
by Mechanical Dispersion Extraction.
1 . Scone
1.1 ; This method -is designed to determine those "unambiguous
priority pollutants" associated with the consent decree
that are solvent extractable and amenable to gas
chronatography. Tables I, II, and III are a surmary of
compounds that should be extracted at an 80-100%
efficiency. It is a "C/MS method intended for qualitative
and seni-quantitative determination of these compounds.
Although this approach has not been sufficiently tested
through extensive experimentation, it is .based on.
laboratory experience and is presently considered to be a
reasonable analytical approach for these; organic materials
in sed inent
1.2 This Tnethod is not applicable to those very volatile
'pollutants listed in Table iv.
2. Special Apparatus and Materials
2.1 Mechanical dispersion device.
2.2 Cen.trifuge - capable of handling 100-al centrifuge tubes.
2.3 Separatory funnels - 2-liter v;ith teflon stopcock.
2.4 Sieve, 20 nesh.
3. ? r o c e d u r e
1.1 'Teigh 20.0-g of \re t, well-nixed scdinent into a 100-nl
cent r if uge t;ib e. If the sediment contains grit larger, than
20 nesh, it is necessary to extrude the sanple through a
-------
20 mesh sieve in order to prevent danage to the mechanical
dispersion device. Add 20-nl acetonitrile and insert the
dispersion device into the sample. Disperse the sediment
into the solvent for 1 nin. Centrifuge and decant the
sblvent into a 2-liter separatory funnel containing 1300-nl
of a 2 percent aqueous solution of sodium sulfate
previously adjusted to ph 11 with 6 H NaOH. Repeat the
dispersion twice, using a 20-nl aliquot each tine, and
combine the acetonitrile washings in the separator;/ funnel.
Caution! the dispersion should be carried out in-a fune
hood to avoid exposure to acetonitrile.
3.2 Extract the aqueous acetonitrile solution in the separatory
funnel with 5.0-nl of hexane for 2 nin. Drain the aqueous
layer into a 2-liter Srlenmeyer flask and pour the hexane
extract through a short column of anhydrous sodium sulfate
prerinsed with hexane. Collect the dried extract in a 500-
nl Kuderna-Danish (K-D) flask fitted with a 10-nl ampul..
P.epeat the extraction and drying steps twice, combining the
extrac ts.
3.3 Evaporate the extract to 5 to 10-ral in. a 500-nl K-D
apparatus fitted with a 3-ball Snyder colunn and a 10-nl
calibrated receiver tube. allow the K-D to cool to roon
temperature. Remove the receiver and adjust the volume to
10-nl. Label this as the base neutral fraction. If
additional sensitivity is required, add fresh boiling
chips, attach a two-ball micro-Snyder colunn, and carefully
evaporate to 1.0-nl or when active distillation ceases.
-------
3.4 Return the aqueous acetonitrile solution to the separatory
funnel and adjust the ph with 6 N HC1 to ph2 or less.
Extract three tines with 60-nl of hexane each tine.
Combine the extracts, dry, and concentrate as described
above. Label this as the acid fraction.
3.5 Analyze both extracts according to "Sampling and Analysis
Procedures for Screening of Industrial Effluents for
Priority Pollutants" Y.4J .. Should the acetonitrile
partition used in this procedure not sufficiently remove
interferences, florisil(2J , alunina (5 \ , and. silica
gel (2! column chromatbgraphic clean-up and separation
techniques can be employed. Sulfur can be removed by
treatment ?/ith mercury./2J .
3.6 Standard quality control assurance protocols? should be
employed, including blan!;s, duplicates and' dosed samples,
as described in the "Analytical Quality Control
Handbook" ,/. 7J . Dosing can be accomplished in injecting 1-
20-ul of a standard solution into the homogenized sedinent
contained in the centrifuge tube.
4. Reporting of Data
4.1 Report results in ug/Ug on a dry weight ' basis using the
percent moisture values determined earlier. Report all
quality control data with the analytical results for the
samnles
-------
Analysis of Fish for General Or^anics by-Solvent Extraction
Scone
1.1 This method is designed to determine those "unarab ijjuous
priority pollutants" associated with the Consent
Decree f '* J that are solvent extractable and anena^hle to
gas chronatography. These compounds are listed in Tables
.II and III. It is a GC/MS method intended for qualitative
and seni-quantitative determination of these cor?pounds.
Although this approach has not been sufficiently' tested
through extensive experimentation,. it is based on
laboratory experience and is presently the best analytical
(.
approach for these organic-materials.in fish.
'Special Apparatus and Haterials
2.1 Tissunizer ST1T-182EM (available from TeV.nar Company, P.O.
Box 37292, Cincinnati, Ohio 45222) or equivalent.
2.2 Centrifuge - capable of handling 100-nl centrifuge Cubes
2.3 Separatory funnels ~ 2-liter with teflon stopcock.
2.4 Organic free water - prepared by passing distilled water
through an activated carbon colurin.
Procedure
3.1 T?eigh 20.0-g of ground, h oraogeneous fish and add to a 100-
nl centrifuge tube. Add 20-r,l of acetonitrile an-J insert
the tissuniser into the sample. Turn en tissunizer aad
disperse the fish into the solvent for 1 nin. 'Centrifuge
and decant the solvent into a 2-liter scparatory funnel
containing 1300-nl of a 2 percent aqueous solution of
-------
so.iiuTn sulfate. "epeat the dispersion f.-'ice, usinr; a 20-nl
aliquot each tine, and conbinc the acetonitrila in the
separa tory funnel.
Caution: the dispersion should be carried out in a fune
hood to avoid exposure to acetonitrile.
3.2 Adjust the ph of the sodium sulfate acetonitrile solution
with nN NaO'l to ph 11 or greater. Use -nultirange ph paper
for the neasurenent. Extract the aqueous acetonitrile
solution with 60-nl of he;:ane. Shake the separatory funnel
for 2 nin. Drain the aqueous layer into a -2-liter
erlenraeyer flash and pour t'he hexane e:;tract through a
short column of prerinsed anhydrous sodlun sulfate.
Collect the dried extract in. a 500-nl Xuderna-Oanish (£-0 )
flask fitted with a 10-nl anpul. repeat the extraction and
drying steps t'Jice, conbinins the extracts. Evaporate the
extract to 5 to 10-nl in a 500-nl "-n apparatus fitted with
a 2-hall Snyder colunn and a 10-nl. Analyze by GC/MS. If
additional sensitivity is required, add fresh boiling
chips, attach a two-ball micr.o-Snyder colunn, and carefully
evaporate to 1.0-nl or when active distillation ceases.
3.3 Return the aqueous acetonitrile solution to the separatory
funnel and adjust the nT! with 61! HC1 to pH 2 or less.
Extract three tines with 60-nl of her.ane each tine.
Conbine the extracts, dry, and concentrate as described
above. Analyze by <~-C/MS.
MOTE: Should the partition used in this procedure not
sufficiently remove the lipid naterial, gel perneation can
-------
be enployed. However, special expensive equipnant is
necessary for this pr oc ed ur e .. r fi .J .
3.4 Standard quality assurance protocols should be enployed,
including. blanks, duplicates, and dosed samples, as
described in the "Analytical Quality Control
Handbook" Y 7 J . DosiriR can be acconplished by "injecting 1
to 20-ul of a. standard solution into, the honogenized tissue
contained in a centrifuge tube.
Reporting of Data
A.I Report results in ug/kg on a TTC t tissue basis. Report all
>
quality control data with the analytical results for the
sanples.
-------
Analysis of f.edinent for Volatile Or panics
b y H e a d S n a c e An a 1 y s e s
1. Scope
1.1 This method is designed to deternine those "unambiguous
'priority pollutants," associated with the Consent
Decree ( f- \ that are anenah-le to head space analyses.
These compounds are listed in Table IV. It is a GC/TTS
method intended for quali'ta tive and semi-quantitative
deternination of these compounds. The head space analyses
and the liquid-1 iquid extraction nethod.s are conplec_en tary
to one another. There is an overlap between the two: sane
conpounds can be recovered by either method. The
efficiency of recovery depends on the vapor pressure and
water solubility of the conpounds .involved. Generally, the
overlap involves compounds boiling between 130 and 150 C
with a water solubility of approximately 2 percent. Then
compounds are efficiently recovered by both methods, the
chromatojraphy determines the method of choice. The
-------
1.3 Although the above approach has not been sufficiently
tested through the extensive experinentat ion, it is based
on laboratory experience and is presently considered to be
the best analytical approach for volatile organic materials
i n s e d i n e n t s
2 . ' Special AP par at irs and Materials
2.1 Oas-tight syringe - 5-cc.
2.2 Head spa'ce standard solutions - Prepare two standard
nethanol solutions of the conpounds listed in Table I? at
the 50-rif>/ul and 2.50-ng/ul concentrations. The 'standard
».
solutions should be stored a.t less than 0 C. Solutions
should be allowed to uarn to room temperature before
dosing. Fresh standards should be prepared weekly.
Procedures for preparing standards are outlined in the
purge and trap section of Reference 4.
2.3 Vials, 125 nl "Hypo-Vials" (Pierce Chenical Co., £12995),
or Equivalent.
2.4 Septa, "Tuf-Bond" (Pierce 212720), or equivalent.
2.5 Seals, aluninun, (Pierce '13214), or equivalent.
2.5 Crinper, hand, (Pierce "13212), or equivalent.
3. Procedures
3.1 Place 10.0-g each of the well-nixed, wet untreated sediment
sanple into five separate 125-nl septur. seal vials.
3.2 Dose one sanple vial through the septun *rith 10-ul of the
50-ng/ul standard nethanol solution. ^ose a second vial
with 10-ul of the 2.50-ng/ul standard.
-------
3.3 vlace the two dosed sample vials and one non-dosed sample
o
into a 90 ;c water bath for 1 hour. Store the two remairiinrj
o
snTriples, near A .C for possible future analyses-
c
3.4 Uhile maintaining the sample at 90 C, withdraw 7.0-ml of the head
gas with a jjas tight syringe and analyze Hy injecting into
a CC , operating- under the conditions recommended in
Ref erence ./. 4 ]. .
3-5 Analyze all three' samples in exactly the sane manner.
Subtract the peak areas of "compounds found in the undosed
sample from the corresponding conpounds contained in the
dosed sanples. Construct a calibration curve fron the
corrected dosed data; quantify the unknown.
MOTE: If the calculated sample concentration is greater
than the concentration of the dosed standard used.in the
dosing step, it is necessary to prepare additional
standards in order to bracket the unknown.
3.5 Standard quality assurance protocols should be employed,
including blanks, duplicates, and dosed sanples, as
described in the "Analytical Quality Control
Handbook" ( ?) .
4. Reporting, of Data
4.1 leport all results in up,/kg on a dry weight basis using the
percent moisture values determined earlier. P.eport all
quality control data with the analytical results for the
sann1es
-------
kn a lysis of ?ish for Volatile Or panics by Head. Space Analyses
Scone
1.1 This nethod is designed to determine those ".unambiguous
priority pollutants" associated with the Consent
Decree f 4j that are amenable to head s.pace analyses.
"These compounds are listed in Table IV. It is a GC-MS
method intended for qualitative and seni-qnan t itative
determination of these compounds. The heart space analyses *
and the liquid-liquid extraction methods are comp lejrjentary
to one another. There is an overlap between the two: sotie
compounds can be recovered by either method. The
efficiency of recovery depends on the vapor pressure and
water solubility of the compounds involved. Generally, the
0
overlap involves compounds boiling between 130 and 150 C
with a water solubility of approximately 2 percent* TJhea
compounds are efficiently recovered by both methods, the
chromatojraphy determines the method of choice. The CC
conditions selected for the head space method are,
generally, not suitable for the determination of compounds
eluting later than chlorobenzene.
1.2 Although the above approach has not been sufficiently
tested through extensive experimentation, it is based on
laboratory experience and is presently considered to be the
best analytical approach for volatile organic materials in
fish.
Special Apparatus and Materials
-------
2.1 Sonifier Cell Disrupter "-350 with nicroprobe ( naiufac Cured
by Brawson Sonic Power Co., Panbury, Connecticut) or
equival en t .
2.2 Gas-tight syringe - 5-cc.
2.3 Organic free water - Prepared by passing distilled water
through an 'activated carbon column.
2.4 Head space standard solutions - Prepare three standard
nethanol solutions of the compounds listed in Table I? at
the 50-ng/ul, 150-ng/ul, and SOQ-ng/ul concentrations. The
standard solutions should be stored at less than 0*0.
Solutions should be al loved to warn to room tesiperature
before dosing. "Fresh standards should be prepared . weeMy«
Procedures for preparing standards .are outlined in the
purge and trap section of Reference
Procedure
3.1 Renove four of the sanple vials containing 10-0-g of
homogenized fish from the freezer. Open the vials and ad.i
10-hl of organic free water to each while the fish is still
frozen. Sonify the fish for 30-scc at naximun probe power.
Immediately reseal the vials.
3.2 Dose one sanple vial through the septun below the water
level with 10-ul of the 50-ng/ul standard nethanol
solution. Dose a second vial x?ith 10-ul of the 150-n.^/ul
standard and a third vial with 10-ul of the 300-n3/ul
standard.
o
3.3 Place all four sanple vials into a 90 C water bath for 1 h.
-------
3.4 Uhile naintaining the snnple at 90°C, withdraw 2.0-nl of
the head gas with a -?,as ti^ht syringe a'nd analyze by
injecting into, a GC , operating under thft conditions'
reconnended in Re f erence £ 4 J, .
NOTE: Specific GC detectors can be substituted for the £S.
3.5 Analyze the undosed sanple first, followed by the 50-ng/ul
dosed sample. If no compounds of interest are found in the
undosed sample and _the dosed sample produces peaks Co
indicate recovery of the protocol conpounds, do not analyze
the remaining samples. Calculate lower linits of detection
based on the response obtained fron the dosed saaple* If
conpounds are observed in the undosed sample, analyze the
two renaining dosed samples in -exactly the sane rian.aec»
Subtract the peak areas of conpounds found in the undosed
sanple fron the corresponding conpounds contained in t£;e
dosed data; quantify the unknown.
NOTE: If the calculated sanple concentration is greater
than the concentration of of the dosed standard used in the
dosing step, it is necessary to prepare additioaal
standards in order to bracket the unknown. Utilize the
renaining sanple in the freezer for this purpose.
3.6 Standard quality assurance protocols should be ennloyed,
including blanks, duplicates, and dosed sannlss, as
described in the "Analytical Quality Control
Handbook" (7 j -
4. p.e oort ing of Data
-------
4.1 Report all results in u^/kr» on a wet tissue basis. Report
all q.uality control ^ata' with the analytical results for
the satin 1 e s .
-------
Analysis of Sediment for Cyanide
1. Scope and Application
1.1 This method is used for the determination of cyanide in
sediments. Insoluble cyanide complexes are dissolved in
10% sodium hydroxide. The cyanide, as hydrocyanic acid
(HCK), is released from the sanple by ueans of a reflux-
distillation and absorbed in a scrubber containing sodiun
hydroxide solution. The cyanide in the absorbing solution
is then determined b}' volumetric tltratiou,
colorine tr ically or no ten.tione t r ical ly .
1.2 For cyanide levels exceeding 0.2 ng per 200-nl of. absorbing
liquid, the silver nitrate titriinetric nethod is used. For
cyanide levels below this value, the colorlnetric procedure
is used. The probe nathod na:/ be used for concentrations
of 0.001 to 200-ng per 200-rnl absorbing liquid.
2. Sanple Preparation
2.1 Although a dry sanple is preferred, a wet sanple .nay also
be taken for analysis. In either case the sedinent' sannles
must be well-mixed to insure a representative aliquot.
3. Interferences
3.1 Interferences are eliminated or lessened by using the
distillation procedure.
3.2 Fatty acids t/ill distill and form soap under the alkaline
titration conditions. Therefore acidification and
extraction with iso-octane, hexane, or chloroform is
recommend ed.
-------
3.3 Annonia and thiosulfate interfer with the electrode method
yielding higher rieasur einents of cyanide. ion activity than
are actually present.
Pro par at ion of Calibration Curve
4.1 The calibration curve is prepared as described in step P . °
Deferences.
4.2 The standards mist contain the sane concentration of **aOH
(7.1) as the sample. .
4.3 At least one standard should be treated as outlined below.
4.4 The calibration curve is prepared by plotting the
absorbance or the n.v reading versus the cyanide
concentration.
Sann le Procedure
5.1 Place a weighed portion of the well-nixed sedinent (1 to
10-3) in an ROO-nl beaker w.ith 500-nl of 10% N-aOH solution
and stir for 1 hour. .
5.2 Transfer the . mixture to a 1 liter foiling flash. Rinse the
beaker with several portions of dcionized distilled water
and add to the boiling flask.
5.3 Add 50-nl of 5'' ITaO'T solution to the absorbing tube and
dilute if necessary with deionised distilled water to "
obtain an adequate depth of liquid in the absorber.
Connect the boiling flask, condenser, absorber, and. trap in
the distillation train as shown in Figure 1, Reference f! .
5.4 Add 50-nl of cone. "-,SOx. slowly thru the air inlet
tube. Rinse with distilled water. Add 20-nl of v.s Cl^
(510-g/l) solution thru the air inlet tube and
-------
again rinse with distilled water. Continue with stops ". £
and 3.5 (Reference 8). If the colorinetric nethod is used,
continue thru G.7 (Reference 3.)
5.5 Record the absorbance or nv reading and determine the
cyanide concentration frotn the calibration curve.
£. nua1i ty As surance
6.1 Initially denonstratc quantitative recovery with each
distillation digestion apparatus by conparing distilled.
aqueous standards to non-distilled aqueous standards. Each
day, distill at least one standard to confirm distillation
efficiency and purity of reagents.
6.2 At least 15% of the cyanide analyses should consist of
duplicate and spiked samples. Duality control linits
should be established and confirmed as described in Chapter
6 of the "Analytical Quality Control Handbook."
Reference f 7 I .
7. Reporting oj Data
7.1 Report cyanide concentrations on a dry weight basis as
follows: less than 1.0-ng/kg, to- the nearest 0.01-ns/kg;
1.0-rng/kg and. above, to two significant figures.
7.2 Report all quality control data with the analytical results
for the sa-iples.
-------
Analysis of Fish for Cyanide
1 . 1coDe and Apnlicat ion
.1.1 This method is used for t.he determination of cyanide in
fish. All samples nust. be distilled prior to the
analytical determination. Tor cyanide levels exceeding 0.2
ng/200-ral of absorbing liquid, the silver nitrate
titrinietric method is used. 7or cyanide levels belo'..1 tliis
value, the colorinetric procedure is used.
2 . Sanple Preparation
2.1 A 5-g portion of the frozen, ground fish (see "Sample
Handling") is used for the analysis- The sample should be
thawed before the analysis begins.
3. Preparation of Ca lib ration Curve
3.1 The calibration curve is prepared fron values for portions
of spiked fish tissue distilled in the nanner used for the
tissue sample being analyzed. For preparation of the
calibration standards, choose and weigh a 50-g portion of
fish and blend in a Uaring blender (or equivalent) with 10-
nl of 10" VaOH and sufficient deionized distilled water to
bring the volume of the nixture to 500-nl.
3.2 Using a volumetric pipet which has had the tip removed,
withdraw eight 50-nl portions and place in a series of 1
liter boiling flasks. Seven of the flasks should bo spifced
with progressively larger volumes of the cy.anide standard
as given in 3.3, Reference S. Adjust the final volume .of
each flask to 500-nl with deionized distilled water.
-------
3-3 Add 50-nl of 5'i MaOH solution to t'ne ah so rbii!?, tube and
"i
dilute, if necessary, with deionized distilled water to
obtain an adequate depth of liquid in the absorber.
.Connect the boiling flask, condenser, absorber, and trap in
the train as shown in Figure 1, Preference 3.
3.4 Continue with step 8.2 through 3.7, Reference 8.
3.5 The -calibration curve is prepared by plotting the
absorbance versus the cyanide concentration. The blank
absorbance value must be subtracted from each value before
plotting the curve.
4. Sanple Procedure
4.1 Place a weighed portion of the ground fish (approximately
5-g) in a blender with 100-nl of deionized distilled water
and 1-ral of 5% NaOH solution.
4.2 Blend until a homogeneous mixture is obtained and transfer
to a 1-liter boiling flask. P.inse the blender with several
portions of deionized distilled water totaling 400-tal ami
add to the boiling flask.
4.3 Add 50-nl of 5% MaOH solution to the absorbing tube and
dilute if necessary with deionized distilled water to
obtain an adequate depth of liquid in the absorber.
Connect the boiling flask, condenser, absorber, and trap in
the distillation train as shown in Figure 1 and continue
with step 8.2 through .1.7, Reference 8.
4.4 Read the absorbance and deternine the cyanide concentration
fron the calibration curve.
5 . Qual if/ As sura nee
-------
Initially, demonstrate quantitative recovery with .each
distillation disestion apparatus by comparing distilled
aqueous standards to non-dis t il ler1 aqueous standards. Fach
day, distill at least one standard to confirn distillation
efficiency and purity of reagents.
5.2 At least 15 of the cyanide analyses should consist of
duplicate and spiked sanples. Quality control limits
should be established and confirmed as described in Chapter
6 of the "Analytical Quality Control flandbooh"
Reference..^?) .' . '
?>.epo r ting of T) a t a
6.1 Report cyanide concentrations as follows: less thaTv 1.0-
ci3/kg, to the nearest 0.01-ng; 1.0-ng/!:g and above, to two
significant figures.
6.2 Report all quality control data with the analytical results
for the sanples.
-------
Analysis of Sedinent for Phenols
1 Scone and A.pp I ica tion
1.1 This net hod is used for the determination of pher.olics in
sediments. All samples nust be distilled prior to the
determination of phenols, using the procedure j»iven on page"
57*, Reference 9. Use method 510 B for samples that
contain less than 1-n^ phenol/kg and method 510 C for
s.inples that contain rtore than l-n» phenol/kg.
1.2 The 4-anino-antipyrine nethod does not deternine. those
parasubstituted phenols in uhich the substitution is an
alkyl, aryl, nitro, benzoyl,.nitroso, or aldehyde group.
--? Sam nl e Preservation and Preparation
2.1 Biological degradation is inibited by cooling the sample to
»
AC. If the sanple cannot be analyzed with 24 hours it
should be frozeri-
2.2 A 5-g portion of the wet, or air dried sediment is used for
the analysis. If the sanple has been frozen it should be
thawed before the analysis begins.
3 . Pr er>ara tion of Cal ib rn tion Curve
3.1 The calibration curve is prepared as described on p.579,
/».a.3 (Ref . Q) for samples containing less than l-ng/hs and
p. 591 for sanples above l-nr»/V.j;.
3.2 Record the absorbance of the standards and plot the values
against nicrosrans of phenol.
4. G.innle^rocedi.ire
-------
A.I Place a 5-g portion of the I/P. t, or air dried sedinent into
a 200-rnl beaker with 100-nl of distilled water. *'ix r/ell
- and lower the pi! to 4-0 with (1 + ?) H -, PO,. . using a
pT^neter.
4.2 Add 5-nl of 10 CuSO,. solution, nix and transfer to -a
1-liter distilling flask.
4-3 Rinse the beaker with several portions of distilled water
and add to the distilling flask. Adjust the vo'lirae in the
flask to 500-nl.
4.4 Using a 50^-nl graduated cylinder as a receiver, begin the
distillation as described on p. 577, nethod 510 A; 4b,
Reference^.
4.5 Continue with the procedure using either the Chlbroforn
Extraction "ethod 510 B, p. 577, Reference 9 or the T>irect
Photometric 'Tetho-1 510 C, p. 5BO, Reference . 9.
4.*> ^Record the absorbance and deternine the siicrograns of
phenol fron the appropriate calibration curve.
Ouality Assurance .
5.1 Demonstrate quantitative recovery with each distillation
apparatus by comparing aqueous distilled standards to non-
distilled standards. Each day, . distill at least one
standard to confirm the distillation efficiency and purity
of reagents .
5.2 At least 15C^ of the phenol analyses should consist of
duplicac an .1 spiked smples. Ouality control linits should
be established and confirmed as described in Reference 7.
Reporting of Tat a
-------
T.I Report phenol concentrations on a dry weight basis as
follows: ...
Method 5 I'O 7? , to the nearest 113/kg
Method 510 C, for less than 1.0-us/kg to the nearest
Q.01-u«5 and for l.O-njj/ks and above to tvo
siginificant figures. . i
t
G.2 Report all qunLity control data './hen reporting results of
sample analysis.
-------
Analysis of Fish for Phenol
Scope a P. ri Ap plica tip n
1.1 This method is used for the determination of phenolics in
fish. All samples nust. be distilled prior to the
deterrainatiori using the procedure given on pase 576.
Reference ?. Use netho4 510 ?, for samples that contain
less than 1-ng pheool/kr» and nethod 510 C for samples that
contain nore than l-nj phenol/kg.
1.2 The 4-anino-antipyrinn nethod does not determine those
para-substituted phenols in which the substitution is an
alkyl , aryl, nitro, benzoyl, nitrosd, or aldehyde group.
Sa?rpl e Prer»arati on
2.1 A 5-g portion of the frozen, ground fish (see "Sample
Handling") is used for the analysis. The sanple should be
thawed before the analysis begins.
Preparation of Ca1ibration Curve
3.1 The calibration curve is pr-epared fron values for portions
of spiked fish tissue distilled in the nanner used for as
the tissue samples being analysed. For preparation of the
calibration standards, choose and weigh a 50-g portion of
fish and blend in a Waring blender (or equivalent) with
sufficient deionizad distilled water to brine.the total
volume of the nixture to 5QO-nl
3.2 Transfer a 50-nl portion of mixture to a beaker usin^ a
volumetric pipet which has had the tip renoved an-.i
determine the volume of (14- 9) -'.-.PO^. required to
-------
lower the pH to 4'. (\ usin^ cither nethyl orange indicator
or a pH neter. This volune of (1 + 9) H ., PQ^. is to
be added to each 50-nl portion of fish mixture prior to the
distillatiOTi step which follows.
3.3 transfer 50-ral portions, of the blended fish nixture to the
distillation apparatus as shown in Figure 31S:1, p. 241,
Reference 9, adding the volune of K.,pCU. (determined
above) to lower the pH to 4.0. Add 5-nl of a lOf, CuSCU.
solution and appropriate volunes of the standard phenol
solution (Reference 9, p. 579, 3C), to each distillation
flask. A blank and seven standards should be distilled for
preparation of the calibration curve. Adjust the voluna in
the distillation flasV. to 500-nl. Use a 500-nl graduated
cylinder as a receiver.
NOTE: The nininun detectable quantity is 1-ug/l phenol in
a 500ml distillate.
3.4 Begin the distillation and continue until a distillate
volune _of 450-ml is obtained. Stop the distillation and
add 50-ral deionized distilled water to the distillation
flask after boiling has ceassd . Continue the distillation
until a total of 500-nl has been collected. If the
distillate is turbid, acidify with (1 + 9) H^pO^
and repeat the distilation as described.
3.5 Continue with the procedure as given in the chloroform.
extraction nethod 510 E, p. 577, Deference 0. ".eat? the
absorbance of the standards against a reagent blank at a
-------
etift tv. of A6°-nn. Plot absorbance against nicrc-rrans
of phenol for the calibration curve.
3.6 Alternatively, follow the direct photometric nethod (510 C,
p. 530, Reference 9), for those sanples in which the phenol
'concentration exceeds l-n^/k^.
Sanpl e P r o c e d u r .e
4.1 Place a weighed portion of the ground fish (approximately
5-g) in a blender with 100-nl of distilled water. Slend
until a honogeneous mixture is obtained and transfer to a
1-liter boiling flask. .
4.2 Rinse- the blender with several portions of distilled water
and add to the distilling flask. Add a volune of (1 -} 9)
Ti^PO^* to brin« the pH of the mixture to 4.0 (the
sarie volune as that deternined for preparation of the
calibration standards can be used)= Add 5-nl of 10*
CuSO^a . solution and adjust the total volune to
approxina tely 5.00-nl. Use a 500-nl graduated cylinder as a
receiver.
4.3 Begin the distillation and continue as described in 3.4
through 3ft above. Read the absorbance and deternine the
us of phenol fron the calibration curve*
Duality Assurance .
5.1 r>enonstrate quantitative recovery with each distillation
apparatus by comparing aqueous distilled standards to non-
distilled standards. Hach day, distill at least one
standard to confirm the.distillation efficiency an;1 purity
ofreagents.
-------
5.2 At least 15"' of the phenol analyses should consist of
duplicate and spiked sanples. ^unlity control limits
should be established and confirmed as described in
Reference / 7\ .
?.enortinq of Data
6.1 Report phenol concentrations as follows:
Method 510 B, to the nearest uj/kg
Method 510 C, for less than l.O-ug/kg to the nearest
O.Ol-uj and for 1.0-113/kg and above to two
significant figures.
5.2 Report all quality control data t/her. reporting results of
sanple analysis.
-------
Analysis of ^edinerit for Mercury
1. Scope and Application
1.1 This nethod is used for the deternination of total nercury
(organic and inorganic) in sedinent. A weighed portion of
the sanple is digested with aqua regia for 2 ninutes at
95.C followed by oxidation with potassiun p crnangariate -
Mercury is subsequently measured by the cold vapor
technique
1.2 The range of the method is 6.2 to 5-ug/g but nay he
extended above or below the normal range by increasing or
decreasing sanple size or through instrunent and recorder
control.
1.3 For a complete description of the method the reader is
/
referred to "Methods for Chenical Analysis of Vater and
Haste ", (") pages 134-138.
'2 . Sanpl e Preparation
2.1 Although a wet sanple nay be taken for analysis, a dry
sanple provides for ease of handling, better honogeniety,
and better storage.
3. Preparation o f Calibration Curve
3.1 The calibration curve is prepared using distilled water
standards, treated in the sana manner as the sedinent
sanples being analysed. The calibration procedure is
described on p. 135, Reference 3.
-------
3.2 Th?. calibration curve is prepared by p Totting the peak
height versus the mercury concentration- Inf. petal: height
of the blank is subtracted fron each of the other values.
4. Sanpl e Procedure
4.1 ' Ueif»h 0.2 to 0 . 3-g portions of the dry sanple and place in
the bottom of a SOD ' bottle. (If a wet saapl.e is to be
analyzed a proportionately larger sample nust be taken.)
Add 5-nl of distilled water and 5-rnl of aqua rejia and
9 '
place the bottle in a water bath maintained at 95 C for 2
ninutes.
4.2 Cool, add 50-nl distilled water, 15-nl of potassium
pernansanate solution and return the bottle to' the t/ater
bath for an additional 30-ninutos. Add additional
KM/nC^. . >, as necessary, to naintain oxidizing
conditions
k» 3 Continue with the procedure as described in step 8.1,
Reference ft.
5 . Calculation .
5.1 Measure the peak height of the unknown froT the chart and
read the nercury value fron the standard curve.
5.2 Calculate the mercury concentration in the sample by the
formula
u.q 'I.q in aliquot
U3 Hg/7,ran= wt . of aliquot in ^
5-3 P.eport nercury concentrations on a dry weight basis as
follows:
-------
Below 0.1-u3/
-------
Analysis of Fish for Mercury
1 . Scopc and Application
1.1 This method is used for determination of total mercury
(organic and inorganic) in fish. A weighed protion of the
o .
sample is digested with sulfuric and nitric acid at . 58 C
followed by overnight oxidation with potassiun permanganate
at 'rooa temperature. Uercury is subsequently Treasured by
the conventional cold vapor technique. -
1.2 The range of the method is 0.2 to 5-ug/s but nay be
extended above or below the nornal instrument an4 recorder
control.
2_^ Sample Preparation
2.1 The sanple nay be prepared as described under "Sanple
Handling" or the special rnetal procedure niay be used. A
0.2 to 0.3-s portion should be taken for each analysis.
The sanple should not be allowed to thaw before weighing.
3. Preparation of Calib ration Curve
3.1 The calibration curve is prepared from values for portions
of spiked fish tissue treated in the nariner used for -the
tissue samples being analyzed. For preparation of the
calibration standards, choose a 5-*"portion of fish and
blend in a Uaring blender.
3.2 "eTtiove equal and accurately weighed portions (0.2g) with, a
spatula and transfer to each of six dry BOD bottles. Add
4-ml of cone. HSd2$bSO$d4 3b and 1-nl of cone. HI-IO^ . and
-------
plac-e in watar bath at 53 .C until t'ne tissue is completely.
dissolved (30 to 60-nin).
3.3 Cool, and transfer 0-, 0.5-, 1.0-, 2.0-, 5.0- and 10.0- nil
aliquots of the working nercury so lut ion containing 0 to
o
'l.Q-uj; of nercury to the 30D bottles. Cool to . 4 -C in an
ice bath and cautiously add 15-nl of potassiun permanganate
solution. Allow to stand overnight at roori te.nnerature
under oxidisinr* conditions.
3.4 Add enough distilled water to-brinp the total volume to
approxinately 125-nl. Add 6-nl of sodium chloride-
hydroxylanine sulfate solution to reduce the . escess
pernangante.
3.5 Uait at least 30-sec after the addition of hydroxylanine .
Treating each bottle individually, add 5-nl of the stannous
sulfate solution and immediately attach the bottle to the
aeration apparatus.
3.5 Continue with the procedure as given on page 121, Reference
8, The calibration curve is prepared by plotting the peal-.
height versus the nercury concentration. The peak height
of the blank is subtracted fron each of the other values.
Sanple Procedure
4.1 i-?Righ 0.2 to 0.3-portions of the sample and place in the
botton of a dry BOH bottle. Care must be taken that none
of the sample adheres to the side of the bottle. Add 4-nl
1-nl of cone. HfVOa . and place
^
th
conpletely dissolved {30 -to 60-ninutes) .
o
'in water bath maintained at 58 .C until the tissue is
-------
4.2 Cool to A .C in an ice bath and cautiously add 5-nl of
potassium permanganate solution in 1-nl increments. Add an
additional 1 0-nl or more of perraangante, as necessary to
maintain oxidizing conditions. Allow to stand overnight at
room temperature (see NOTE). Continue as described under
*» / ' .
_> 'i
NOTE: As an alternate to the overnight digestion, the
solub liza tion of the tissue nay be carried out in a water
<3 ' ' '
bath at 80. .C for 30-nin. The sample is then cooled aad 15-
nl of potassiun permanganate solution added cautiousl7. At
this point the sanple is returned to the water bath and
digested for an additional 90-nin at SQ.'.C . vlO * If this
method is followed, the calibration standards nust also be
treated in this nanner . Continue as described .under 3»4.
5 . Ca 1 cul a t ion
5.1 Measure the peak height of the unknown from the chart and
read the nercury value from the standard curve.
5.2 Calculate the nercury .concentration in the sample bv the
fornula
u !> Her in aliquot
US Hg/gran= wt. of aliquot in g?ns
5.3 Report aercury concentrations as follows:
Below 0.1-ug/gn, ^0.1-ug; between 0.1 and I-U^/STI, to
nearest 0.01-ug; between 1 and 10-ug/gn, to nearest
O.l-ug; above lO-ug/gn, to nearest u,3«
5. Duality Assurance
-------
6.. 1 Standard quality assurance protocols should he enployed,
including blanks, duplicates and spiked sanplcs as
described in the "Analytical Quality Control
Handbook"(?)
6.2 Report all quality control data r/hen reporting results of
sample analyses*
7. ? r e' c i s i o n a n d Ac curacy
7.1 The following standard deviations on replicate fish sanples
were recorded at the indicated levels: 0.19 ug/gn 0.02,
+ " 4-
0.74-us/gn -0.05, and Z-l-tig/gn TO.OS. The coefficients of
variation at these levels v/ere 11.9", 7.0", and. 3.^%,
respectively. Recovery of nercury at these levels, added
as nethyl mercuric chloride, was 1.12%, 93%, and P-6S,
respectively.
-------
Analysis of Sfi^incnts for ifetals
(Sb, *e, Cd, Cr, Cu, Pb, Hi, Ag , 71, &.Zn)
1. Scope and Application
1.1 . This nethod is designed to deternine those priority
pollutants in sediients as listed in the Consent
Decree£4) that are classified as heavy metals otr
considered toxic as they exist in their elenental fora and
associated confounds The pollutants include antimony,
berylliun, cadnitin, chroniun, copper, lead, nickel, silver;
thalliun, and?, inc.
2. Sunnary o £ Method
.2.1 The sedinent is prepared for analysis by drying and
grinding the sanple. A representative portion is subjected
to a wet oxidation-digestion after which, atonic absorption
- either direct aspiration, or a flaneless technique-is
used to neasure the concentration of the pollutant.
2.2 For a discussion of basic principles, general .operating
parameters, preparation of standards and calibaration, and
the nethod of standard addition, the reader is referred to
"Methods for Chemical Analysis of Uater and T?astcs" f*J
pages 73-91, and the individual analyses sheets as follow:.
-------
Page references to "Methods for Chemical Analysis of Water
of Water 'and'Wastes, 1974
ELEMENT. AS
PARE 146
Pro.sevatlon and
Tie Cd Cr Cu Mi Pb Sb
99 101 105 108 141 112 94
Handling
Tl Zn
149 155
3.1 The sanple should be stored at 4 C 'if the analysis can be
carried out within 7 days of collection. For longer
periods the samples should be frozen. An alternative is to
dry the sanple as soon as possible, grind it with a nortar
and pestle reno-'irig rocks, sticks, and other foreign
n '.
obj ects and store the sedinent in a vial or other suitable.
LX
container.
3.2 Dust in the laboratory environnent, impurities in reagents
and impurities on laboratory apparatus which the sanple
contacts, are all sources of potential contanination. All
glassware should be thoroughly washed with detergent and
tap water, rinsed with 1:1 nitric acid, tap water, and
finally deionized distilled water in that order. NOTE:
Chronic acid nay be useful to renove organic deposits fron
glassware; however, the analyst should be cautioned that
the glassware nust be thorou^hl}/ r'insed with water to
renove the last trace of chroniun. This is especially
important if chroniun is to be included in the analytical
schene. A commercial prod uc t--?!OCH10" I '^--available fror>.
-------
Go da;: Labor a tor iss , 6 Yarick, '-ev York, HY 10013, can be
used in place of chronic acid.
4 . 5 a rip 1 e Pr epar a t ion
4.1' Dry a representative portion of the well 'nixed sanple (10
o
to 2 5-g) at 60.C until all.-moist tire, has been.rerioved.
4.2 Grind the dry sample uith a nortar and pestle, reaovitijrj,
sticks, stones, and other foreign naterial. Store the
sanple in glass or plastic vials removing aliquots as
needed.
5. Procedure ' .
5.1 Weigh 1.00-s of the '/ell nixed sedinent into a 250-nl
TSr 1 enTneyer flash and add 50-nl deionized T*ater, 0.5-tnl
H--T03 (sp. gr. 1.42) and 5-nl of HC1 (sp. gr. 1^.10) to
eachflask.
5.2 F.eat the sanpies, blanks and standards on a hot plate
o
maintained -at approximately 95 C. until the volume has
been reduced to 15 to 20-nl, naking certain that the
sanpies do not boil.
5.3 Cool and clarify the sample by centr if urja t ion or by
filtration through !7hatnan Mo. 42 filter panter or
equivalen t.
5.4 Dilute the sanple to 100-ril or soie appropriate volune
based on the concentration present.
5.5 Proceed with tha appropriate method for the atonic
absorption analysis of the netals of interest using either
direct aspiration or furnace techniques.
-------
5.5.1.Because of the adequate sensivity for copper and zinc
by direct aspiration AA and the probable
concentration levels of those two netals in
sediments, direct aspiration should be enployed. The
furnace technique is preferred for the -deterttiziation
of the remaining metals because of their expected lox?
concentration. T*hen using the furnace technique, the
operating parameters and instructions as specified by
the particular instrument manufacturer should be
followed. If the concentration detected by the
furnace procedure is above the working range of the
standard curve, the sample should be either diluted.
and reanalyzed or analyzed by direct aspiration. The
method for standard additions should be enployed vhen.
need ed .
5. Ca1culation
6.1 Fron the ^alues read off the appropriate calibration curve,
calculate the concentration of each netal pollutant in thft
sedinent as follows:
ng/1 of constituent volune of prepared
f prepared sample 7i sanple in ml
ng/kg = .
weight of dry sample in 2
7 . Quality As suranc e
7.1 Standard quality assurance protocols should be enployed,
including blanks, duplicates, and does samples as described
in the "Analytical Quality Control Handbook". (7)
-------
7.2 Report all quality control ;lata whun report inf. results of
sanple analyses. .
-------
Analysis of Fish for Petals
Sc
1.1 This nethod is designed to determine in whole fish those
priority pollutants listed in the Consent Decree fft } that
are classified as heavy netals or considered toxic as they
exist in their elemental form and associated compounds.
The pollutants include antimony, arsenic, berylliun,
cadniun, chroniura, copper, lead, nickel, seleniun, silver,
thalliun, and zinc.
Sun nary of Me thod .."'- .
2.1 The fish is prepared for analysis by being chopped into
small pieces, homogenized in a blender with dry ice, and
solubilized by either dissolution after dry ashing or a wet
oxidation digestion. After sample preparation, atonic.
absorption - either direct aspiration, gaseous hydride, or
a flaneless technique - is used to measure the
concentration of the pollutant.
Preservation an d T'and lin£
3.1 Although an aliquot of the ground fish as prepared under
!-
"Sarinle Handling" raay be used for the netals determination,
it nay be more desirable to prepare an individual fish to
avoid possible metal contamination from the rjrindp.r. Dust
in the laboratory environment, inpurities in reagents, and
impurities on laboratory apparatus that the sample contacts
are all sources of potential contamination. Ml glassware
should be thoroughly washed with detergent and tap water,
-------
r insed wi th 1:1 nitric acid, then tap water, and finally
deionized dis tilled water.
MOTE: Chronic acid nay be useful to reriovc organic
deposits fron glassware; however, the analyst should be
cautioned that the glassware ?iust be thoroughly rinsed with
water to remove the last trace of chroniura. This is
especially important if 'chroniun is to be included in the'
analytical schene. A. connercial produc t--"OCKno?:IS
available from Codax Laboratories, 6 Yarick Street, 'let/
York, NY 10013, can be used in place of chronic acid .
S a n n 1 e Ho-iog eniza.tion
A.I, If a fish sanple other than that prepared under "Sanple
Handling" is to be used for netals analyses, unvrap and
weigh the frozen fish at the tine of processing. Select a
fish that weighs between 50 and 300-g. If ah analysis is
required for a. fish^ 300-g, a 50-g representative portion
nust be taken fron the sanple after it has been pretreated
as described in "Sanple Handling" on page 1 of this
docunent and proceed' to step 4.3.
£.2 After weighing, the fish should be chopped into
approximately 1-in. or snaller chunks with a neat cleaver
of a knife and nallet (2 to 3-lb). Snaller pieces ensure
efficient grinding.
4.3 Place crushed or pelleted dry ice into the blender
container. The weight of dry ice should be entual to, or
«reat.er then, the weight of the fish.
-------
4.4 Turn on the blender for 10-snc to pulverize the ice and
chill the blender.
4.5 Add the pieces of fish and blend at high spcad until the
nixture is homogeneous. This usually requires 2 to. 5-
' ninutes. Add nore dry ice if needed.
4.6 Pour the horiogenate into a plastic bag and close the bag
with a rubber band. Do not seal the bag tightly to allot/
C0$d2$b escape.
o
4.7 Place the bag in the freezer (-12 C for at least 16-h)
until ready to proceed with the digestion step. -
NOTE: If desired, the blender blades can be notified in.
order to ha-'e the leading edge of the blades (the sharpened
edge) turned down so that, as it rotates, the blade will
throw the material upwards. 3tainless steel blades riay be
a possible source of nickel and chrociiur*. contamination and
» should be noted if detected. If a tantalun blade is
available, it should be substituted for the stainless
steel. ' .
The hole in the blender lid should be enlarged.
sufficiently to allow the evolved gas to escape (1/2 in-
quart-size, 1 in-galIon-size) . Hold a cloth or labuipe
over this hole when blending to prevent loss of the saaple
material. A glove should be worn to prevent possible
freezing of the skin by escaping 2as-
?.e ar: en 13
5.1 ~>eioni-zed distilled water: Prepare by passing distilled
water through a nixed bed of cation and ariion
-------
resins. Use deionized distilled water for the preparation
of .all reagents., and cal ih rat ion standards and as dilution
wa'ter
5.2 Nitric acid (cone.): If metal impurities are present,
distill reagent grade nitric acid in a borosilicate glass
distillationapparatus.
5.3 Sulfuric acid, ACS grade (95.5 * to 95.5%).
5.4 Sulfuric acid - 20" v/v solution. Carefully add 200-nl of
concentrated ^-^SO^ to 500-tal of watrir. Cool and
dilute to 1-liter with water.
5.5 hydrochloric acid, ACS grade (37% to 33TO-
5.6 Hydrogen Peroxide, 50°' stabilized ACS grade.
5.7 Dry.ice (frozen carbon dioxide), pellet form preferred.
6 . Ap n a r a t u s
. 6.1 Blender, faring, two-speed, stainless steel blade or
tantalun blade if available, glass container capacity 1000-
nl, or equivalent equipnent.
& o
6.2 Drying oven - Controllable with the range, of lOOv to 150 :C
4- °
with less that i5 C variation. Check calibration of oven
tenperature control to ensure accurate ashing teriperatures.
Furnace nust be operated in suitable fune hood.
c c
6.3 Hot plate, controllable within the range of SO: to 400 C.
Hot plate nust be operated in fur.e hood.
7. Procedure
Except for mercury, which'requires a cold vapor technique,
the pollutants can be divided into two p.roups for continued
p'roc.es r>in«> .
-------
OROfJ? I: "e, Cd, Cr , Cn, ?h, ?M , AC, T1, and Zn .
GROUP II: As and Se .
Group I is digested by a dry ashing process fll) with the use of
. an ashing aid; Croup II is prepared utilizing a wet ashing
process.
7.1 Group I - "e tals
7.1.1 Renove the homogenized sample from the freezer and
weigh appro;:ina t ely 10-g into a tared, 100-ril tall,
form, Pyrex beaker. Subtract the beaker weight fron
- the total and record the.wo.t sample weight. "
7.1.2 Add 25-nl of 20% sulfuric acid. Mix each sanple
thoroughly with a glass stirring rod ensuring all
sample material is wetted by the acid. Rinse the
stirring rod with water into the ashing vessel and
cover the sanple with a ribbed watch glass.
o
7.1.3 Dry the samples in an oven or furnace at 110+5 -.C
until a charred viscous sulfuric acid/sanple residue
remains. Usually 12 to 16-h (overnight) is
sufficient. Transfer the ashing vessels containing
the dried samples to a cold, clean 'nuffle furnace
which is provided with good external ventilation
(fur.e hood), ensuring that the sanple remains covered
during the transfer. Initially set the furnace at
o
125 C and increase the temperature approximately
every hour in 50 increments up to 275 .C. Hold the
temperature at 275?C for 3-h. Finally, increase the
tenpe-rature to 450°C.(at 50° per hour) and hold for
-------
12 to 1 6-h (overnight). ?.e-iove the covered ashing
vessels frora the furnace and allow to cool to roon
teiaperature in a clean, draft-free area.
7.1.4 After initial overnight ashing, soie residual carbon
nay remain in the sanples. Treat each sanple ash
with 0.5-ml of water and 1-nl of concentrated nitric
acid (whether or not they are already white).
Evaporate carefully just to dryness on a warn
hotplate (in a fune hood). Place the ashing .vessels
(covered with watch glasses) in a cool nuffle furnace
and raise the tenperature to 300 C and hold for
exactly 30-ciin. Tlenove each covered sanple ash from
the furnace and allow to cool as before. If residual
carbon renains, repeat the nitric acid treatment
until 'a carbon-free white ash is obtained. The
covered ashing vessels containing the ash nay be
stored in a dessicator or in a laminar flow clean
hood .
NOTE: Copious carbon residues (i.e., black ashes)
after overnight ashing nay indicate inefficient or
uneven heating within the furnace. P.outinc
calibration of the furnace is advised.
7.1.5 Add 0.5-nl of nitric acid and 10-inl of water to each
cool ashing vessel, then warn gently on a hotplate at
80 to 90^:0 for 5 to 10-nin to effect dissolution of
the ash. A snail amount of insoluble white
siliceous-like residue nay ronain nndissoIved; do not
-------
filter the residue because of the possibility of
contamination. Quantitatively t'ransfer the contents
of each ash in.7, vessel into a 100-^.1 volunetric flash,
dilute to volume vith water, and shake thoroughly.
Allow any residue to settle to the botton of the
flask (about 2-hr). no not shake the saraple further
before taking an aliquot. The sanple is no'/ ready
for analysis.
NOT?:;: The presence of a precipitate other than the
insoluble siliceous-1ike material nay result in lo*7
or eratic results for Pb. Precipitate formation can
.result frori heating tho sanples too lon» or at too
high a tenperattire after nitric acid treatncnt of the
ash. Precipitate fornation nust be avoided by
maintenance of appropriate ashing temperatures.
7.1.6 The prepared sanple should be analysed by AA using
either direct aspiration or furnace techniques. For
a discussion of basic principles, the nethod of
standard addition, the chelation/solvent e?ctraction
procedures, general instrumental operating
parameters, and preparation of standards and
calibration see the section on "Atonic Absorption
Methods",- pa^es 73-^1, Reference P, , and. -the
individual analyses sheets (on pa?, es as listed
b e1ow) .
-------
ELF^'E^T AS 3e Cd Cr Cu ?-Ti °b 3b Tl 7.n
PAGE 146 99 101 105 103 .141 112 94 149 155
7 1 . 7 ' Because of the adequate sensitivity by conventional
flane A A. and the expected concentration levels of
cadniun, copper, and zinc in the sample, t'hese three
elements should be analyzed by direct aspiration.
The furnace technique is preferred for the analysis
of the other Group I metals because, of their expected
low concentrations. T?hen using the furnace
technique, the operating parameters and instructions
as specified by the particular instrument
manufacturer should be followed. If the
concentration detected by the furnace procedure is
beyond the working ran^e of the standard curve, the
%
sanple should he either diluted an.d reanalyzed or
analyzed by direct aspiration. The nftthod o 'c.
standard additions should be ennloyed when needed.
If the sanple natrir: is so complex that sanple
dilution followed by furnace analysis cannot be used,
or if the use of the chelation/solvent extraction
technique for concentration of .\g , r±, '"b, and Tl is
preferred, the procedure as describee starting on
pa^e HO, Fleferoncfi " , should, bo utilized.
7.2 r, r o u n II - ''e t als
-------
^.2.1 7p.nove the 'homogenized sample fron the freezer an.-1.
wei.3'n approximately 5-g into a tared, .125-nl conical
Weaker. Subtract t'xc beaker weight fron the totnl
.and record the '.ret sample weight.
7:2.2 Add 5-nl of cone. H!TO 3 . Then slowly add 6-nl of
cone. K^SO>_ - aP^ cover with a watch glass.
7.2.3 Place beaker on hot plate and var-m slightly.
Continue heating until the nixture becomes dark or a.
possible reducing condition is evident. Do not allow
the ni;:ture to char. Renove beaker fron hotplate and
allow to cool.
I'lOTE: Renove beaker if foaming becomes excessive.
7.2.4 Add an additional 5-nl of cone. TMC^ ., cover with
a t-;atch glass, and return beaker to hot plate.
Re peat step 7.2.3.
7.2.5 Uhen nixture again turns brown, cool, and slowly add
5-nl of 50" hydrogen pero::ide. Cover with watch
glass and heat gently until the initial reaction has
ceased. If the solution bccorics dark, repeat .the
peroxide addition, several tines if necessary, and
heat to SO3 fumes. If charring, occurs, add
further 1-nl portions of hydro3en peroxide until the
funinn sulfuric aci-i remains colorless or very light
yellow. (If at any sta^e it seems that the sulfuric
acid nay approach .dryness, cool, add 2 to 3-nl of
sulfuric acid, and continue).
-------
7.2.6 Cool, add 40-nl of cone. 'TCI and dilute to 100-nl
with deionised d.istilled water. The sanple is now
ready for analysis.
7.2.7 The Croup II netals should be analyzed by AA using
;
the gaseous hydride technique. The apparatus setup,
standard preparation and calibration, and analysis
procedure that is to be followed is given starting on
page 159, Reference 9. Fron the prepared sanple a.
2c»-nl aliquot should be withdrawn and the analysis
continued as described in section 3 d , page. 162,.
Reference 9.
Calculation
S.I "sing the values fron the appropriate calibration curve
calculate the concentration of each netal pollutant in the
fish as follows:
If. the concentration of standards in thn calibration curve.
is plotted as ng/1,
ng/1 of constituent volume of prepared
in prepared sanple X sanple in nl
ug/gran = ; .
weight of wet sanple in 5
If the concentration of standards in the calibration curve
is plotted as us/1,
ug/1 of constituent
in prepared sanple X volume of prepared
1000 sanple in -nl
u!» / 3 r a P. =
weight of wet sanple in 3
-------
Duality Assurance
i
9.1 Standard- quality assurance protocols should. be employed,
including blanks, duplicates, and dosed sanples, as
described in the "Analytical Quality Control
Handbook"
9.2 P.eport all quality control data >7hen reporting results of
sample anal3/ses..
-------
Analysis of Sediment for Arsenic and Sclcniun
1 . Scope and Appl icr.t ion
1.1 This method is to be used for the determination of..arsenic
and seler.iun in sediment. A weighed portion of the wet,
well-nixed sedi-ent is digested with TINO^ "-'.' and
n^SQ^L folloxred by treatment with 'T^ O^ '"'."'. f'2 .
Arsenic and seleniun are subsequenfly dcternined by the
gaseous hydride technique.
1.2 The range, of the method is to u
-------
char, "enova the beaker fron the hotplate anr. allo'.* to
cool .
4.4 Add an additional 5-nl of cone. "'TO^ , cover with a
watch glass, and return beaker to hot plate. P.epcat step
-.6.3.
4.5 'Then mixture ajjai-n turns brown, cool, and slowly add 5-nl
of 50^ hydrogen peroxide. Cover with watch gla'ss and heat
3ently until the initial reaction has ceased. If the
solution becones dark, repeat the peroxide addition,
several tines if necessary, and heat to SOa funes. If
charring occurs, add additional 1-nl portions of hydroson
peroxide until the funing sulfuric acid renains colorless
or very light yellow. (If at any staje the sulfuric acid
approaches dryness, cool, add 2 to 3-nl of additional
sulfuric acid, and continue.)
4.^ Cool, add 40-nl of cone. HC1 and dilute to 100-nl with
deionized distilled water. The sanple is now ready for
analysis by the gaseous hydride technique.
4.7 The apparatus setup, standard preparation and calibration,
and analytical procedure to be followed is jjiven beginning
on page 150, Peference 9. A 25-nl aliquot should be
withdrawn fron the prepared sanple and the analysis
continued as described in section 3-d,-pa*e 1^2, "eference
?
5 . Calibration
5.1 Calculate the concentration of arsenic and seleniun present
in ng/ks on a dry weight basis.
-------
Quality Assurance . . .
Standard quality assurance protocols should he employed,
including .blanks, duplicates, and spiked sar.ples ns describe^ in
the "Analytical Quality Control handbook".
-------
1. "Method for Organochlorinc Pesticides in Industrial Effluents,"
U. S. Environmental Protection Ajjency, Environmental Monitoring
and Support Laboratory ,. Cincinnati , Ohio, 45268, 1973.
2. "Method for Polychlorinated ""iphenyls in Industrial Effluents,"
U. S. Environmental Protection Agency, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio, 45268, 1973. .
3. Federal Register, Volume 41, number 232, p. 527SO, Uednesday,
December 1, 1976. . *
4. "fjanplins and Analysis Procedures for Screening of Industrial
Effluents fo'r Priority Pollutants," U. S. Environnental
Protection Agency, Environnental T^onitoring and Support
Laboratory, rev. April, 1977.
5. Boyle, H. TT. et al . , Adv. Chen. Ser., 60. 207 (1^66).
6. Stalling, D. L.; Tindle, P.. C.; Johnson, J. L.; "Cleanup of
Pesticide and Polychlorinated Biphenyl Residues in Fish Extracts
by Gel Perraeation Ch roaa togr aphy . " JAOAC. 5 3 , 32-3-1?. (1972).
7. Handbook fo Analyticcal Quality Control in T.'ater and T'astetra ter
Laboratories. U.S. Environnental Protection Agency, Technology
Transfer. (1972).
0. "Methods for Chen.ical Analysis of Hater and Pastes", U. S.
Environnental Protection Agency, Technology Transfer. (1974).
9. "Standard i'ethods for the Examination of T7ater and !Tasteuater" ,
lAth edition (1975).
^^. Bishop, J. R.., "Mercury in Pish.," Ontario TIater Resources Conn.,
Toronto, Ontario, Canada, 1971.
-------
11. Jones, J. T'.; Oajan, T:. .1. ; F-oyer, T'.. TJ . ; Tiorino, J. A.; "Dry
Ash - Voltannetric Heternination of Ca-'lniun, Copper, Lea.-!, and
Zinc in Foods". JAOAC, 50, 826. (1977).
-------
TABLE I
Priority Pollutants Analyzed by Soxhlet Extraction
Pesticides
Aldrin
a-BHC
b-BHC
d-BHC
g-BHC
Chlordane
PCBs
Aroclor 1016
Aroclor 1221
Aroclor 1232
Non-polar Neutrals
Acenaphthylene
Acenaphthene
Isophorone
Fluroene
Phenanthrene
Anthracene
Dimethylphthalate
Diethylphthaiate
Fluoranthene
Pyrene
Naphthalene
Chrysene
ODD
DDE
DDT
Dieldrin
a-Endosulfan
b-Endosulfan
Aroclor 1242
Aroclor 1248
1,3-dichlorobenzene
1,4-dichlorobenzene
Hexachlorethane
1,2-dichlorobenzene
Hexachlorobut ad i ene
1,2,4-trichlorobenzene
2,6-dinitrotoluene
Hexachlorobenzene
4-broraophenyl phenyl
ether
Bis (2-chloroethoxy)
methane
2-chloronaphtha1ene
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor -
Heptachlor epoxide
Toxaphene
Aroclor 1254
Aroclor 1260
Bis (2-ethylhexyl) phthalate
Benzo (a) anthracene
Benzo (b) fluoranthene
Benzo (k) fluroanthene
Benzo (a) pyrene |
Indeno (1,2,3-cd) pyrene j
Dibenzo (a,h) anthracene j
Benzo (ghi) perylene
4-chlorophenyl phenyl ether j
2,3,7.8-tetrachlorodibenzo-p-dioxir ''
Di-n-butylphthalate :
Butyl benzylphthalate
-------
- 1.1 -
Table IT. Base-neutral Extractables
RRTV Limit of
(hexachloro- Detection
Compound tfair.e
1 , 2-'Hchlorobcn7.cnc
1 , -1 -«.'. tchlo robcnzene
hcxaciiloroethanc
1 , 2-c'.ichlorobenzene
bis (2-cliloroisopropyl)
atS'.er
he^is'.ilorobutadicno
1,2, -1-trichlorobenzerie
r.aoi!>:)ialnne .
bis (2-chloroethyl) ether
he::nchlorocyclopentadicna
nitrobenzene
bin (2-chloi:oethoxy) methane
2-chloron&phthalene
acenoph thy lone
acunaphthono
i.sophorone
fiuorer.e
2 , G-:1 ini Irotoluenc
1 , 2-diphcnylhydrazine
2, '> -clinit.ro toluenes
tl-nitrosodiuhenylamine
hoxachlorobenzene
-------
- 12 -'
Table II Base-neutral Extractables (Cont'd.)
Compound
chrysene
bi s ( 2-ethy Iho.xyl ) phthalate
bcr.zo (a) anthracene
benzo (h) fluornnthene
L-onzo (k) t luoranthene
be«so(.?.)pyrenc
inser:o\ 1 , 2. 3-ccl)pyrer.G
cil-cnzo (a , h) anthracene
be" 20 (g, h, ijperylens
RRT V
(hexachloro-
bensene)
its
(
1.46
i 1,50
1:54
1.66
1.66
1.73
2.07
2.12
2.18
Limit of
Detection
(ng)
40
40
40
40
40
40
100 .
100
100
Characteristic
El ions (P.cl. Int.)
228(100), 229(19), 226(23)
l'.9(JOO), 167(31) , 279(26)
220/100), 229(19), 226(19)
252(100), 253(23), 125(15)
. 252(100), 253(23), 125(16)
252(100), 253(23), 125(21)
276(100), 130(28), 277(27)
278(100), 139(24), 279(24)
276(100), 138(37), 277(25)
CI ions
(Ke thane)
228, 229, 257
149
223, 229, 227
252, 253, 201
252, 253, 231
252, 253, 201
276, 277, 305
278, 279, 307
276, 277, 305
H-nitvosocliir.Qthyla.mine
N-nitsror.orJ.i-n-prcpylarr.ine
;-chic;ro-pher.yl phony 1 ether
er.drin
0, ? '-clichlorobcnzidine
2,3,7 , 3-tetrachlo.rodibanzo-
p-dioxin
bis (ch.1. ororaethyl) other
42(100), 74(08), 44(21)
130(22), 42(64), 101(12)
204(100), 206(34), 141(29)
252(100), 254(66), 126(16)
322(100), 320(90), 59(95)
45(100), 49(14), 51(5)
* 13 SF-2250 oo 100/120 mesh Supclcoport in a 6' x 2 mm id. glass column;. He @ 30 ml/min;
Procjram: 50 for 4 min, then 8 /min to 260 and hold for 15 min.
**" Conditioning of column with base is required.
-------
. - 13 -
Tabla in Acdd
Limit of
RRT' * Detection
Compound Nnmo (2-nitrophenol)
2-chlorophunol
phenol-
2 , '! -clicliiorophcnol
2-nitrophsnol
p-cliloro-m-ciresol
2,4,6-trichlorophenol
2 , ! -dimcthylphenol
2, 4-clir.it rophcnol
4 , 6-dir.itro-o-cresol
<-r.itrcphenol
psntachlorophonol
deaterated anthracene (dlO)
0.
0.
0.
1.
1.
. 1.
' 1.
1.
1.
1.
1.
1.
63
66
96
00
05"
14
32 .
34 . .
42
43
64
68
il SP-2250 on 100/120 mesh Supelcoport in a
Program: 50 for 4 min, then 8 /min to' 260
(ng)
100
100
100
100
100
100
100
2 vg
2 ug
100
100
40
6 ' x 2 mm
and hold
*
Characteristic
El ions (Rcl. Int.)
120(100),
94(100),
162(100),
. 139(100),
142(100) ,
196(100),
122(100),
104(100),
198(100),
65(100),
266(100),
188(100),
.C4(54),
65(17),
164(50)
65(35),
107(00)
198(92)
107(90)
63(59),
182(35)
139(45),
264(62)
94(19)',
130(31)
66(19)
, 98(G1) .
109(8)
, 144(32)
, 200(26)
, 121(55)
154(53)
, 77(2:8)
109(72)
, 268(63)
80(18)
id. glass column;. He @ 30 ir.l/min
for 15 min.
CI
ionn
(Methone)
129,
95,
163,
140,
143,
197,
123,
105,
199,
140,
267,
109,
i
131,
123,
165,
160,
171,
199,
151,
213,
227,
1G8,
265,
217
157
135
167
122
103
201
163
225
239
122
269
-------
- 17 -
Table
Compound
chloromethane
bromome thane
vinyl chloride
chloroethane
methylene "chloride
,1-dichloroethane
chloroform
1, 2-dichloroethane
1 / 2-dichloropropane
trichloroethylene
Characteristic Ions of Volatile
El /ons (Relative
intensity)
50(100) ; 52(33)
romethane 85(100); 87(33);
101(13); 103(9)
94(100); 96(94)
62(100) ; 64(33)
64(100) ; 66(33)
ride 49 (100) ; 51 (33) ;
84(86).; 86(55)
Dinethane 101 (100) ; 103 (66)
lylene 61(100) ; 96(80) ;
nane(IS) 49(100); 130(88);
128(70); 51(33)
nane 63(100) ; 65(33) ;
85(8) ; 98(7) ; 100
Loroethylene 61 (100) ; 96 (90) ;
83(100) ; 85(66)
nane 62(100); 64(33);
98(23); 100(15)-
Dethane 98 (100) ; 99 (66) ;
117(17); 119(16)
Loride 117(100); 119(96)
Bthane 83(100); 85(66);
127(13) ; 129(17)
/I ether 79(100') ; 81(33)
Dpane 63(100) ; 65(33) ;
112(4); 114(3)
loropropene 75(100): 77(33)
ene " 95(100); 97(66) ;
130(90) ; 132(85)
sthane 129(100); 127(78)
1 . 208(13) ; 206(10)
ropropene 75(100); 77(33)
Organics
Ion used to
quantify
50
101
94
62
64
84
M01
98(53) 96
12»
83(13); .
(4) 63
98(57) -.96
. 83
' 98
97
; 121(30) 117
127
79
112
75
130
127
75
-------
Compound
1,1,2~trichlorcethane
benzene
2-chloroethylvinyl ether
2-bromo-l-chloroprcpane(IS)
broir.of orm . ' ; -
1,1,2,2-tetrachloroethene
1,1,2,2-tetrachloroethane
l,4-dichlorobutane(IS)
toluene
chlorobenzene
hylbanzene
ctcrolein
acrylonitrile
TABLE IV
El /ifons (Relative
intensity)
83(95); 35(60); 97(100);
99(63); 132(9); 134(8)
78(100)
63(95); 65(32);. 106(18)
77(100); 79(33);156(5)
171(50};173(100) ; 175(50);
250(4); 252(11); 254(11);
256(4)
129(64); 131(62);
164(78); 166(100)
83(100) ; 85(66); 131(7);
133(7); 166(5); 168(6)
55(100); 90(30); 92(10)
91(100); 92(78)
112(100); 114(33)
91(100); 106(33)
26(49); 27(100);
55(64); 56(83)
26(100); 51(32);
52(75); 53(99)
Ion used to
quantify
97
78
106
77
173
164
55
92
112
106
56
53
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