&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
. EPA/600/R-92/121
November 1992
Methods for the
Determination of
Chemical Substances in
Marine and Estuarine
Environmental Samples
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EPA/600/R-92/121
November 1992
Methods for the Determination of Chemical Substances in
Marine and Estuarine Environmental Samples
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Printed on Recycled Paper
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Disclaimer
This manual has been reviewed by the Environmental Monitoring Systems Laboratory -
Cincinnati, U.S. Environmental Protection Agency, and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or recommendation
for use.
Revision 1.0 November 1992
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Foreword
Environmental measurements are required to determine the quality of ambient waters
and the character of waste effluents. The Environmental Monitoring Systems Laboratory -
Cincinnati (EMSL-Cincinnati) conducts research to:
• Develop and evaluate analytical methods to identify and measure the concentration
of chemical pollutants in marine and estuarine waters, drinking waters, surface
waters, ground waters, wastewaters, sediments, sludges, and solid wastes.
• Investigate methods for the identification and measurement of viruses, bacteria, and
other microbiological organisms in aqueous samples and to determine the responses
of aquatic organisms to water quality.
• Develop and operate a quality assurance program to support the achievement of data
quality objectives in measurements of pollutants in marine and estuarine waters,
drinking waters, surface waters, ground waters, wastewaters, sediments, and solid
wastes.
• Develop methods and models to detect and quantify responses in aquatic and
terrestrial organisms exposed to environmental stressors and to correlate the
exposure with effects on chemical and biological indicators.
This EMSL-Cincinnati publication, "Methods for the Determination of Chemical Sub-
stances in Marine and Estuarine Environmental Samples" was prepared as the continuation
of an initiative to gather together under a single cover a compendium of standardized
laboratory analytical methods for the determination of nutrients, metals and organics in
marine matrices. It is the goal of this initiative that the methods that appear in this manual will
be multilaboratory validated. We are pleased to provide this manual and believe that it will
be of considerable value to many public and private laboratories involved in marine studies
for regulatory or other reasons.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
HI
Revision 1.0 November 1992
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Abstract
This manual contains seven methods for determination of nutrients, metals, and
chlorophyll. Methods 353.4, revision 1.2, and 365.5, revision 1.3, for the measurement of
nitrite + nitrate and orthophosphate, respectively, appeared in the 1991 interim manual.
Since then they have undergone multiiaboratory validation studies. Method 365.5 performed
well in the study and multiiaboratory data are presented in the revision of the method that
appears here. The performance of Method 353.4 in the study indicated that the cadmium
reduction column chemistry and maintenance require further investigation. The method has
been retained in this manual sothatfurthertesting can continue using a standardized method
description.
Method 440.0 for measurement of total particulate carbon and nitrogen is based upon
a well established combustion technique. Procedures for partitioning the organic and
inorganic fractions of carbon are also presented. A multiiaboratory study is in progress, and
the results will be included in a subsequent revision of the method.
The three metals methods represent current state-of-the-science in metals measure-
ments. Two of the methods are graphite furnace atomic absorption techniques and the third
uses inductively coupled plasma mass spectrometry. Single laboratory performance data
are included in the methods. Although few laboratories currently have the instrumentation
capabilities to perform all of these methods, it is extremely important to present them in order
to stimulate the development of laboratory capability before multiiaboratory studies can be
conducted.
Method 445.0 is for the determination of chlorophyll a and the pheopigments using
fluorescence detection. This method has been used for many years for low level measure-
ment of chlorophyll. The method was evaluated using two natural water samples of primarily
green and blue-green algae.
The numbering of methods was correlated'with previous EMSL-Cmcinnati methods
whenever possible. The metals methods are 200 series, the nutrients nitrite + nitrate and
orthophosphate are 300 series, and the particulate carbon and nitrogen, and chlorophyll
methods are 400 series.
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Contents
Disclaimer
Foreword
Abstract
Acknowledgments
Introduction ,
Page
....ii
...iii
...iv
...vi
....1
Method
Number Title
200.10 Determination of Trace Elements in
Marine Waters by On-Line Chelation
Preconcentration and Inductively
Coupled Plasma - Mass Spectrometry
200.12 Determination of Trace Elements in
Marine Waters by Stabilized Temperature
Graphite Furnace Atomic Absorption
200.13 Determination of Trace Elements in
Marine Water by Off-Line Chelation
Preconcentration with Graphite Furnace
Atomic Absorption
353.4 Determination of Nitrite + Nitrate in
Estuarine and Coastal Waters by
Automated Colorimetric Analysis
365.5 Determination of Orthophosphate in
Estuarine and Coastal Waters by
Automated Colorimetric Analysis
440.0 Determination of Carbon and Nitrogen
in Sediments and Particulates of Estuarine/
Coastal Waters Using Elemental Analysis
445.0 In Vitro Determination of Chlorophyll a and
Pheophytin a in Marine and Freshwater
Phytoplankton by Fluorescence
Revision
1.6
1.0
1.0
1.3
1.4
1.4
1.1
Revision 1.0 November 1992
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Acknowledgments
This methods manual was prepared by the Inorganic Chemistry Branch of the Chemistry
Research Division, Environmental Monitoring Systems Laboratory - Cincinnati (EMSL-
Cincinnati). Individual manuscripts were prepared by the combined efforts of many people.
The nutrient methods were prepared by Carl Zimmermann and Carolyn Keefe at the
Chesapeake Biological Laboratory, University of Maryland, with the editorial assistance of
Jerry Bashe and Stephen Long of Technology Applications, Inc. Additional contributions by
James Longbottom, EMSL-Cincinnati, and Kenneth Edgell, The Bionetics Corporation, in the
preparation and distribution of quality control samples and statistical evaluation of the data
are very much appreciated.
The chlorophyll method evaluation was aided by the technical contributions of Gary
Collins and Cornelius Weber both in EMSL-Cincinnati. Their expertise was and continues to
be greatly appreciated. We would also like to thank John Macauley, Environmental Research
Laboratory, Gulf Breeze, Florida, who provided 400 chlorophyll samples from Lake
Pontchatrain, Louisiana. Those samples have allowed issues beyond chlorophyll measure-
ment by fluorescence detection to be explored.
Diane Schirmann and Patricia Hurr provided invaluable assistance in manuscript
production. Diane has no doubt read this manual more times than anyone else involved in
its production. Their contributions were significant, and we thank them.
The overall USEPA effort to standardize analytical methods for use in the marine
environment was identified as a need and championed by the USEPA regions. The staff at
Region 2 and Region 3 were, and continue to be, instrumental in identifying resources for this
project. They provided insight from the regional perspective and served as technical
advisors. Their reviews and comments to these methods were invaluable.
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Introduction
During 1988, the coastal regions, led by Regions 2 and 3, began organizing and
compiling a list of analytical methods research needs specific to the marine and estuarine
monitoring community. In January 1990, after conducting an extensive survey, the 10
Regional Environmental Services Division Directors produced a document that contained
seven marine and estuarine analytical methods research needs. This document was widely
distributed and has been the basis for ordering research priorities. In May 1990, Regions 2
and 3, with support from the Office of Marine and Estuarine Protection (since renamed Office
of Wetlands, Oceans, and Watersheds), held a workshop in Annapolis, Maryland, to bring
together investigators from the marine and estuarine monitoring programs, representatives
and experts from the private sector, and others with an interest in the marine environment.
Their goals were to establish a network for technical exchange, restate analytical methods
needs, and set a course of action. Toward that end, four workgroups were formed: (1)
Nutrients, Demand, and Chlorophyll; (2) Metals; (3) Organics; and (4) Biologicals. Each
workgroup was "charged with the collection, assembly, review, and evaluation of existing
analytical methods and standard reference materials (SRMs) in saline water, sediments and
biologicals." When methods or SRMs were identified, the workgroups were to present
recommendations to the Office of Research and Development (ORD) for funding and further
investigation. Nutrient methods and SRMs received the highest priority for immediate work.
In March 1991, William L. Budde (Director, Chemistry Research Division) and Larry
Lobring (Chief, Inorganic Chemistry Branch) participated in a meeting at Region 2 with
Barbara Metzger (Director, Environmental Services Division, Region 2), members of her
staff, Claudia Walters (Chesapeake Bay Program, Region 3), Bettina Fletcher (Regional
Operations, HQ), and Rich Pruell (Environmental Research Laboratory, Narragansett). The
purpose of the meeting was to discuss priorities and planning for analytical methods research
and development. The following immediate priorities were named from the seven priority
items established by the regions in January 1990: (1) orthophosphate, nitrite + nitrate,
paniculate nutrients, and preservation studies; (2) nutrient reference materials; and (3)
chlorophyll.
Larry Lobring, as Principal Investigator within ORD for the Marine Methods Initiative,
subcontracted through Technology Applications, Inc., the Chesapeake Biological Laboratory
(CBL) at the University of Maryland to evaluate the orthophosphate and nitrite + nitrate
methods. CBL performed single-laboratory validation of the methods, wrote them in EMSL-
Cincinnati format, and aided in the design and execution of the multilaboratory validation
studies. In September 1991, an interim manual containing these two nutrient methods was
delivered by EMSL-Cincinnati to all interested parties for review and comment.
During the last year, reviews of the nutrient methods have been duly noted, results of the
two multilaboratory validation studies have been evaluated, key personnel have changed
within EMSL-Cincinnati, and a subsequent meeting between EMSL-Cincinnati and the
regions has reestablished priorities and renewed commitments by both parties to the mission
of this initiative. William L. Budde, who replaced Larry Lobring as Principal Investigator,
appointed Elizabeth J. Arar as the lead investigator for the nutrient methods, John T. Creed
as lead investigator for the metals methods, and James W. Eichelberger as the lead
investigator for the organics methods development effort. This team is responsible for current
research in this area and the methods in this manual.
vii
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The principal aim of this manual is to bring together under one cover a suite of analytical
methods specifically adapted or developed for the examination of marine and estuarine
environmental samples. Three of the methods presented here are adaptations of analytical
techniques that, for many years, have been used routinely by the marine community.
Hallmarks of the methods that appear in this manual, however, are the integrated quality
control/quality assurance requirements, the use of standardized terminology, and the use of
the Environmental Monitoring Management Council (EMMC) methods format. The manda-
tory demonstration of laboratory capability and the continuing checks on method perfor-
mance ensure the quality and comparability of data reported by different laboratories and
programs. Another distinction of this manual is the eventual multilaboratory validation study
of each method.
Multilaboratory validation studies testthe ruggedness of methods, provide single-analyst
and multilaboratory precision and accuracy statements and method detection limits that are
"typical" of what most laboratories can achieve. Methods that reach this level of evaluation
have been thoroughly investigated by a single laboratory and have usually been informally
adopted as standard methods by the analytical community. Method 365.5, "Determination of
Orthophosphate in Estuarine and Coastal Waters by Automated Colorimetric Analysis," a
widely accepted method in the marine community, performed quite well in a multilaboratory
study. Atable has been added to the method to summarize single-analyst and multilaboratory
precision and accuracy of the method for three water matrices. As a result of the study, pooled
method detection limits for Orthophosphate in a wide range of water salinities have also been
added to the method.
On the other hand, Method 353.4, "Determination of Nitrite + Nitrate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis," did not give acceptable multilaboratory
results, and it must return to the development phase. Method 353.4, despite its wide
acceptance and routine use in the marine community, failed the ruggedness test when 50%
of the participating laboratories in the multilaboratory study returned unacceptable data.
Their data suggest that the cadmium reduction column chemistry and maintenance require
further investigation. The method, nonetheless, appears in this manual with appropriate
caveats for the user so that further testing can continue using a standardized method
description.
Method 440.0 for paniculate carbon and nitrogen uses a well established combustion
technique and is currently undergoing multilaboratory validation. The results from that study
will be incorporated into the next revision of this manual.
Method 445.0 for the in vitro determination of chlorophyll a and the pheopigments using
fluorescence detection was evaluated using primarily freshwater phytoplankton samples.
We do not believe this prohibits its inclusion in a marine methods manual since the analytical
steps are the same regardless of algae classification. An effort was made to include a review
of the current pertinent literature on chlorophyll measurement. A visible spectrophotometric
method for chlorophyll a, b, and c and the carotenoids is not included in this edition of the
manual because more research is required for a thorough evaluation of this method.
The three metals methods presented here represent current state-of-the-science in
metals measurement and are suitable for low-level concentrations in high salinity waters. The
two methods that use the chelation preconcentration chromatography system offer dete ction
limits roughly an order of magnitude lower than their conventional counterpart methods. As
the instrumentation for these techniques becomes more prevalent in analytical laboratories,
the methods will undergo multilaboratory validation studies.
This manual should be viewed as a living document, with methods for organics, nutrients,
and metals continually being added, updated, revised, and validated. There is also much
viii
Revision 1.0 November 1992
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work to be done in assuring the provision of SRMs and quality control samples to the marine monitoring
community. The energy to sustain this long-term effort comes from the commitment of personnel in Regions
2 and 3 and in EMSL-Cincinnati to the goals set by the coastal regions in 1990. We encourage users of the
methods in this manual to share their experiences with us and to obtain new editions of the manual as they
become available.
The methods in this manual are not intended to be specific for any single USEPA regulation, compliance
monitoring program, or specific study. In the past, manuals have been developed and published that respond
to specific regulations, such as the Safe Drinking Water Act (SDWA), or to special studies, such as the
Environmental Monitoring and Assessment Program (EMAP) Near Coastal Demonstration Project. These
methods are, however, available for incorporation into regulatory programs that require the measurement of
nutrients and metals in marine waters.
Elizabeth J. Arar, William L Budde, and Larry B. Lobring
Chemistry Research Division
November 1992
ix
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Method 200.10
Determination of Trace Elements in Marine Waters by
On-Line Chelation Preconcentration and Inductively
Coupled Plasma - Mass Spectrometry
Stephen E. Long
Technology Applications, Inc.
and
Theodore D. Martin
Inorganic Chemistry Branch
Chemistry Research Division
Revision 1.6
November 1992
Edited by
John T. Creed
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
200.10-1
Revision 1.6 November 1992
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Method 200.10
i.
Determination of Trace Elements in Marine Waters by On-Line Chelation
Preconcentration and Inductively Coupled Plasma - Mass Spectrometry
1.0 Scope and Application
1.1 This method describes procedures for
preconcentration and determination of total recoverable
trace elements in marine waters, including estuarine
water, seawater, and brines.
1.2 Acid solubiiization is required prior to the determi-
nation of total recoverable elements to facilitate break-
down of complexes or colloids that might influence trace
element recoveries. This method should only be used for
preconcentration and determination of trace elements in
aqueous samples.
1.3 This method is applicable to the following ele-
ments:
Element
Chemical Abstracts Service
Registry Numbers (CASRN)
Cadmium
Cobalt
Copper
Lead
Nickel
Uranium
Vanadium
(Cd)
(Co)
(Cu)
(Pb)
(Ni)
(U)
(V)
7440-43-9
7440-48-4
7440-50-8
7439-92-1
7440-02-0
7440-61-1
7440-62-2
1.4 Method detection limits (MDLs) forthese elements
will be dependent on the specific instrumentation em-
ployed and the selected operating conditions. However,
the MDLs should be essentially independent of the matrix
because elimination of the matrix is a feature of the
method. Reagent water MDLs, which were determined
using the procedure described in Section 9.2.4, are listed
in Table 1.
1.5 A minimum of 6-months experience in the use of
commercial instrumentation for inductively coupled
plasma mass spectrometry (ICP-MS) is recommended.
2.0 Summary of Method
2.1 This method is used to preconcentrate trace ele-
ments using an iminodiacetate functionalized chelating
resin.1-2 Following acid solubiiization, the sample is buff-
ered prior to the chelating column using an on-line
system. Groups 1 and II metals, as well as most anions,
are selectively separated from the analytes by elution
with ammonium acetate at pH 5.5. The analytes are
subsequently eluted into a simplified matrix consisting of
dilute nitric acid and are determined by ICP-MS using a
directly coupled on-line configuration.
2.2 The determinative step in this method is
MS.3-5 Sample material in solution is introduced by
pneumatic nebulization into a radiofrequency plasma
where energy transfer processes cause desolvation,
atomization and ionization. The ions are extracted from
the plasma through a differentially pumped vacuum
interface and separated on the basis of their mass-to-
charge ratio by a quadrupole mass spectrometer hav-
ing a minimum resolution capability of 1 amu peak width
at 5% peak height. The ions transmitted through the
quadrupole are registered by a continuous dynode elec-
tron multiplier or Faraday detector and the ion informa-
tion is processed by a data handling system. Interfer-
ences relating to the technique (Section 4) must be
recognized and corrected. Such corrections must in-
clude compensation for isobaric elemental interferences
and interferences from polyatomic ions derived from the
plasma gas, reagents or sample maitrix. Instrumental
drift must be corrected for by the use of internal standard-
ization.
3.0 Definitions
3.1 Calibration Blank (CB) — A volume of reagent
water fortified with the same matrix as the calibration
standards but without the analytes, internal standards, or
surrogate analytes.
3.2 Calibration Standard (CAL) — A solution pre-
pared from the primary dilution standard solution or stock
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate
the instrument response with respect to analyte concen-
tration.
3.3 Instrument Detection Limit (IDL) — The mini-
, mum quantity of analyte or the concentration equivalent
that gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance
line, etc.
3.4 Instrument Performance Check Solution
(IPC) — A solution of one or more method analytes,
surrogates, internal standards, or othertest substances
used to evaluate the performance of the instrument
system with respect to a defined set of criteria.
3.5 Internal Standard (IS)—Apureanalyte(s) added
to a sample, extract, or standard solution in known
amount(s) and used to measure the relative responses
of other method analytes and surrogates that are compo-
nents of the same sample or solution. The internal
standard must be an analyte that is not a sample compo-
nent.
200.10-2
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3.6 Laboratory Fortified Blank (LFB)—An aliquot of
reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
is in control and whether the laboratory is capable of
making accurate and precise measurements.
3.7 Laboratory Fortified Sample Matrix (LFM) —
An aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample'
matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background
concentrations.
3.8 Laboratory Reagent Blank (LRB)—An aliquot of
reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, the
reagents, or the apparatus.
3.9 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instru-
ment response to an analyte is linear.
3.10 Material Safety Data Sheet (MSDS) — Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
3.11 Method Detection Limit (MDL)—The minimum
concentration of an analyte that can be identified, mea-
sured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.12 Quality Control Sample (QCS) — A solution of
method analytes of known concentrations that is used to
fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.13 Stock Standard Solution (SSS) — A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
3.14 Total Recoverable Analyte (TRA) — The con-
centration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following treat-
ment by refluxing with hot dilute mineral acid(s) as
specified in the method.
3.15 Tuning Solution (TS)—A solution that is used to
adjust instrument performance prior to calibration and
sample analyses.
4.0 Interferences
4.1 Several interference sources may cause inaccura-
cies in the determination of trace elements by ICP-MS.
These are:
4.1.1 Isobaric elemental interferences—Are caused by
isotopes of different elements that form singly or doubly
charged ions of the same nominal mass-to-charge ratio
and that cannot be resolved by the mass spectrometer in
use. All elements determined by this method have, at a
minimum, one isotope free of isobaric elemental interfer-
ence. The analytical isotopes recommended for use with
this method are listed in Table 1.
4.1.2 Abundance sensitivity— Is a property defining the
degree to which the wings of a mass peak contribute to
adjacent masses. The abundance sensitivity is affected
by ion energy and quadrupole operating pressure. Wing
overlap interferences may result when a small ion peak
is being measured adjacent to a large one. The potential
for these interferences should be recognized and the
spectrometer resolution adjusted to minimize them.
4.1.3 Isobaric polyatomic ion interferences—Are caused
by ions consisting of more than one atom that have the
same nominal mass-to-charge ratio as the isotope of
interest and that cannot be resolved by the mass spec-
trometer in use. These ions are commonly formed in the
plasma or interface system from support gases or sample
components. Such interferences must be recognized,
and when they cannot be avoided by the selection of
alternative analytical isotopes, appropriate corrections
must be made to the data. Equations for the correction of
data should be established at the time of the analytical
run sequence as the polyatomic ion interferences will be
highly dependent on the sample matrix and chosen
instrument conditions.
4.1.4 Physical interferences — Are associated with the
physical processes that govern the transport of sample
into the plasma, sample conversion processes in the
plasma, and the transmission of ions through the plasma-
mass spectrometer interface. These interferences may
result in differences between instrument responses for
the sample and the calibration standards. Physical inter-
ferences may occur in the transfer of solution to the
nebulizer (e.g., viscosity effects), at the point of aerosol
formation and transport to the plasma (e.g., surface
tension), or during excitation and ionization processes
within the plasma itself. Internal standardization may be
effectively used to compensate for many physical inter-
ference effects.6 Internal standards ideally should have
similar analytical behavior to the elements being deter-
mined.
4.1.5 Memory interferences— Result when isotopes of
elements in a previous sample contribute to the signals
measured in a new sample. Memory effects can result
from sample deposition on the sampler and skimmer
cones and from the buildup of sample material in the
plasma torch and spray chamber. The site where these
200.10-3
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effects occur is dependent on the element and can be
minimized by flushing the system with a rinse blank
between samples. Memory interferences from the che-
lating system may be encountered especially after ana-
lyzing a sample containing high concentrations of the
anaiytes. A thorough column rinsing sequence following
elution of the anaiytes is necessary to minimize such
interferences.
4.2 A principal advantage of this method is the selec-
tive elimination of species giving rise to polyatomic spec-
tral interferences on certain transition metals (e.g., re-
moval of the chloride interference on vanadium). As the
majority of the sample matrix is removed, matrix induced
physical interferences are also substantially reduced.
4.3 Low recoveries may be encountered in the
preconcentration cycle if the trace elements are
complexed by competing chelators in the sample or are
present as colloidal material. Acid solubilization pretreat-
ment is employed to improve analyte recovery and to
minimize adsorption, hydrolysis, and precipitation ef-
fects.
5.0 Safety
5.1 Each chemical reagent used in this method should.'
be regarded as a potential health hazard and exposure to
these reagents should be as low as reasonably achiev-
able. Each laboratory is responsible for maintaining a
current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this
method.7-8 A reference file of material data handling
sheets should also be available to all personnel involved
in the chemical analysis.
5.2 Analytical plasma sources emit radiofrequency
radiation in addition to intense UV radiation. Suitable
precautions should be taken to protect personnel from
such hazards.
5.3 The acidification of samples containing reactive
materials may result in the release of toxic gases, such as
cyanides or sulfides. Acidification of samples should be
performed in a fume hood.
5.4 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.5 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Equipment and Supplies
6.1 Preconcentration System—System containing
no metal parts in the analyte flow path, configured as
shown in Figure 1. •
6.1.1 Column—Macroporous iminodiacetate chelating
resin (Dionex Metpac CC-1 or equivalent).'
6.1.2 Sample loop— 10-mL loop constructed from nar-
row bore, high-pressure inert tubing, Tefzel ethylene
tetra-fluoroethylene (ETFE) or equivalent.
6.1.3 Eluent pumping system (P1) — Programmable
flow, high pressure pumping system, capable of deliver-
ing either one of two eluents at a pressure up to 2000 psi
and a flow rate of 1-5 mL/min.
6.1.4 Auxiliary pumps — On line buffer pump (P2),
piston pump (Dionex QIC pump or equivalent) for deliv-
ering 2M ammonium acetate buffer solution; carrierpump
(P3), peristaltic pump (Gilson Minipuls or equivalent) for
delivering 1% nitric acid carrier solution; sample pump
(P4), peristaltic pump for loading sample loop.
6.1.5 Control valves — Inert double stack, pneumati-
cally operated four-way slider valves with connectors.
6.1.5.1 Argon gas supply regulated at 80-100 psi.
6.1.6 Solution reservoirs— Inert containers, e.g., high
density polyethylene (HOPE), for holding eluent and
carrier reagents.
6.1.7 Tubing— High pressure, narrow bore, inert tubing
(e.g., Tefzel ETFE or equivalent) for interconnection of
pumps/valve assemblies and a minimuim length for con-
nection of the preconcentration system to the ICP-MS
instrument.
6.2 Inductively Coupled Plasma - Mass Spectrom-
eter
6.2.1 Instrument capable of scanning the mass range 5-
250 amu with a minimum resolution capability of 1 amu
peak width at 5% peak height. Instrument may be fitted
with a conventional or extended dynamic range detection
system.
6.2.2 Argon gas supply (high-purity grade, 99.99%).
6.2.3 A mass-flow controller on the nebulizer gas supply
is recommended. A water-cooled spray chamber may be
of benefit in reducing some types of interferences (e.g.,
polyatomic oxide species).
6.2.4 Operating conditions—Because of the diversity of
instrument hardware, no detailed instrument operating
conditions are provided. The analyst is advised to follow
the recommended operating conditions provided by the
manufacturer.
6.2.5 If an electron multiplier detector is being used,
precautions should be taken, where necessary, to pre-
vent exposure to high ion flux. Otherwise changes in
instrument response or damage to the multiplier may
result. Samples having high concentrations of elements
beyond the linear range of the instrument and with
isotopes falling within scanning windows should be di-
luted prior to analysis.
6.3 Labware — For the determination of trace ele-
ments, contamination and loss are of critical concern.
Potential contamination sources include improperly
200.10-4
Revision 1.6 November 1992
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cleaned laboratory apparatus and general contamina-
tion within the laboratory environment. A clean laboratory
work area, designated for trace element sample han-
dling, must be used. Sample containers can introduce
positive and negative errors in the determination of trace
elements by (1) contributing contaminants through sur-
face desorption or leaching or (2) depleting element
concentrations through adsorption processes. For these
reasons, borosilicate glass is not recommended for use
with this method. All labware in contact with the sample
should be cleaned prior to use. Labware may be soaked
overnight and thoroughly washed with laboratory-grade
detergent and water, rinsed with water, and soaked for 4
hr in a mixture of dilute nitric and hydrochloric acids,
followed by rinsing with ASTM type I water and oven
drying.
6.3.1 Griffin beakers, 250-mL, polytetrafluoroethylene
(PTFE) or quartz.
6.3.2 Storage bottles — Narrow mouth bottles, Teflon
FEP (fluorinated ethylene propylene), or HOPE, 125-mL
and 250-mL capacities.
6.4 Sample Processing Equipment
6.4.1 Air displacement pipetter — Digital pipet system
capable of delivering volumes from 10 to 2500 \iL with an
assortment of metal-free, disposable pipet tips.
6.4.2 Balances—Analytical balance, capable of accu-
rately weighing to ± 0.1 mg; top pan balance, accurate to
± 0.01 g.
6.4.3 Hotplate — Corning PC100 or equivalent.
6.4.4 Centrifuge — Steel cabinet with guard bowl,
electric timer and brake.
6.4.5 Drying oven — Gravity convection oven with
thermostatic control capable of maintaining 105°C+5°C.
6.4.6 pH meter— Bench mounted or hand-held elec-
trode system with a resolution of ± 0.1 pH units.
7.0 Reagents and Standards
7. i Water— For all sample preparation and dilutions,
ASTM type I water (ASTM D1193) is required.
7.2 Reagents may contain elemental impurities that
might affect the integrity of analytical data. Because of
the high sensitivity of this method, ultra high-purity re-
agents must be used unless otherwise specified. To
minimize contamination, reagents should be prepared
directly in their designated containers where possible.
7.2.1 Acetic acid, glacial (sp. gr. 1.05).
7.2.2 Ammonium hydroxide (20%).
7.2.3 Ammonium acetate buffer 1M, pH 5.5— Add 58
mL (60.5 g) of glacial acetic acid to 600 mL of ASTM type
water. Add 65 mL (60 g) of 20% ammonium hydroxide
and mix. Check the pH of the resulting solution by
withdrawing a small aliquot and testing with a calibrated
pH meter, adjusting the solution to pH 5.5 ± 0.1 with small
volumes of acetic acid or ammonium hydroxide as nec-
essary. Cool and dilute to 1 L with ASTM type I water.
7.2.4 Ammonium acetate buffer 2M, pH 5.5— Prepare
as for Section 7.2.3 using 116 mL (121 g) glacial acetic
acid and 130 mL (120 g) 20% ammonium hydroxide,
diluted to 1000 mL with ASTM type I water.
Note: The ammonium acetate buffer solutions may be
further purified by passing them through the
chelating column at a flow rate of 5.0 mL/min.
With reference to Figure 1, pump the buffer
solution through the column using pump P1, with
valves A and B off and valve C on. Collect the
purified solution in a container at the waste outlet.
Following this, elute the collected contaminants
from the column using 1.25M nitric acid for 5 min
at a flow rate of 4.0 mL/min.
7.2.5 Nitric acid, concentrated (sp.gr, 1.41).
7.2.5.1 Nitric acid 1.25M — Dilute 79 mL (112 g) cone.
nitric acid to 1000 mL with ASTM type I water.
7.2.5.2 Nitric acid 1 %—Dilute 10 mL cone, nitric acid to
1000 mL with ASTM type I water.
7.2.5.3 Nitric acid (1 +1)—Dilute 500 mLconc. nitric acid
to 1000 mL with ASTM type I water.
7.2.5.4 Nitric acid (1 +9) — Dilute 100 mLconc. nitric acid
to 1000 mL with ASTM type I water.
7.2.6 Oxalic acid dihydrate (CASRN 6153-56-6), 0.2M
— Dissolve 25.2 g reagent grade C2H2O4-2H2O in 250 mL
ASTM type I water and dilute to 1000 mL with ASTM type
I water. Caution - Oxalic acid is toxic; handle with care.
7.3 Standard Stock Solutions— May be purchased
from a reputable commercial source or prepared from
ultra high-purity grade chemicals or metals (99.99 -
99.999% pure). All salts should be dried for 1 h at 105°C,
unless otherwise specified. (Caution- Many metal salts
are extremely toxic if inhaled or swallowed. Wash hands
thoroughly after handling.) Stock solutions should be
stored in plastic bottles. The following procedures may
be used for preparing standard stock solutions:
Note: Some metals, particularly those that form surface
oxides require cleaning prior to being weighed.
This may be achieved by pickling the surface of
the metal in acid. An amount in excess of the
desired weight should be pickled repeatedly,
rinsed with water, dried, and weighed until the
desired weight is achieved.
7.3.1 Cadmium solution, stock 1 mL = 1000 jig Cd:
Pickle cadmium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5 mL (1+1) nitric acid,
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Revision 1.6 November 1992
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heating to effect solution. Cool and dilute to 100 mL with
ASTM type I water.
7.3.2 Cobalt solution, stock 1 mL = 1000 jig Co: Pickle
cobaltmetal in (1+9) nitricacid to an exact weight of 0.100
g. Dissolve in 5 mL (1+1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I
water.
7.3.3 Copper solution, stock 1 mL= 1000 fig Cu: Pickle
copper metal in (1+9) nitric acid to an exact weight of
0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
I water.
7,3.4 Indium solution, stock 1 mL = 1000-jig in: Pickle
Indium metal in (1+1) nitric acid to an exact weight of ,
0.100 g. Dissolve in 10 mL (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
I water.
7.3.5 Lead solution, stock 1 mL= 1000 jig Pb: Dissolve
0.1599 g PbNO3 in 5 mL (1 +1) nitric acid. Dilute to 100 mL
with ASTM type I water.
7.3.6 Nickel solution, stock 1 mL= 1000 jig Ni: Dissolve
0.100 g nickel powder in 5 mL cone, nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
1 water.
7.3.7 Scandium solution, stock 1 mL = 1000 ^g Sc:
Dissolve 0.1534 g Sc2O3 in 5 mL (1 +1) nitric acid, heating
to effect solution. Cool and dilute to 100 mL with ASTM
type I water.
7.3.8 Terbium solution, stock 1 mL = 1000 p.g Tb: Dis-
solve 0.1176 g Tb«O7 in 5 mL cone, nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
I water.
7.3.9 Uranium solution, stock 1 mL = 1000 ng U: Dis-
solve 0.2110 g UO2(NCg2.6H O (Do Not Dry) in 20 mL
ASTM type I water. Add 2 mL (1 +1) nitric acid and dilute
to 100 mL with ASTM type I water.
7.3.10 Vanadium solution, stock 1 mL = 1000 ng V:
Pickle vanadium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5 mL (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100 mL with
ASTM type I water.
7.3.11 Yttrium solution, stock 1 mL = 1000 ng Y: Dis-
solve 0.1270 g Y2O3 in 5 mL (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
I water.
7.4 Multielement Stock Standard Solution—Care
must be taken in the preparation of multielement stock
standards that the elements are compatible and stable.
Originating element stocks should be checked for impu-
rities that might influence the accuracy of the standard.
Freshly prepared standards should be transferred to acid
cleaned, new FEP or HOPE bottles for storage and
monitored periodically for stability. A multielement stock
standard solution containing the elements, cadmium,
cobalt, copper, lead, nickel, uranium, and vanadium (1
mL = 10 ng) may be prepared by diluting 1 mL of each
single element stock in the list to 100 ml. with ASTM type
I water containing 1 % (v/v) nitric acid.
7.4.1 Preparation of calibration standards — Fresh
multielement calibration standards should be prepared
weekly. Dilute the stock multielement standard solution
in 1% (v/v) nitric acid to levels appropriate to the re-
quired operating range. The element concentrations in
the standards should be sufficiently high to produce
good measurement precision and to accurately define
the slope of the response curve. A suggested mid-range
concentration is 10 ng/L.
7.5 Blanks—Fourtypes of blanks are required forthis
method. A calibration blank is used to establish the
analytical calibration curve, and the laboratory reagent
blank is used to assess possible contamination from the
sample preparation procedure. The laboratory fortified
blank is used to assess the recovery of the method
analytes and the rinse blank is used between samples to
minimize memory from the nebulizer/spray chamber
surfaces.
7.5.1 Calibration blank— Consists of 1 % (v/v) nitric acid
in ASTM type I water (Section 7.2.5.2).
7.5.2 Laboratory reagent blank (LRB)—-Must contain all
the reagents in the same volumes as used in processing
the samples. The LRB must be carried through the entire
sample digestion and preparation scheme.
7.5.3 Laboratory FortifiedBlank (LFB)—Toan aliquot of
LRB, addaliquots from the multielement stock standard
(Section 7.4) to produce a final concentration of 10 ng/L
for each analyte. The fortified blank must be carried
through the entire sample pretreatment and analytical
scheme.
7.5.4 Rinse Blank (RB) — Is a 1% (v/v) nitric acid
solution that is delivered to the ICP-MS between samples
(Section 7.2.5.2).
7.6 Tuning Solution — This solution is used for
instrument tuning and mass calibration prior to analysis
(Section 10.2). The solution is prepared by mixing nickel,
yttrium, indium, terbium, and lead stock solutions (Sec-
tion 7.3) in 1 % (v/v) nitric acid to produce a concentration
of 100 jig/L of each element.
7.7 Quality Control Sample (QCS) — A quality con-
trol sample having certified concentrations of the analytes
of interest should be obtained from a source outside the
laboratory. Dilute the QCS if necessary with 1% nitric
acid, such that the analyte concentrations fall within the
proposed instrument calibration range.
7.8 Instrument Performance Check (IPC) Solution
— The IPC solution is used to periodically verify instru-
ment performance during analysis. It should be prepared
by combining method analytes at appropriate concentra-
200.10-6
Revision 1.6 November 1992
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tions to approximate the midpoint of the calibration curve.
The I PC solution should be prepared from the same
standard stock solutions used to prepare the calibration
standards and stored in a FEP bottle. Agency programs
may specify or request that additional instrument perfor-
mance check solutions be prepared at specified concen-
trations in order to meet particular program needs.
7.9 Internal Standards Stock Solution, 1 ml = 100
\ig.—Dilute 10 ml of scandium, yttrium, indium, terbium,
and bismuth stock standards (Section 7.3) to 100 mL with
ASTM type I water, and store in a Teflon bottle. Use this
solution concentrate for addition to blanks, calibration
standards and samples (Method A, Section 10.5), or
dilute by an appropriate amount using 1%(v/v) nitricacid,
if the internal standards are being added by peristaltic
pump (Method B, Section 10.5).
Note: Bismuth should not be used as an internal stan-
dard using the direct addition method (Method A,
Section 10.5) as it is not efficiently concentrated
on the iminodiacetate column.
8.0 Sample Collection, Preservation, and
Storage
8.1 Prior to the collection of an aqueous sample,
consideration should be given to the type of data re-
quired, so that appropriate preservation and pretreat-
ment steps can be taken. Acid preservation should be
performed at the time of sample collection or as soon
thereafter as practically possible. The pH of all aqueous
samples must be tested immediately prior to aliquoting
for analysis to ensure the sample has been properly
preserved. If properly acid preserved, the sample can be
held up to 6 months before analysis.
8.2 For the determination of total recoverable ele-
ments in aqueous samples, acidify with (1+1) nitric acid
(high purity) at the time of collection to pH < 2; normally,
3 mL of (1 +1) acid per liter of sample is sufficient for most
samples. The sample should not be filtered prior to
analysis.
Note: Samples that cannot be acid preserved at the
time of collection because of sampling limitations
or transport restrictions, or are > pH 2 because of
high alkalinity should be acidified with nitric acid
to pH <2 upon receipt in the laboratory. Following
acidification, the sample should be held for 16 h
and the pH verified to be < 2 before withdrawing
an aliquot for sample processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container and acid as used in sample collec-
tion.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability and the
periodic analysis of laboratory reagent blanks, fortified
blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to
maintain performance records that define the quality of
the data generated.
9.2 Initial Demonstration of Performance (Manda-
tory)
9.2.1 The initial demonstration^ performance is used to
characterize instrument performance (determination of
linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) priorto samples being analyzed
by this method.
9.2.2 Linear calibration ranges—The upper limit of the
linear calibration range should be established for each
analyte. Linear calibration ranges should be determined
every six months or whenever a significant change in
instrument response is expected.
9.2.3 Quality control sample (QCS) — When beginning
the use of this method, on a quarterly basis or as required
to meet data-quality needs, verify the calibration stan-
dards and acceptable instrument performance with the
preparation and analyses of a QCS (Section 7.7). If the
determined concentrations are not within ± 10% of the
stated values, performance of the determinative step of
the method is unacceptable. The source of the problem
must be identified and corrected before either proceed-
ing with the initial determination of method detection
limits or continuing with ongoing analyses.
9.2.4 Metho.d detection limit (MDL) — MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.9 To determine MDL
values, take seven replicate aliquots of the fortified
reagent water and process through the entire analytical
method. Perform all calculations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
MDL = (t) x (S)
where: t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
Note: If the relative standard deviation (BSD) from the
analyses of the seven aliquots is < 15%, the
concentration used to determine the analyte MDL
may have been inappropriately high forthe deter-
mination. If so, this could result in the calculation
of an unrealistically low MDL. If additional confir-
mation of the MDL is desired, reanalyze the
seven replicate aliquots on two more
200.10-7
Revision 1.6 November 1992
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nonconsecutive days and again calculate the
MDL values for each day. An average of the three
MDL values for each analyte may provide for a
more appropriate MDL estimate. Concurrently,
determination of MDL in reagent water repre-
sents a best case situation and does not reflect
possible matrix effects of real world samples.
However, successful analyses of LFMs (Section
9.4) can give confidence to the MDL value deter-
mined in reagent water. Typical single laboratory
MDL values using this method are given in Table
1.
MDLs should be determined every six months, when a
new operator begins work or whenever there is a signifi-
cant change in the background or instrument response.
9.3 Assessing Laboratory Performance (Manda-
tory)
9.3.1 Laboratory reagent blank (LRB)—The laboratory
must analyze at least one LRB (Section 7.5.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and
the samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
9.3.2 Laboratory fortified blank (LFB)—The laboratory
must analyze at least one LFB (Section 7.5.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that analyte
is judged out of control, and the source of the problem
should be identified and resolved before continuing analy-
ses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance againstthe required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most- recent 20-30 data
points. Also, the standard deviation (S) data should be
used to established an ongoing precision statement for
the level of concentrations included in the LFB. These
data must be kept on file and be available for review.
9.3.4 Instrument performance check (IPC) solution —
For all determinations the laboratory must analyze the |
IPC solution (Section 7.8) and a calibration blank imme-
diately following daily calibration, after every tenth sample
(or more frequently, if required) and at the end of the
sample run. Analysis of the IPC solution and calibration
blank immediately following calibration must verify that
the instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration
within ± 15%. If the calibration cannot be verified within
the specified limits, reanalyze the IPC solution. If the
second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be discon-
tinued, the cause determined and/or in the case of drift
the instrument recalibrated. All samples following the last
acceptable IPC solution must be reanalyzed. The analy-
sis data of the calibration blank and IPC solution must be
kept on file with the sample analyses data.
9.3.5 The overall sensitivity and precision of this method
are strongly influenced by a laboratory's ability to control
the method blank. Therefore, it is recommended that the
calibration blank response be recorded for each set of
samples. This record will aid the laboratory in assessing
both its long- and short-term ability to control the method
blank.
9.4 Assessing Analyte Recovery and Data Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and the
quality of the data. Taking separate aliquots from the
sample for replicate and fortified analyses can in some
cases assess these effects. Unless otherwise specified
by the data user, laboratory or program, the following
laboratory fortified matrix (LFM) procedure (Section 9.4.2)
is required.
9.4.2 The laboratory must add a known amount of each
analyte to a minimum of 10% of the routine samples. In
each case the LFM aliquot must be a duplicate of the
aliquot used for sample analysis and for total recoverable
determinations added prior to sample preparation. For
water samples, the added analyte concentration must be
the same as that used in the laboratory fortified blank
(Section 9.3.2).
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is less than
25% of the unfortified sample concentration. Percent
recovery may be calculated in units appropriate to the
matrix, using the following equation:
R=(cs-c)x100
S
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
S = concentration equivalent of analyte
added to sample.
200.10-8
Revision 1.6 November 1992
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9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range and the laboratory per-
formance for that analyte is shown to be in control
(Section 9.3), the recovery problem encountered with the
LFM is judged to be either matrix or solution related, not
system related.
9.4.5 If analysis of LFM sample(s) and the test routines
above indicate an operative interference and the LFMs
are typical of the other samples in the batch, those
samples that are similar must be analyzed in the same
manner as the LFMs. Also, the data user must be
informed when a matrix interference is so severe that it
prevents the successful analysis of the analyte or when
the heterogeneous nature of the sample precludes the
use of duplicate analyses.
9.4.6 Where reference materials are available, they
should be analyzed to provide additional performance
data. The analysis of reference samples is a valuable tool
for demonstrating the ability to perform the method
acceptably.
10.0 Calibration and Standardization
10.1 Initiate proper operating configuration of ICP-MS
instrument and data system. Allow a period of not less
than 30 min for the instrument to warm up. During this
process conduct mass calibration and resolution checks
using the tuning solution. Resolution at low mass is
indicated by nickel isotopes 60,61,62. Resolution at high
mass is indicated by lead isotopes 206, 207, 208. For
good performance adjust spectrometer resolution to pro-
duce a peak width of approximately 0.75 amu at 5% peak
height. Adjust mass calibration if it has shifted by more
than 0.1 amu from unit mass.
1.0.2 Instrument stability must be demonstrated by ana-
lyzing the tuning solution (Section 7.6) a minimum of five
times with resulting relative standard deviations of abso-
lute signals for all analytes of less than 5%.
10.3 Prior to initial calibration, set up proper instrument
software routines for quantitative analysis and connect
the ICP-MS instrument to the preconcentration appara-
tus. The instrument must be calibrated forthe analytes of
interest using the calibration blank (Section 7.5.1) and
calibration standard (Section 7.4.1) prepared at one or
more concentration levels. The calibration solutions should
be processed through the preconcentration system us-
ing the procedures described in Section 11.
10.4 Demonstration and documentation of acceptable
initial calibration is required before any samples are
analyzed. After initial calibration is successful, a calibra-
tion check is required at the beginning and end of each
period during which analyses are performed and at
requisite intervals.
70.4.7 After the calibration has been established, it
must be initially verified for all analytes by analyzing the
I PC (Section 7.8). If the initial calibration verification
exceeds ± 10% of the established I PC value, the analysis
should be terminated, the source of the problem identi-
fied and corrected, the instrument recalibrated, and the
new calibration verified before continuing analyses.
70.4.2 To verify that the instrument is properly cali-
brated on a continuing basis, analyze the calibration
blank (Section 7.5.1) and I PC (Section 7.8) after every 10
analyses. The results of the analyses of the standards
will indicate whether the calibration remains valid. If the
indicated concentration of any analyte deviates from the
true concentration by more than 15%, reanalyze the
standard. If the analyte is again outside the 15% limit, the
instrument must be recalibrated and the previous 10
samples reanalyzed. The instrument responses from the
calibration check may be used for recalibration pur-
poses.
10.5 Internal Standardization - Internal standardiza-
tion must be used in all analyses to correct for instrument
drift and physical interferences. For full mass range
scans, a minimum of three internal standards must be
used. Internal standards must be present in all samples,
standards and blanks at identical levels. This may be
achieved by directly adding an aliquot of the internal
standards to the CAL standard, blank or sample solution
(Method A), or alternatively by mixing with the solution
prior to nebulization using a second channel of the
peristaltic pump and a mixing coil (Method B). The
concentration of the internal standard should be suffi-
ciently high that good precision is obtained in the mea-
surement of the isotope used for data correction and to
minimize the possibility of correction errors if the internal
standard is naturally present in the sample. Internal
standards should be added to blanks, samples and
standards in a like manner, so that dilution effects result-
ing from the addition may be disregarded.
Note: Bismuth should not be used as an internal stan-
dard using the direct addition method (Method A,
Section 10.5) because it is not efficiently concen-
trated on the iminodiacetate column.
11.0 Procedure
11.1 Sample Preparation - Total Recoverable Ele-
ments
77.7.7 Add 2- mL (1 +1) nitric acid to the beaker contain-
ing 100 mL of sample. Place the beaker on the hot plate
for solution evaporation. The hot plate should be located
in a fume hood and previously adjusted to provide evapo-
ration at a temperature of approximately but no higher
than 85°C. (See the following note.) The beaker should
be covered with an elevated watch glass or other neces-
sary steps should be taken to prevent sample contamina-
tion from the fume hood environment.
Note: For proper heating, adjust the temperature con-
trol of the hot plate such that an uncovered Griffin
beaker containing 50 mL of water placed in the
200.10-9
Revision 1.6 November 1992
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center of the hot plate can be maintained at a
temperature approximately but no higher than
85°C. (Once the beaker is covered with a watch
glass the temperature of the water will rise to
approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20 mLby gentle heating at 85°C. Do Not Boil. This
step takes about 2 h for a 100-mL aliquot with the rate of
evaporation rapidly increasing as the sample volume
approaches 20 mL (A spare beaker containing 20 mL of
water can be used as a gauge.)
11.1.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the sample
forSO mln. (Slight boiling may occur, but vigorous boiling
must be avoided.)
11.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
11.1.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight, the
sample contains suspended solids, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination
from filtration.) The sample is now ready for analysis.;
Because the effects of various matrices on the stability of
diluted samples cannot be characterized, all analyses
should be performed as soon as possible after the
completed preparation.
11.2 Prior to first use, the preconcentration system
shoutdbethoroughly cleaned and decontaminated using
0.2M oxalic acid.
11.2.1 Place approximately 500-mL 0.2M oxalic acid in
all the eluent/solution containers and fill the sample loop
with 0.2M oxalic acid using the*sample pump (P4) at a
flow rate of 3-5 mL/min. With the preconcentration sys-
tem disconnected from the ICP-MS instrument, use the
pump program sequence listed in Table 2 to flush the
complete system with oxalic acid. Repeat the flush se-
quence three times.
11.2.2 Repeatthe sequence described in Section 11.2.1
using 1.25M nitric acid and again using ASTM type I
water in place of the 0.2M oxalic acid.
11.2.3 Rinse the containers thoroughly with ASTM type
I water, fill them with their designated reagents (see
Figure 1) and run through the sequence in Table 2 once
to prime the pump and all eluent lines with the correct
reagents.
11.3 Initiate ICP-MS instrument operating configura-
tion. Tune the instrument for the analytes of interest
(Section 10).
11.4 Establish instrument software run procedures for
quantitative analysis. Because the analytes are eluted
from the preconcentration column in a transient manner,
it is recommended that the instrument software is config-
ured in a rapid scan/peak hopping mode. The instrument
is now ready to be calibrated.
11.5 Reconnect the preconcentration system to the
ICP-MS instrument. With valves A and B in the off
position and valve C in the on position, load sample
through the sample loop to waste using pump P4 for 4
min at 4 mL/min. Switch on the carrier pump (P3) and
pump 1% nitric acid to the nebulizer of the ICP-MS
instrument at a flow rate of 0.8-1.0 mL/min.
11.6 Switch on the buffer pump (P2), and pump 2M
ammonium acetate at a flow rate of 1.0 mL/min.
11.7 Preconcentration of the sample may be achieved
by running through an eluent pump program (P1) se-
quence similar to that illustrated in Table 2. The exact
timing of this sequence should be modified according to
the internal volume of the connecting tubing and the
specific hardware configuration used.'
11.7.1 Injectsample—With valves A, B, and C on, load
sample from the loop onto the column using 1M ammo-
nium acetate for 4.5 min at 4.0 mL/min. The analytes are
retained on the column, while the majority of the matrix is
passed through to waste.
11.7.2 Elute analytes — Turn off valves A and B and
begin eluting the analytes by pumping 1.25M nitric acid
through the column at 4.0 mL/min, then turn off valve C
and pump the eluted analytes into the ICP-MS instrument
at 1.0 mL/min. Initiate ICP-MS software data acquisition
and integrate the eluted analyte profiles.
11.7.3 Column Reconditioning— Turn on valve C to
direct column effluent to waste, and pump 1.25M nitric
acid, 1M ammonium acetate, 1.25M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min. During this process, the next sample can be
loaded into the sample loop using the sample pump (P4).
11.8 Repeat the sequence described in Section 11.7 for
each sample to be analyzed. At the end of the analytical
run leave the column filled with 1M ammonium acetate
buffer until it is next used.
11.9 Samples having concentrations higher than the
established linear dynamic range should be diluted into
range with 1% HNO3 (v/v) and reanalyzed.
12.0 Data Analysis and Calculations
12.1 Analytical isotopes and elemental equations rec-
ommended for sample data calculations are listed in
Table 3. Sample data should be reported in units of pg/
L. Do not report element concentrations below the deter-
mined MDL.
12.2 Fordatavalueslessthan 10,twosignificantfigures
should be used for reporting element concentrations. For
data values greater than or equal to 10, three significant
figures should be used.
200.10-10
Revision 1.6 November 1992
-------
12.3 Reported values should be calibration blank sub-
tracted. If additional dilutions were made to any samples,
the appropriate factor should be applied to the calculated
sample concentrations.
12.4 Data values should be corrected for instrument
drift by the application of internal standardization. Cor-
rections for characterized spectral interferences should
be applied to the data.
12.5 The QC data obtained during the analyses provide
an indication of the quality of the sample data and should
be provided with the sample results.
13.0 Method Performance
13.1 Experimental conditions used for single laboratory
testing of the method are summarized in Table 4.
13.2 Data obtained from single laboratory testing of the
method are summarized in Tables 5 and 6 for two
reference water samples consisting of National Research
Council Canada (NRCC) Estuarine Water (SLEW-1) and
Seawater (NASS-2). The samples were prepared using
the procedure described in Section 11.1.1. For each
matrix, three replicates were analyzed and the average
of the replicates was used to determine the sample
concentration for each analyte. Two further sets of three
replicates were fortified at different concentration levels,
one set at 0.5 |ig/L, the other at 10 ng/L The sample
concentration, mean percent recovery, and the relative
standard deviation of the fortified replicates are listed for
each method analyte. The reference material certificate
values are also listed for comparison.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity ortoxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that place pollution pre-
vention as the management option of first choice. When-
ever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation
(e.g., Section 7.8). When wastes cannot be feasibly
reduced at the source, the Agency recommends recy-
cling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 115516th Street N.W., Wash-
ington, D.C. 20036, (202)872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management, consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. Siraraks, A., H. M. Kingston, and J. M. Riviello,
Anal Chem. ,62,1185 (1990).
2. Heithmar, E. M., T. A. Hinners, J. T. Rowan, and J.
M. Riviello, Anal Chem., 62, 857 (1990).
3. Gray A. L. and A. R. Date, Analyst, 108,1033
(1983).
4. Houk, R. S., et al. Anal Chem., 52, 2283 (1980).
5. Houk, R. S., Anal. Chem., 58, 97A (1986).
6. J. J. Thompson and R. S. Houk, Appl. Spec., 41,
801 (1987).
7. OSHA Safety and Health Standards, General Indus-
try, (29 CFR1910), Occupational Safety and Health
Administration, OSHA 2206, (Revised, January
1976).
8. Safety in Academic Chemistry Laboratories, Ameri-
can Chemical Society Publication, Committee on
Chemical Safety, 3rd Edition, 1979.
9. Code of Federal Regulations 40, Ch. 1, R. 136
Appendix B.
200.10-11
Revision 1.6 November 1992
-------
17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Total Recoverable Method Detection Limits
for Reagent Water
Element
Cadmium
Cobalt
Copper
Lead
Nickel
Uranium
Vanadium
Recommended
Analytical Mass
111
59
63
206.207.208
60
238
51
MDL'
ng/L
0.041
0.021
0.023
0.074
0.081
0.031
0.014
'Determined using 10-mL sample loop.
Table 2. Eluent Pump Programming Sequence for Preconcentration of Trace
Elements
Time
(mln)
0.0
4.5
5.1
5.5
7.5
8.0
10.0
11.0
12.5
Flow
(mL/mln)
. 4.0
4.0
1.0
1.0
4.0
4.0
4.0
4.0
0.0
Eluent
1M ammonium acetate
1.25M nitric acid
1 .25M nitric acid
1.25M nitric acid
1 .25M nitric acid
1M ammonium acetate
1.25M nitric acid
1 M ammonium acetate
Valve
A,B
ON
ON
OFF
OFF
OFF
OFF
OFF
OFF
OFF
Valve
C
ON
ON
ON
OFF
ON
ON
ON
ON
ON
Table 3. Recommended Analytical Isotopes and Elemental Equations for Data Calculations
Element
Cd
Co
Cu
Pb
Ni
U
V
Isotope
106.108.J7J.114
59
63,65
206,207,208
60
238
51
Elemental Equation
(1.000)("1C)-(1.073)[(108CH0.712)('°6C)]
(1.000}(59C)
(1.000)(
-------
Table 4. Experimental Conditions for Single Laboratory
Validation
Chromatography
Instrument
Preconcentration column
ICP-MS Instrument Conditions
Instrument
Plasma forward power
Coolant flow rate
Auxiliary flow rate
Nebulizer flow rate
Internal standards
Data Acquisition
Detector mode
Mass range
Dwell time
Number of MCA channels
Number of scan sweeps
Dionex chelation system
DionexMetPac CC-1
VG PlasmaQuad Type I
1.35kW
13.5 L/min
0.6 L/min
0.78 L/min
Sc, Y, In, Tb
Pulse counting
45-240 amu
160 (is
2048
250
Table 5. Precision and Recovery Data for Estuarine Water (SLEW-1)
Analyte
Cd
Co
Cu
Pb
Ni
U
V
Certificate
(ug/L)
0.018
0.046
1.76
0.028
0.743
Sample
Cone.
(|ig/L)
<0.041
0.078
1.6
<0.074
0.83
1.1
1.4
Spike
Addition
(ng/U
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Average
Recovery
(%)
94.8
102.8
106.0
100.2
100.0
96.7
100.0
RSD
(%)
9.8
4.0
2.7
4.0
1.5
7.4
3.2
Spike
Addition
(H9/L)
10
10
10
10
10
10
10
Average
Recovery
(%)
99.6
96.6
96.0
106.9
102.0
98.1
97.0
RSD
(%)
1.1
1.4
4.8
5.8
2.1
3.6
4.5
— No certificate value
Table 6. Precision and Recovery Data for Seawater (NASS-2)
Analyte
Cd
Co
Cu
Pb
Ni
U
V
Certificate
(H9/L)
0.029
0.004
0.109
0.039
0.257
3.00
Sample
Cone.
(W/L)
<0.041
<0.021
0.12
<0.074
0.23
3.0
1.7
Spike
Addition
(M9/1-)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Average
Recovery
(%)
101.8
98.9
95.8
100.6
102.2
94.0
104.0
RSD
(%)
1.0
. 3.0
2.3
8.5
2.3
0.7
3.4
Spike
Addition
(ng/U
10
10
10
10
10
10
10
Average
Recovery
(%)
96.4
99.2
93.1
92.1
98.2
98.4
109.2
RSD
(%)
3.7
1.7
0.9
2.6
1.2
1.7
3.7
— No certificate value
200.10-13
Revision 1.6 November 1992
-------
2M NH4OAc
I I
1 M NH..OAC 1 .25 M Nitric Acid
P3
1% Nitric Acid
Mixing 1'ee
Figure 1. Configuration of Preconcentration System.
Off
On
200.10-14
Revision 1.6 November 1992
-------
Method 200.12
Determination of Trace Elements in Marine Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption
John T. Creed and Theodore D. Martin,
Inorganic Chemistry Branch
Chemistry Research Division
Revision 1.0
November 1992
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
200.12 - 1
Revision 1.0 November 1992
-------
Method 200.12
Determination of Trace Elements in Marine Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption
1.0 Scope and Application
1.1 This method provides procedures for the determi-
nation of total recoverable elements by graphite furnace
atomic absorption (GFAA) in marine waters, including
estuarine, ocean and brines with salinities of up to 35 ppt.
This method is applicable to the following analytes:
Analyte
Chemical Abstracts
Service Registry
Numbers (CASRN)
Arsenic
Cadmium
Chromium
Copper
Lead
Nickel
Selenium
(As)
(Cd)
(Or)
(Cu)
(Pb)
(Ni)
(Se)
7440-38-2
7440-43-9
7440-47-3
7440-50-8
7439-92-1
7440-02-0
7782-49-2
1.2 For determination of total recoverable analytes in
marine waters, a digestion/extraction is required prior to
analysis.
1.3 Method detection limit and instrumental operating
conditionsforthe applicable elements are listed in Tables
1 and 2. These are intended as a guide and are typical of
a commercial instrument optimized for the element.
However, actual method detection limits and linear work-'.
ing ranges will be dependent on*the sample matrix,
instrumentation and selected operating conditions.
1.4 Users of the method data should state the data
quality objectives prior to analysis. The ultra-trace metal
concentrations typically associated with marine water
may preclude the use of this method based on its sensi-
tivity. Users of the method must document and have on
file the required initial demonstration performance data
described in Section 9.2 prior to using the method for
analysis.
2.0 Summary of Method
2.1 Nitric acid is dispensed into a beaker containing an
accurately weighed or measured, well-mixed, homoge-
neous aqueous sample. Then, for samples with undis-
solved material, the beaker is covered with a watch glass
and heated, made up to volume, centrifuged or allowed
to settle, and the sample is then analyzed.
2.2 The analytes listed in this method are determined
by stabilizedtemperature platform graphite furnace atomic
absorption (STPGFAA). In STPGFAA, the sample and
the matrix modifier are first pipetted onto the platform or
a device which provides delayed atomization.
The furnace chamber is then purged with a continuous
flow of a premixed gas (95% argon - 5% hydrogen) and
the sample is dried at a relatively low temperature (about
120°C) to avoid spattering. Once dried, the sample is
pretreated in a char or ashing step which is designed to
minimize the interference effects caused by the concomi-
tant sample matrix. After the char step, the furnace is
allowed to cool prior to atomization. The atomization
cycle is characterized by rapid heating of the furnace to
a temperature where the metal (analyte) is atomized from
the pyrolytic graphite surface into a stopped gas flow
atmosphere of argon containing 5% hydrogen. (Only
selenium is determined in an atmosphere of high purity
argon.) The resulting atomic cloud absorbs the element-
specific atomic emission produced by a hollow cathode
lamp (HCL) or an electrodeless discharge lamp (EDL).
Following analysis, the furnace is subjected to a cleanout
period of high temperature and continuous argon flow.
Because the resulting absorbance usually has a nonspe-
cific component associated with the actual analyte ab-
sorbance, Zeeman background correction is required to
subtract from the total signal the component which is
nonspecific to the analyte. In the absence of interfer-
ences, the background-corrected.absorbance is directly
related to the concentration of the analyte. Interferences
relating to STPGFAA (Section 4.0) must be recognized
and corrected. Suppressions or enhancements of instru-
ment response caused by the sample matrix must be
corrected for by the method of standard addition (Section
11.3).
3.0 Definitions
3.1 Calibration Blank (CB) — A volume of reagent
water fortified with the same matrix as the calibration
standards, but without the analytes, internal standards,
or surrogate analytes.
3.2 Calibration Standard (CAL) — A solution pre-
pared from the primary dilution standard solution or stock.
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate
the instrument response with respect to analyte concen-
tration.
3.3 Field Reagent Blank (FRB) — An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sam-
pling site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other
interferences are present in the field environment.
200.12-2
Revision 1.0 November 1992
-------
3.4 Instrument Detection Limit (IDL) — The mini-
mum quantity of analyte or the concentration equivalent
which gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance
line, etc.
3.5 Instrument Performance Check Solution (IPC)
— A solution of one or more method analytes, surro-
gates, internal standards, or other test substances used
to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.6 Laboratory Duplicates (LD1 and LD2) — Two
aliquots of the same sample taken in the laboratory and
analyzed separately with identical procedures. Analyses
of LD1 and LD2 indicates precision associated with
laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.7 Laboratory Fortified Blank (LFB)—Analiquotof
reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.8 Laboratory Fortified Sample Matrix (LFM)—An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background
concentrations.
3.9 Laboratory Reagent Blank (LRB)—An aliquot of
reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, the
reagents, or the apparatus.
3.10 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instru-
ment response to an analyte is linear.
3.11 Material Safety Data Sheet (MSDS) — Written
information provided by vendors concerning achemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
3.12 Matrix Modifier (MM)—A substance added to the
instrument along with the sample in order to minimize the
interference effects by selective volatilization of either
analyte or matrix components.
3.13 Matrix Performance Check (MFC) — A solution
of method analytes used to evaluate the laboratory's
ongoing capabilities in analyzing high salinity samples.
The reference material NASS-3 or its equivalent is forti-
fied with the same analytes at the same concentration as
the LFB. This provides an ongoing check of furnace
operating conditions to assure the analyte false positives
are not being introduced via elevated backgrounds.
3.14 Method Detection Limit (MDL)—The minimum
concentration of an analyte that can be identified, mea-
sured and reported with 99% confidence that the analyte
concentration is greater than zero.
3.15 Quality Control Sample (QCS) — A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.16 Standard Addition — The addition of a known
amount of analyte to the sample in order to determine the
relative response of the detector to an analyte within the
sample matrix. The relative response is then used to
assess either an operative matrix effect or the sample
analyte concentration.
3.17 Stock Standard Solution (SSS) — A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
3.18 Total Recoverable Analyte (TRA) — The con-
centration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following treat-
ment by refluxing with hot dilute mineral acid(s) as
specified in the method.
4.0 Interferences
4.1 Several interference sources may cause inaccura-
cies in the determination of trace elements by GFAA.
These interferences can be classified into three major
subdivisions: spectral, matrix, and memory.
4.2 Spectral interferences are caused by absorbance
of light by a molecule or atom which is not the analyte of
interest or emission from black body radiation. '
4.2.1 Spectral interferences caused by an element only
occur if there is a spectral overlap between the wave-
length of the interfering element and the analyte of
interest. Fortunately, this type of interference is relatively
uncommon in STPGFAA because of the narrow atomic
line widths associated with STPGFAA. In addition, the
use of appropriate furnace temperature programs and
high spectral purity lamps as light sources can minimize
the possibility of this type of interference. However,
molecular absorbances can span several hundred na-
nometers producing broadband spectral interferences.
This type of interference is far more common in STPGFAA.
The use of matrix modifiers, selective volatilization, and
background correctors are all attempts to eliminate un-
wanted nonspecific absorbance. Table 2 contains typical
background absorbances associated with the analysis of
200.12 - 3
Revision 1.0 November 1992
-------
the MPC solution (NASS-3) which has a salinity of 35 ppt.
These background absorbances were obtained using
the suggested matrix modifiers and the appropriate fur-
nace charring conditions. Figure 1 is a plot of integrated
background absorbance vs. char temperature for Ni, Cd,
Pb, and Se. Figure 1 indicates that the background
absorbance in a saline matrix is strongly affected by the
char temperature. Therefore, char temperature optimi-
zation is acritical part of the successful analysis of metals
in saline water by GFAA. The elevated backgrounds
associated with ocean water can produce false positives.
For this reason, the char temperature profiles shown in \
Figure 1 should be constructed for each analyte prior to
using this method for saline water analysis.
Note: False analyte positives can be generated by large
backgrounds. Figure 2 is an atomization profile for Pb
using a 800°C chartemperature. The background shown
in the figure has exceeded the capabilities of the Zeeman
corrector. This profile can be used as a guide in screening
otheranalyses which may have background absorbances
which exceed the Zeeman capability. The background
profile is characterized by a smooth baseline in the
beginning of the atomization cycle followed by a sharp
increase. During this sharp increase the background
peak profile may remain relatively smooth, but when the
background exceeds the Zeeman correction capability,
the background profile will appear extremely erratic. The
atomic profile is also erratic during this part of the atomi-
zation cycle. These types of background/atomic profiles
obtained during atomization result in false positives.
Since the nonspecific component of the total absorbance
can vary considerably from sample type to sample type,
to provide effective background correction and eliminate
the elemental spectral interference of palladium on cop-
per and iron on selenium, the exclusive use of Zeeman
background correction is specified in this method.
4.2.2 Spectral interferences are also caused by black
body radiation produced during the atomization furnace
cycle. This black body emission reaches the photomulti-
plier tube, producing erroneous results. The magnitude
of this interference can be minimized by proper furnace
tube alignment and monochromator design. In addition,
atomization temperatures which adequately volatilize
the analyte of interest without producing unnecessary
black body radiation can help reduce unwanted back-
ground emission produced during atomization.
4.3 Matrix interferences are caused by sample com-
ponents which inhibitthe formation of free atomic analyte
atoms during atomization. In this method the use of a
delayed atomization device which provides a warmer
gas phase environment during atomization is required.
These devices provide an environment which is more
conducive to the formation of free analyte atoms and
thereby minimize this type of interference. This type of
interference can be detected by analyzing the sample
plus a sample aliquot fortified with a known concentration
of the analyte. If the determined concentration of the
analyte addition is outside a designated range (Section
9.4.3), a possible matrix effect should be suspected. In
addition, the matrix can produce analyte complexes
200.
Revision 1.0 November 1992
which are lost via volatilization during the char. These
losses will result in poor recovery of the analyte within the
matrix and should be corrected by adjusting the char
temperature.
4.4 Memory interferences result from analyzing a
sample containing a high concentration of an element
(typically a high atomization temperature element) which
cannot be removed quantitatively in one complete set of
furnace steps. The analyte which remains in the furnace
can produce false positive signals on subsequent
sample(s). Therefore, the analyst should establish the
analyte concentration which can be injected into the
furnace and adequately removed in one complete set of
furnace cycles. If this concentration is exceeded, the
sample should be diluted and a blank analyzed to assure
the memory effect has been eliminated before reanalyz-
ing the diluted sample.
4.5 Specific Element Interferences. The matrix ef-
fects caused by the saline water can be severe. In order
to evaluate the extent of the matrix suppression as a
function of increasing salinity a plot of normalized inte-
grated absorbance vs. microliters NASS-3 (Reference
Material from the National Research Council of Canada)
is constructed. Figure 3 is a plot of relative response of
As, Se, Cd, Ni, Cu, and Pb in waters containing salinity of
3.5 ppt (1 \il NASS-3) to 35 ppt (10 \± NASS-3). Figure
3 indicates that the matrix effects caused by the increas-
ing salinity are minor for Pb, Cu, and Ni. The relative
responses of Pb, Ni, and Cu shown in Figure 3 are within
± 5% of the 1% HNO standard or zero \iL of matrix.
Figure 3 indicates that the increasing salinity does cause
a substantial matrix interference for Se and Cd. This
suppression must be compensated for by methods of
standard addition or the use of matrix matched standards
where applicable.
4.5.1 Cadmium: The background level associated with
the direct determination of Cd in NASiS-3 exceeds the
Zeeman background correction. Therefore, NH4NO is
used as a matrix removing modifier in addition to the Pd/
Mg(NO3)2.1 Figure 4 is a plot of the relative Cd response
vs. the amount of seawater on the platform. A similar
response profile is observed in a solution containing
10,000 ppm NaCI. Therefore, in well-characterized
samples of known salinity it is possible to effectively
matrix match the standards with NaCI and perform the
analysis directly using matrix matched standards, thereby
avoiding the time consuming method of standard addi-
tions. If the matrix matched standards are going to be
used, it is necessary to document that the use of NaCI is
indeed compensating for the suppression. This docu-
mentation should include a response plot of increasing
matrix vs. relative response similar to Figure 4.
4.5.2 Selenium: The background levesl associated with
the direct determination of Se in NASS-3 exceeds the
Zeeman correction capability. Therefore, HNO3 is used
as a matrix removing modifier in addition to the Pd/
Mg(NO3)? for the determination of Se in saline waters.
Figure 5 is a plot of relative response vs. the amount of
seawater on the platform. A similar suppression is ob-
12-4
-------
served in a solution containing 10,000 ppm NaCI. There-
fore, in well-characterized samples of known salinity it is
possible to effectively matrix match the standards with
NaCI and perform the analysis directly using matrix
matched standards, thereby avoiding the time consum-
ing method of standard additions. If the matrix matched
standards are going to be used, it is necessary to docu-
ment that the use of NaCI is indeed compensating for the
suppression. This documentation should include a re-
sponse plot of increasing matrix vs. relative response
similar to Figure 5.
4.5.3 y4rsen/c:The elevated char temperatures possible
with the determination of As minimize the interferences
produced by the marine water background levels. Figure
3 is a plot of relative response vs. the amount of seawater
on the platform. Figure 3 indicates a matrix suppression
on As caused by the seawater. Although this suppression
does cause a slight bias as shown in the recovery data in
Table 3, the suppression does not warrant the method of
standard additions (MSA) given the recovery criteria of
75-125%forl_FMs.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible
for maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals speci-
fied in this method.2'5 A reference file of material data
handling sheets should also be made available to all
personnel involved in the chemical analysis. Specifically,
concentrated nitric and hydrochloric acids present vari-
ous hazards and are moderately toxic and extremely
irritating to skin and mucus membranes. Use these
reagents in a fume hood whenever possible and if eye or
skin contact occurs, flush with large volumes of water.
Always wear safety glasses or a shield for eye protection,
protective clothing, and observe proper mixing when
working with these reagents.
5.2 The acidification of samples containing reactive
materials may result in the release of toxic gases, such as
cyanides or sulfides. Acidification of samples should be
done in a fume hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 The graphite tube during atomization emits in-
tense U V radiation. Suitable precautions should be taken
to protect personnel from such a hazard.
5.5 The use of the argon/hydrogen gas mixture during
the dry and char steps may evolve a considerable amount
of HCI gas. Therefore, adequate ventilation is required.
6.0 Equipment and Supplies
6.1 Graphite Furnace Atomic Absorption
Spectrometer
6.1.1 The GFAA spectrometer must be capable of pro-
grammed heating of the graphite tube and the associated
delayed atomization device. The instrument must be
equipped with Zeeman background correction and the
furnace device must be capable of utilizing an alternate
gas supply during specified cycles of the analysis. The
capability to record relatively fast (< 1 s) transient signals
and evaluate data on a peak area basis is preferred. In
addition, a recirculating refrigeration unit is recommended
for improved reproducibility of furnace temperatures.
6.1.2 Single element hollow cathode lamps or single
element electrodeless discharge lamps along with the
associated power supplies.
6.1.3 Argon gas supply (high-purity grade, 99.99%) for
use during the atomization of selenium, for sheathing the
furnace tube when in operation, and during furnace
cleanout.
- 6.1.4 Alternate gas mixture (hydrogen 5% - argon 95%)
for use as a continuous gas flow environment during the
dry and char furnace cycles.
6.1.5 Autosampler capable of adding matrix modifier
solutions to the furnace, a single addition of analyte, and
completing methods of standard additions when re-
quired.
6.2 Analytical balance, with capability to measure to
0.1 mg, for preparing standards, and for determining
dissolved solids in digests or extracts.
6.3 A temperature adjustable hot plate capable of
maintaining a temperature of 95°C.
6.4 An air displacement pipetter capable of delivering
volumes ranging from 100 to 2500 \iL with an assortment
of high quality disposable pipet tips.
6.5 Labware — All reusable labware (glass, quartz,
polyethylene, PTFE, FEP, etc.) should be sufficiently
clean for the task objectives. Several procedures found
to provide clean labware include washing with a deter-
gent solution, rinsing with tap water, soaking for 4 h or
more in 20% (v/v) nitric acid or a mixture of HCI and
HNO3, rinsing with reagent water and storing clean.
Chromic acid cleaning solutions must be avoided be-
cause chromium is an analyte.
Note: Glassware having ground glass stoppers, etc.
should be avoided because the ground glass surface is
difficult to clean properly and can contain active sites
which adsorb metals.
6.5.1 Glassware — Volumetric flasks, graduated cylin-
ders, funnels and centrifuge tubes (glass and/or metal-
free plastic).
5.6 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
200.12-5
6.5.2 Assorted calibrated pipettes.
6.5.3 Griffin beakers, 250-mL with 75-mm watch glasses
Revision 1.0 November 1992
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and (optional) 75-mm ribbed watch glasses.
6.5.4 Narrow-mouth storage bottles, FEP (fluorinated
ethylene propylene) with screw closure, 125-mL to 1-L
capacities.
6.5.5 One-piece stem FEP wash bottle with screw clo-
sure, 125-mL capacity.
7.0 Reagents and Standards
7.1 Reagents may contain elemental impurities which
might affect analytical data. Only high-purity reagents
that conform to the American Chemical Society specifi-
cations8 should be used whenever possible. If the purity
of a reagent is in question, analyze for contamination. All
acids used for this method must be of ultra high-purity
grade or equivalent. Suitable acids are available from a
number of manufacturers. Redistilled acids prepared by
sub-boiling distillation are acceptable.
7.2 Nitric acid, concentrated (sp.gr. 1.41) - HNO3.
7.2.1 Nitric acid (1+1) — Add 500 ml concentrated
HNO3 to 400 mL reagent water and dilute to 1 L
7.2.2 Nitric acid (1 +5)—Add 50 mL concentrated HNO3
to 250 mL reagent water.
7.2.3 Nitric acid (1 +9)—Add 10 mL concentrated HNO3
to 90 mL reagent water.
7.3 Reagent water. All references to water in this
method refer to ASTM Type I grade water.7
7.4 Ammonium hydroxide, concentrated (sp. gr. 0.902).
7.5 Matrix Modifier, dissolve 300 mg palladium (Pd)
powder in concentrated HNO3 (1 mL of HNO3, adding 10
jiL of concentrated HCI if necessary). Dissolve 200 mg of
Mg(NO3)2-6H2O in ASTM Type I water. Pour the two
solutions together and dilute to 100 mL with ASTM Type
I water.
Note: It is recommended that the matrix modifier be
analyzed separately in order to assess the contribution of
the modifier to the absorbance of calibration and reagent
blank solutions.
7.6 Standard stock solutions may be purchased or
prepared from ultra-high purity grade chemicals (99.99 to
99.999% pure). All compounds must be dried for 1 h at
105°C, unless otherwise specified. It is recommended
that stock solutions be stored in FEP bottles. Replace
stock standards when succeeding dilutions for prepara-
tion of calibration standards cannot be verified.
Caution: Many of these chemicals are extremely toxic if
inhaled or swallowed (Section 5.1). Wash hands thor-
oughly after handling.
Typical stock solution preparation procedures follow for
1-L quantities, but for the purpose of pollution prevention,
the analyst is encouraged to prepare smaller quantities
when possible. Concentrations are calculated based
upon the weight of the pure element or upon the weight
of the compound multiplied by the fraction of the analyte
in the compound.
From pure element,
Concentration = weight (mg)
volume (L)
From pure compound,
Concentration = weight (mg) x gravimetric factor
volume (L)
where:
gravimetric factor = the weight fraction of the analyte
in the compound.
7.6.1 Arsenic solution, stock, 1 mL = 1000 jig As: Dis-
solve 1.320 g of As2O3 (As fraction = 0.7574), weighed
accurately to at least four significant figures, in 100 mL of
reagent water containing 10.0 mL concentrated NH4OH.
Warm in solution gently to effect dissolution. Acidify the
solution with 20.0 mL concentrated HNO3 and dilute to
volume in a 1-L volumetric flask with reagent water.
7.6.2 Cadmium solution, stock, 1 ml. = 1000 jig Cd:
Dissolve 1.000 g Cd metal, acid cleaned with (1+9)
HNO3, weighed accurately to at least four significant
figures, in 50 mL (1+1) HNO3 with heating to effect
dissolution. Let solution cool and dilute with reagent
water in a 1-L volumetric flask.
7.6.3 Chromium solution, stock, 1 mL = 1000 jig Cr:
Dissolve 1.923 g CrO (Cr fraction = 0.5200), weighed
accurately to at least four significant figures, in 120 mL
(1 +5) HNO3. When solution is complete, dilute to volume
in a 1 -L volumetric flask with reagent water.
7.6.4 Copper solution, stock, 1 mL = 1000 |iig Cu: Dis-
solve 1.000 g Cu metal, acid cleaned with (1+9) HNO3,
weighed accurately to at least four significant figures, in
50.0 mL (1 +1) HNO3 with heating to effect dissolution. Let
solution cool and dilute in a 1-L volumetric flask with
reagent water.
7.6.5 Lead solution, stock, 1 mL= 1000 ng Pb: Dissolve
1.599 g Pb(NO3) (Pb fraction = 0.6256), weighed
accurately to at feast four significant figures, in a mini-
mum amount of (1+1) HNO3. Add 20.0 mL (1+1) HNO3
and dilute to volume in a 1 -L volumetric flask with reagent
water.
7.6.6 Nickel solution, stock, 1 mL = 1000 jig Ni: Dissolve
1.000 g of nickel metal, weighed accurately to at least
four significant figures, in 20.0 mL hot concentrated
HNO3, cool, and dilute to volume in a 1 - L volumetric flask
with reagent water.
7.6.7 Selenium solution, stock, 1 ml_ = 1000 ng Se:
Dissolve 1.405 g SeO (Se fraction = 0.7116), weighed
accurately to at least four significant figures, in 200 mL
reagent water and dilute to volume in a 1-L volumetric
flask with reagent water.
7.7 Preparation of Calibration Standards — Fresh
calibration standards (CAL Solution) should be prepared
200.12 - 6
Revision 1.0 November 1992
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weekly, or as needed. Dilute each of the stock standard
solutions to levels appropriate to the operating range of
the instrument using the appropriate acid diluent. The
element concentrations in each CAL solution should be
sufficiently high to produce good measurement precision
and to accurately define the slope of the response curve.
The instrument calibration should be initially verified
using a I PC sample (Section 7.9).
7.8 Blanks—Fourtypes of blanks are required forthis
method. A calibration blank is used to establish the
analytical calibration curve, the laboratory reagent blank
(LRB) is used to assess possible contamination from the
sample preparation procedure and to assess spectral
background, the laboratory fortified blank is used to
assess routine laboratory performance, and a rinse blank
is used to flush the instrument autosampler uptake sys-
tem. All diluent acids should be made from concentrated
acids (Section 7.2) and ASTM Type I water.
7.8.1 The calibration blank consists of the appropriate
acid diluent in ASTM Type I water. The calibration blank
should be stored in a FEP bottle.
7.8.2 The laboratory reagent blanks must contain all the
reagents in the same volumes as used in processing the
samples. The preparation blank must be carried through
the entire sample digestion and preparation scheme.
7.8.3 The laboratory fortified blank (LFB) is prepared by
fortifying an aliquot of the laboratory reagent blank with
all analytes to provide a final concentration which will
produce an absorbance of approximately 0.1 for each
analyte. The LFB must be carried through the complete
procedure as used for the samples.
7.8.4 The rinse blank is a 0.1% HCI and 0.1% HNO3
solution used to flush the autosampler tip and is stored in
the appropriate plastic containers.
7.9 Instrument Performance Check (IPC) Solution—
The IPC solution is used to periodically verify instrument
performance during analysis. It should be prepared in the
same acid mixture as the calibration standards by com-
bining method analytes at appropriate concentrations to
approximate the midpoint of the calibration curve. The
IPC solution should be prepared from the same standard
stock solutions used to prepare the calibration standards
and stored in a FEP bottle. Agency programs may specify
or request that additional instrument performance check
solutions be prepared at specified concentrations in
order to meet particular program needs.
7.10 Quality Control Sample (QCS) — For initial and
periodic verification of calibration standards and instru-
ment performance, analysis of a QCS is required. The
QCS must be obtained from an outside source different
from the standard stock solutions and prepared in the
same acid mixture as the calibration standards. The
concentration of the analytes in the QCS solution should
be such that the resulting solution will provide an absor-
bance reading of approximately 0.1. The QCS solution
should be stored in a FEP bottle and analyzed as needed
to meet data-quality needs. A fresh solution should be
prepared quarterly or as needed.
200.
7.11 Matrix Performance Check (MPC) — The MPC
solution is used to periodically evaluate the laboratory/
instrument performance in saline samples. It should be
prepared in the same acid mixture as the calibration
standards by combining method analytes at appropriate
concentrations in a seawater matrix (NASS-3, or its
equivalent) to produce an absorbance of 0.1. The MPC
solution should be prepared from the same standard
stock solutions used to prepare the calibration standards
and stored in a FEP bottle. The MPC sample should be
analyzed after every 10 samples to assure saline matrix
is not producing false positives.
8.0 Sample Collection, Preservation and
Storage
8.1 Prior to collection of an aqueous sample, consider-
ation should be given to the type of data required. Acid
preservation should be performed at the time of sample
collection or as soon thereafter as practically possible.
The pH of all aqueous samples must be tested immedi-
ately prior to aliquoting for analysis to ensure the sample
has been properly preserved. If properly acid-preserved,
the sample can be held up to 6 months before analysis.
8.2 For determination of total recoverable elements in
aqueous samples, acidify with (1 +1) nitric acid at the time
of collection to pH <2. Normally, 3 mL of (1 +1) nitric acid
(ultra high purity) per liter of sample is sufficient for most
ambient water samples. The sample should not be fil-
tered prior to analysis.
Note: Samples that cannot be acid-preserved atthe time
of collection because of sampling limitations or transport
restrictions, or are > pH 2 because of high alkalinity
should be acidified with nitric acid to pH <2 upon receipt
in the laboratory. Following acidification, the sample
should be held for 16 h and the pH verified to be<2 before
withdrawing an aliquot for sample processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container and acid as used in sample collec-
tion.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability, and the
periodic analysis of laboratory reagent blanks, fortified
blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to
maintain performance records that define the quality of
the data thus generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of
linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) prior to samples being analyzed
by this method.
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9.2.2 Linear dynamic range (LDR) —The upper limit of
the LDR must be established for the wavelength utilized
for each analyte by determining the signal responses
from a minimum of six different concentration standards
across the range, two of which are close to the upper limit
of the LDR. Determined LDRs must be documented and
kept on file. The linear calibration range which may be
used for the analysis of samples should be judged by the
analyst from the resulting data. The upper LDR limit
should be an observed signal no more than 10% below
the level extrapolated from the four lower standards.
New LDRs should be determined whenever there is a
significant change in instrument response, a change in
instrument analytical hardware or operating conditions.
Note: Multiple cleanout furnace cycles may be neces-
sary in order to fully define or utilize the LDR for certain
elements such as chromium. For this reason, the upper
limit of the linear calibration range may not correspond to
the upper operational LDR limit.
Measured sample analyte concentrations that exceed
the upper limit of the linear calibration range must either
be diluted and reanalyzed (with concern for memory
effects Section 4.4) or analyzed by another approved
method.
9.2.3 Quality control sample (QCS) —When beginning
the use of this method, on a quarterly basis or as required
to meet data-quality needs, verify the calibration stan-
dards and acceptable instrument performance with-the,
preparation and analyses of a QCS (Section 7.10). Ifthe
determined concentrations are not within ± 10% of the
stated values, performance of the determinative step of
the method is unacceptable. The source of the problem
must be identified and corrected before either proceed-
ing on with the initial determination of method detection
limits or continuing with ongoing analyses.
9.2.4 Method detection limit (MDL) — MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.8 To determine MDL
values, take seven replicate aliquots of the fortified
reagent water and process through the entire'analytical
method. Perform all calcujations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
MDL = (t) x (S)
where, t - Student's t value for a 99% confidence level
and a standard deviation estimate with n-1 degrees
of freedom [t« 3.14 for seven replicates].
S « standard deviation of the replicate analyses.
Note: Ifthe percent relative standard deviation (% RSD)
from the analyses of the seven aliquots is < 15%, the
concentration used to determine the analyte MDL may
have been inappropriately high for the determination. If
so, this could result in calculation of an unrealistically low
MDL. If additional confirmation of the MDL is desired,
reanalyze the seven replicate aliquots on two more
nonconsecutive days and again calculate the MDL val-
ues for each day. An average of the three MDL values for
each analyte may provide a more appropriate MDL
estimate. Concurrently, determination of MDL in reagent
water represents a best case situation and does not
reflect possible matrix effects of real world samples.
However, successful analyses of LFMs (Section 9.4) and
the analyte addition test described in Section 9.5.1 can
give confidence to the MDL value determined in reagent
water. Typical single laboratory MDL values using this
method are given in Table 2.
MDLs should be determined every six months, when a
new operator begins work or whenever there is a signifi-
cant change in the background or instrument response.
The MDLs reported in Table 2 were determined in forti-
fied NASS-3 samples. It is recommended that a certified
saline matrix such as NASS-3 be used to determine
MDLs.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory reagent blank (LRB)—The laboratory
must analyze at least one LRB (Section 7.8.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and
the samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
9.3.2 Laboratory fortified blank (LFB) —The laboratory
must analyze at least one LFB (Section 7.8.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that analyte
is judged out of control, and the source of the problem
should be identified and resolved before continuing analy-
ses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 85-115%. When sufficient internal perfor-
mance data become available (usually a minimum of 20-
30 analyses), optional control limits can be developed
from the percent mean recovery (x) and the standard
deviation (S) of the mean recovery. These data can be
used to establish the upper and lower control limits as
follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most recent 20-30 data
points. Also, the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
200.12-8
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9.3.4 Instrument performance check (IPC) solution —
For all determinations the laboratory must analyze the
IPC solution (Section 7.9) and a calibration blank imme-
diately following daily calibration, after every tenth sample
(or more frequently, if required) and after the last sample
in the batch is analyzed. Analysis of the IPC solution and
calibration blank immediately following calibration must
verify that the instrument is within + 5% of calibration.
Subsequent analyses of the IPC solution must verify the
calibration within ± 10%. If the calibration cannot be
verified within the specified limits, reanalyze the IPC
solution. If the second analysis of the IPC solution con-
firms calibration to be outside the limits, sample analysis
must be discontinued, the cause determined and/or, in
the case of drift, the instrument recalibrated. All samples
following the last acceptable IPC solution must be reana-
lyzed. Data for the calibration blank and IPC solution
must be kept on file with associated sample data.
9.3.5 Matrix performance check (MFC) solution — For
all determinations, the laboratory must analyze the MPC
solution (Section 7.11) immediately following daily cali-
bration, after every tenth sample (or more frequently, if
required) and after the last sample in the batch is ana-
lyzed. Analysis of the MPC must verify that the instru-
ment is within ± 15% of calibration and confirm that the
matrix is not causing matrix/background interferences. If
the MPC is not within ± 15%, reanalyze the MPC solution.
If the second analysis of the MPC solution is outside the
limits, sample analysis must be discontinued, the cause
determined and/or, in the case of drift, the instrument
recalibrated. All samples following the last acceptable
MPC solution must be reanalyzed. The analysis data for
the calibration blank and MPC solution must be kept on
file with the sample analyses data.
9.4 Assessing Analyte Recovery and Data
Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and the
data quality. Taking separate aliquots from the sample
for replicate and fortified analyses can in some cases
assess these effects. Unless otherwise specified by the
data user, laboratory or program, the following laboratory
fortified matrix (LFM) procedure (Section 9.4.2) is re-
quired. Also, the analyte addition test (Section 9.5.1) can
aid in identifying matrix interferences. However, all
samples must have a background absorbance < 1.0
before the test results obtained can be considered reli-
able.
9.4.2 The laboratory must add a known amount of each
analyte to a minimum of 10% of the routine samples. In
each case the LFM aliquot must be a duplicate of the
aliquot used for sample analysis and for total recoverable
determinations added prior to sample preparation. For
water samples, the added analyte concentration must be
the same as that used in the laboratory fortified blank
(Section 9.3.2).
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is less than
25% of the unfortified sample concentration. Percent
recovery may be calculated in units appropriate to the
matrix, using the following equation:
R = C -C x100
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte
added to sample.
9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range (but is still within the
range of calibration and the background absorbance is <
1.0 abs.) and the laboratory performance for that analyte
is shown to be in control (Section 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related. A flowchart
of the remainder of this section can be found in Figure 6.
This flowchart may clarify the verbal discussion given
below.
If the background absorbance is> 1 abs., the sample and
the LFM should be diluted 1:3 and reanalyzed until the
background absorbance is < 1, at which point a percent
recovery of the LFM should be calculated. If the fortified
analyte in the diluted LFM is found to be < 25% of the
sample concentration or the diluted LFM produces an
atomic signal of < 10 times the MDL, the diluted sample
should be analyzed by methods of standard addition. If
the calculated recovery of the diluted sample is within the
designated range, the sample concentration should be
calculated from the diluted sample. If the calculated
recovery of the diluted sample is outside the designated
range, follow the directions given below. If the back-
ground is reduced and/or the matrix effect is reduced by
dilution, all samples of a similar matrix should be diluted
and analyzed in a similar fashion. The result should be
flagged indicating the methods sensitivity has been re-
duced by the dilution. If dilution is unacceptable because
of data quality objectives the sample should be flagged
indicating the analysis is not possible via this analytical
procedure.
If the analyte recovery on the LFM is < 75% and the
background absorbance is < 1, complete the analyte
addition test (Section 9.5.1) on the original sample (or its
dilution). The results of the test should be evaluated as
follows:
1. If recovery of the analyte addition test (< 85%)
confirms a low recovery for the LFM, a suppressive
matrix interference is indicated and the unfortified
sample aliquot must be analyzed by method of
standard additions (Section 11.3).
2. If the recovery of the analyte addition test is be-
tween 85% to 115%, a low recovery of the analyte
200.12-9
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in the LFM (< 75%) may be related to the heteroge-
neity of the sample, sample preparation or a poor
transfer, etc. Reportthe sample concentration based
on the unfortified sample aliquot.
3. If the recovery of the analyte addition test is less
than recovery calculated for the LFM, matrix sup-
pression is confirmed. The unfortified sample should
be analyzed by MSA (Section 11.3). Significantly
lower recoveries (relative to the LFM) associated
with the anaiyte addition test are unlikely unless the
sample is heterogeneous.
4. If the recovery of the analyte addition test is>115%,
the dramatic change in analyte response should be
verified by fortifying the LFM. The recovery in the
sample and the recovery in the LFM should be
compared. If the recoveries verify the dramatic
response difference, the sample results should be
flagged indicating the sample matrix is not homoge-
neous.
If the analyte recovery in the LFM is > 125% and the
background absorbance is < 1, complete the analyte
addition test (Section 9.5.1) on the unfortified sample (or
its dilution) aliquot.
1. If the percent recovery of the analyte addition test is
> 115% and the LFB does not indicate laboratory
contamination, an enhancing matrix interference
(albeit rare) is indicated, and the unfortified sample
aliquot must be analyzed by method of standard
additions (Section 11.3).
2. If the percent recovery of the analyte addition test is
between 85% to 115%, either random sample con-
tamination of the LFM, an incorrect analyte concen-
tration was added to the LFM prior to sample
preparation, or sample heterogeneity should be
suspected. Report analyte data determined from
the analysis of the unfortified sample aliquot.
3. If the percent recovery of the analyte addition test is
< 85%, a heterogeneous sample with matrix inter-
ference is suspected. This dramatic change in re-
sponse should be verified by performing the analyte
addition test to the LFM. The recovery in the sample
and the recovery in the LFM should be compared.
If the recoveries verify the dramatic response differ-
ence the sample results should be flagged indicat-
ing the sample matrix is not homogeneous.
9.4.5 If the analysis of a LFM sample(s) and the test
routines above indicate an operative interference and the
LFMs are typical of the other samples in the batch, those
samples that are similar must be analyzed in the same
manner as the LFMs. Also, the data user must be
informed when a matrix interference is so severe that it
prevents successful determination of the analyte or when
the heterogeneous nature of the sample precludes the
use of duplicate analyses.
9.4.6 Where reference materials are available, they
should be analyzed to provide additional performance
data. Analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method accept-
ably. It is recommended that NASS-3 or its equivalent be
fortified and used as an MPC.
9.5 Matrix interference effects and the need for MSA
can be assessed by the following test. Directions for
using MSA are given in Section 11.3.
9.5.1 Analyte addition test: An analyte standard added
to a portion of a prepared sample or its dilution should be
recovered to within 85-115% of the known value. The
analyte addition should occur directly to sample in the
furnace and should produce a minimum absorbance of
0.1. The concentration of the analyte addition plus that in
the sample should not exceed the linear calibration range
of the analyte. If the analyte is not recovered within the
specified limits, a matrix effect should be suspected and
the sample must be analyzed by MSA.
10.0 Calibration and Standardization
10.1 Specific wavelengths and instrument operating
conditions are listed in Table 1. However, because of
differences among makes and models of spectropho-
tometers and electrothermal furnace devices, the actual
instrument conditions selected may vary from those
listed.
10.2 Prior to the use of this method, the instrument
operating conditions must be optimised. The analyst
should follow the instructions provided by the manufac-
turer while using the conditions listed in Table 1 as a
guide. The appropriate charring condition for each of the
analytes is a critical part of the metal analysis in saline
waters; therefore, the char temperature profiles should
be determined in a saline water matrix. The appropriate
charring temperature should be chosen so as to minimize
background absorbance while providing some furnace
temperature variation without the loss of analyte. For
analytical operation, the charring temperature is usually
set at least 100°C below the point at which analyte begins
to be lost during the char. Because the background
absorbance can be affected by the atomization tempera-
ture, care should be taken in the choice of an appropriate
atomization temperature. The optimum conditions se-
lected should provide the lowest reliable MDLs and be
similar to those listed in Table 2. Once the optimum
operating conditions are determined, they should be
recorded and available for daily reference. The effective-
ness of these operating conditions are continually evalu-
ated by analyzing the MPC.
10.3 Prior to an initial calibration the linear dynamic
range of the analyte must be determined (Sect 9.2.2)
using the optimized instrument operating conditions. For
all determinations allow an instrument and hollow cath-
ode lamp warm-up period of not less than 15 min. If an
EDL is to be used, allow 30 min for warm-up.
200.12-10
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10.4 Before using the procedure (Section 11.0) to ana-
lyze samples, there must be data available documenting
initial demonstration of performance. The required data
and procedure are described in Section 9.2. This data
must be generated using the same instrument operating
conditions and calibration routine to be used for sample
analysis. These documented data must be kept on file
and be available for review by the data user.
11.0 Procedure
11.1 Aqueous Sample Preparation-Total
Recoverable Analytes
11.1.1 Add 2 ml (1 +1) nitric acid to the beaker contain-
ing 100 ml_ of sample. Place the beaker on a hot plate for
solution evaporation. The hot plate should be located in
a fume hood and previously adjusted to provide evapo-
ration at a temperature of approximately but no higher
than 85°C. (See the following note.) The beaker should
be covered with an elevated watch glass or other neces-
sary steps should be taken to prevent sample contamina-
tion from the fume hood environment.
Note: For proper heating adjust the temperature control
of the hot plate such that an uncovered Griffin beaker
containing 50 ml_ of water placed in the center of the hot
plate can be maintained at a temperature approximately
but no higher than 85°C. (Once the beaker is covered
with a watch glass the temperature of the water will rise
to approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20 ml_ by gentle heating at 85°C. DO NOT BOIL.
This step takes about 2 h for a 100-mL aliquot with the
rate of evaporation rapidly increasing as the sample
volume approaches 20 ml_. (A spare beaker containing
20 mL of water can be used as a gauge.)
11.1.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the sample
for 30 min.
7 7.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
11.1.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight the
sample contains suspended solids, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination
from filtration.) The sample is now ready for analysis.
Because the effects of various matrices on the stability of
diluted samples cannot be characterized, all analyses
should be performed as soon as possible after the
completed preparation.
11.2 Sample Analysis
11.2.1 Prior to daily calibration of the instrument, in-
spect the graphite tube and contact rings for salt buildup,
etc. Generally, it will be necessary to clean the contact
rings and replace the graphite tube daily. The contact
rings are a cooler environment in which salts can deposit
after atomization. A cotton swab dipped in a 50/50
mixture of isopropyl alcohol (I PA) and H2O (such that it is
damp but not dripping) can be used to remove the
majority of the salt buildup. A second cotton swab is
dipped in IPA and the contact rings are wiped down to
assure they are clean. The rings are then allowed to
thoroughly dry and then a new tube is placed in the
furnace and conditioned according to instrument
manufacturer's specifications.
11.2.2 Configure the instrument system to the selected
, optimized operating conditions as determined in Sec-
tions 10.1 and 10.2.
11.2.3 Before beginning daily calibration the instrument
should be reconfigured to the optimized conditions. Ini-
tiate the data system and allow a period of not less than
15 min for instrument and hollow cathode lamp warm up.
If an EDL is to be used, allow 30 min for warm up.
/1.2.4 After the warm up period but before calibration,
instrument stability must be demonstrated by analyzing
a standard solution with a concentration 20 times the IDL
a minimum of five times. The resulting relative standard
deviation of absorbance signals must be < 5%. If the
relative standard deviation is > 5%, determine and cor-
rect the cause before calibrating the instrument.
11.2.5 For initial and daily operation, calibrate the in-
strument according to the instrument manufacturer's
recommended procedures using the calibration blank
(Section 7.8.1) and calibration standards (Section 7.7)
prepared at three or more concentrations within the
usable linear dynamic range of the analyte (Sections 4 4
and 9.2.2).
11.2.6 An autosampler must be used to introduce all
solutions into the graphite furnace. Once the sample and
the matrix modifier are injected, the furnace controller
completes a set of furnace cycles and a cleanout period
as programmed. Analyte signals must be reported on an
integrated absorbance basis. Background absorbances,
background heights and the corresponding peak profiles
should be displayed to the CRT for review by the analyst
and be available as hard copy for documentation to be
kept on file. Flush the autosampler solution uptake sys-
tem with the rinse blank (Section 7.8.4) between each
solution injected.
11.2.7 After completion of the initial requirements of this
method (Section 9.2), samples should be analyzed in the
same operational manner used in the calibration routine.
11.2.8 During sample analyses, the laboratory must
comply with the required quality control described in
Sections 9.3 and 9.4.
71.2.9 For every new or unusual matrix, when practical,
it is. highly recommended that an inductively coupled
plasma atomic emission spectrometer be used to screen
for high element concentration. Information gained from
this may be used to prevent potential damage to the
instrument and to better estimate which elements may
require analysis by graphite furnace.
200.12-11
Revision 1.0 November 1992
-------
11.2.10 Determined sample analyte concentrations that
are £ 90% of the upper limit of calibration must either be
diluted with acidified reagent water and reanalyzed with
concern for memory effects (Section 4.4), or determined
by another approved but less sensitive procedure.
Samples with background absorbances > 1 must be
diluted with appropriate acidified reagent water such that
the background absorbance is < 1 (Section 9.4.4). If the
method of standard additions is required, follow the
instructions described in Section 11.3.
77.2.77 When it is necessary to assess an operative
matrix interference (e.g., signal reduction due to high,
dissolved solids), the test described in Section 9.5 is
recommended.
71.2.12 Report data as directed in Section 12.
11.3 Standard Additions— If the method of standard
addition is required, the following procedure is recom-
mended:
71.3.1 The standard addition technique9 involves pre-
paring new standards in the sample matrix by adding
known amounts of standard to one or more aliquots of the
processed sample solution. This technique compen-
sates for a sample constituent that enhances or de-
presses the analyte signal, thus producing a different
slope from that of the calibration standards. It will not
correct for additive interference, which causes a baseline
shift. The simplest version of this technique is the single-
addition method. The procedure is as follows: Two iden-
tical aliquots of the sample solution, each of volume Vx,
are taken. To the first (labeled A) is added a small volume
Vs of a standard analyte solution of concentration Cs. To
the second (labeled B) is added the same volume Vs of
the solvent. The analytical signals of A and B are mea-
sured and corrected for nonanalyte signals. The,un-
known sample concentration Cx is calculated:
Cx= SBVSCS
-------
lations and Science Policy, 115516th Street N.W., Wash-
ington D.C. 20036, (202)872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section 14.2.
16.0 References
1. Pruszkowska, E., G. Carnrick, and W. Slavin. Anal.
Chem. 55, 182-186,1983.
2. Carcinogens - Working With Carcinogens,
Department of Health, Education, and Welfare, Public
Health Service, Centers for Disease Control, National
Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
4. Safety in Academic Chemistry Laboratories,
American Chemical Society Publication, Committee
op Chemical Safety, 3rd Edition, 1979.
5. Proposed OSHA Safety and Health Standards,
Laboratories, Occupational Safety and Health
Administration, Federal Register, July 24,1986.
6. Rohrbough, W.G. etal. Reagent Chemicals, American
Chemical Society Specifications, 7th edition.
American Chemical Society, Washington, DC, 1986.
7. American Society for Testing and Materials. Standard
Specification for Reagent Water, D1193-77. Annual
Book of ASTM Standards, Vol. 11.01. Philadelphia,
PA, 1991.
8. Code of Federal Regulations 40, Ch. 1, Pt. 136,
Appendix B.
9. Winefordner, J.D., Trace Analysis: Spectroscopic
Methods for Elements, Chemical Analysis, Vol. 46,,
pp. 41-42, 1976.
200.12-13
Revision 1.0 November 1992
-------
17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Furnace Conditions for Determination of Metals in Seawater1
Element
As
Cd
•
Cr
Cu
Ni
Pb
Se
Wavelength (nm)
Slit Width (nm)
193.7
0.7
22&S
0.7
357.9
0.7
3&S
0.7
232A
0.2
2S12
0.7
196.Q
2.0
Method of
Analysis
Direct
Matrix Match
Standard
or
Std. Addition
Direct
Direct
Direct
Direct
Matrix Match
Standard
or
Std. Addition
Modifier2-3
Pd/Mg
Pd/Mg
+
600 jig
NH4N03
Pd/Mg
Pd/Mg
Pd/Mg
Pd/Mg
Pd/Mg
9% HNO3 on
Platform
Furnaces5
Cycle
Dry
Char
Atomization
Dry
Charl
Char 2
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Temp
°C
130
14004
2200
130
350
850
1500
130
1500
2600
130
1300
2600
130
1400*
2600
130
1200
2200
130
1000
2100
Temp
Ramp
1
10
0
1
45
1
0
1
5
0
1
10
0
1
10
0
1
10
0
1
5
0
Hold Time (sec)
60
60
5
60
30
30
5
60
30
5
60
30
5
60
30
7
60
45
5
60
60
5
' 10-uL sample size.
3 S uL of (30 mg Pd Powder and 20 mg Mg(NCg2'6H2O to 10 mL).
a A gas mixture of 5% H? in 95% Ar is used during the dry and char.
4 Sodium emission is visibly exiting from the sample inlet port.
* The furnace program has a cool down step of 20° between char and .atomization
and a dean out step of 2600°C after atomization.
Table 2. MDLs and Background Absorbances Associated with a Fortified NASS-31
Typical
Integrated
MDL5 Background
Element jig/L Absorbances6
Cd 0.1
Cr
Cu 2.8
Ni 1.8
Pb 2.4
Se4 9.5
As4 2.6
1.2
0.2
0.2
0.1
0.4
1.4
0.3
1 Matrix Modifier « 0.015 mg Pd + 0.01 mg Mg{NO3)2.
1 A 5% Hj in Ar gas mix is used during the dry and char steps at 300 mL/min for all elements.
3 10-nL sample size. .
4 An electrodeless discharge lamp was used for this element.
* MDL calculated based on fortifying NASS-3 with metal analytes.
* Backgroundabsorbancesareaffectedbytheatomizationtemperatureforanalysis, therefore, lowering atomization
temperatures may be advantageous if large backgrounds are observed.
- Not Determined.
200.12-14
Revision 1.0 November 1992
-------
Table 3. Precision and Recovery Data for Fortified NASS-3
Element
As
Cd'
Cr
Cu
Pb
Ni
Se1
Certified
Value
M9/L
1.65 ±0.1 9
0.029 ± 0.004
0.175 ±0.010
0.1 09 ±0.011
0.039 ±0.006
0.257 ±0.027
0.024 ± 0.004
Observed
Value
3
c
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
oCd
Ni
400 600 800 1000 1200 1400
Char Temperature °C
1600
1800
Figure 1. Integrated Background Absorbance vs. Char Temperature.
200.12-15
Revision 1.0 November 1992
-------
3.221
»!
Current Atomic
1 Current Backgrd
Time (sec)
Figure 2. Pb Atomization Profile Utilizing a 800°C Char.
5.00
110
100 -
90
80
70
-Si 60
50
40
30
+ Pb
•All samples fortified with 5 fil of Standard
JL
2 4 6
Microliters of Fortified NASS-3
Figure 3. Normalized Integrated Absorbance vs. Microliters of Fortified NASS-3.
10
200.12-16
Revision 1.0 November 1992
-------
110
105
100
95
90
1 85
1
2 80
75
70
+ NASS-3
5 nL of a Cd Standard Added.
• 5
Microliters of Matrix
Figure 4. Cd Response in NASS-3 and 10,000 ppm NaCI.
o
I
CD
-------
(1) Poor Transfer
C2) Sample Heterogeneity
(3) Digestion/Precipitation
(4) Matrix Suppression/Enhancements
(5) Contamination
Report Results on Diluted Sample
IFA = In Furnace Analyte Addition
Report Results on
Unfortified Sample
85% LFM > 125%
Dilute 1:3
Sample & LFM
Compare N, |FA<85
Recoveries
IFAs to LFM
Background
Absorbance
<1.0abs
Start
S) ^>
Report Results on Unfortified Sample
Rgure 6. Matrix Interference Flowchart.
200.12-18
Revision 1.0 November 1992
-------
Method 200.13
Determination ofTrace Elements in Marine Waters by Off-Line Chelation
Preconcentration with Graphite Furnace Atomic Absorption
John T. Creed and Theodore D. Martin
Inorganic Chemistry Branch
Chemistry Research Division
Revision 1.0
November 1992
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
200.13-1
Revision 1.0 November 1992
-------
Method 200.13
Determination of Trace Elements in Marine Waters by Off-Line Chelation
Preconcentration with Graphite Furnace Atomic Absorption
1.0 Scope and Application
1.1 This method describes procedures for
preconcentration and determination of total recoverable
trace elements in marine waters, including estuarine
water, seawater and brines.
1.2 Acid soiubilization is required prior to determina-
tion of total recoverable elements to facilitate breakdown
of complexes or colloids which might influence trace
element recoveries. This method should only be used for
preconcentration and determination of trace elements in
aqueous samples.
1.3 This method is applicable to the following ele-
ments:
Element
Cadmium
Cobalt
Copper
Lead
Nickel
(Cd)
(Co)
(Cu)
(Pb)
(NO
Chemical Abstracts Service
Registry Numbers (CASRN)
7440-43-9
7440-48-4
7440-50-8
7439-92-1
7440-02-0
1.4 Method detection limits (MDLs) forthese elements
will be dependent on the specific instrumentation em-
ployed and the selected operating conditions. MDLs in
NASS-3 (Reference Material, National Research Coun-
cil of Canada) were determined using the procedure
described in Section 9.2.4 and are listed in Table 1.
1.5 A minimum of 6 months experience in graphite
furnace atomic absorption (GFAA) is recommended.
2.0 Summary of Method
2.1 Nitric acid is dispensed into a beaker containing an
accurately weighed or measured, well-mixed, homoge-
neous aqueous sample. The sample volume is reduced
to approximately 20 mL and then covered and allowed to
reflux. The resulting solution is diluted to volume and is
ready for analysis.
2.2 This method is used to preconcentrate trace ele-
ments using an iminodiacetate functionalized chelating
resin.1-2 Following acid soiubilization, the sample is buff-
ered using an on-line system prior to entering the chelat-
ing column. Group I and II metals, as well as most anions,
are selectively separated from the analytes by elution
with ammonium acetate at pH 5.5. The analytes are
subsequently eluted into a simplified matrix consisting of
0.75 M nitric acid and are determined by GFAA.
3.0 Definitions
3.1 Calibration Blank (CB) — A volume of reagent
water fortified with the same matrix as the calibration
standards, but without the analytes, internal standards,
or surrogate analytes.
3.2 Calibration Standard (CAL) — A solution pre-
pared from the primary dilution standard solution or stock
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate
the instrument response with respect to analyte concen-
tration.
3.3 Field Reagent Blank (FRB) — An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sam-
pling site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.4 Instrument Performance Check Solution
(IPC) — A soiution of one or more method analytes,
surrogates, internal standards, or other test substances
used to evaluate the performance of the instrument
system with respect to a defined set of criteria.
3.5 Laboratory Fortified Blank (LFB) — An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.6 Laboratory Fortified Sample Matrix (LFM) —
An aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background
concentrations.
3.7 Laboratory Reagent Blank (LRB) — An aliquot
of reagent water or other blank matricess that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
200.13-2
Revision 1.0 November 1992
-------
ences are present in the laboratory environment, the
reagents, or the apparatus.
3.8 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instru-
ment response to an analyte is linear.
3.9 Matrix Modifier (MM)—A substance added to the
instrument along with the sample in order to minimize the
interference effects by selective volatilization of either
analyte or matrix components.
3.10 Method Detection Limit (MDL)—The minimum
concentration of an analyte that can be identified, mea-
sured and reported with 99% confidence that the analyte
concentration is greater than zero.
3.11 Quality Control Sample—A solution of method
analytes of known concentrations which is used to fortify
an aliquot of LRB or sample matrix. The QCS is obtained
from a source external to the laboratory and different
from the source of calibration standards. It is used to
check laboratory performance with externally prepared
test materials.
3.12 Standard Addition — The addition of a known
amount of analyte to the sample in order to determine the
relative response of the detector to an analyte within the
sample matrix. The relative response is then used to
assess either an operative matrix effect or the sample
analyte concentration.
3.13 Stock Standard Solution (SSS) — A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
3.14 Total Recoverable Analyte (TRA) — The con-
centration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following treat-
ment by refluxing with hot dilute mineral acid(s) as
specified in the method.
4.0 Interferences
4.1 Several interference sources may cause inaccura-
cies in the determination of trace elements by GFAA.
These interferences can be classified into three major
subdivisions: spectral, matrix, and memory. Some of
these interferences can be minimized via the
preconcentration step, which reduces the Ca, Mg, Na
and Cl concentration in the sample prior to GFAA analy-
sis.
4.2 Spectral interferences are caused by absorbance
of light by a molecule or atom which is not the analyte of
interest or emission from black body radiation.
4.2.1 Spectral interferences caused by an element only
occur if there is a spectral overlap between the wave-
length of the interfering element and the analyte of
interest. Fortunately, this type of interference is relatively
uncommon in STPGFAA (Stabilized Temperature Plat-
form Graphite Furnace Atomic Absorption) because of
the narrow atomic line widths associated with STPGFAA.
In addition, the use of appropriate furnace temperature
programs and high spectral purity lamps as light sources
can minimize the possibility of this type of interference.
However, molecular absorbances can span several hun-
dred nanometers, producing broadband spectral inter-
ferences. This type of interference is far more common in
STPGFAA. The use of matrix modifiers, selective volatil-
ization, and background correctors are all attempts to
eliminate unwanted nonspecific absorbance. Because
the nonspecific component of the total absorbance can
vary considerably from sample type to sample type, to
provide effective background correction and eliminate
the elemental spectral interference of palladium on cop-
per and iron on selenium, the exclusive use of Zeeman
background correction is specified in this method.
4.2.2 Spectral interferences are also caused by emis-
sions from black body radiation produced during the
atomization furnace cycle. This black body emission
reaches the photomultiplier tube, producing erroneous
results. The magnitude of this interference can be mini-
mized by proper furnace tube alignment and moriochro-
mator design. In addition, atomization temperatures which
adequately volatilize the analyte of interest without pro-
ducing unnecessary black body radiation can help re-
duce unwanted background emission produced during
atomization.
4.3 Matrix interferences are caused by sample com-
ponents which inhibit formation of free atomic analyte
atoms during the atomization cycle. In this method the
use of a delayed atomization device which provides
warmer gas phase temperatures is required. These
devices provide an environment which is more condu-
cive to the formation of free analyte atoms and thereby
minimize this type of interference. This type of interfer-
ence can be detected by analyzing the sample plus a
sample aliquot fortified with a known concentration of the
analyte. If the determined concentration of the analyte
addition is outside a designated range, a possible matrix
effect should be suspected (Section 9.4).
4.4 Memory interferences result from analyzing a
sample containing a high concentration of an element
(typically a high atomization temperature element) which
cannot be removed quantitatively in one complete set of
furnace steps. The analyte which remains in the furnace
can produce false positive signals on subsequent
sample(s). Therefore, the analyst should establish the
analyte concentration which can be injected into the
furnace and adequately removed in one complete set of
furnace cycles. If this concentration is exceeded, the
sample should be diluted and a blank analyzed to assure
the memory effect has been eliminated before reanalyz-
ing the diluted sample.
4.5 Low recoveries may be encountered in the
preconcentration cycle if the trace elements are
complexed by competing chelators (humic/fulvic) in the
sample or are present as colloidal material. Acid solubi-
lization pretreatment is employed to improve analyte
recovery and to minimize adsorption, hydrolysis and
precipitation effects.
200.13-3
Revision 1.0 November 1992
-------
4.6 Memory interferences from the chelating system
may be encountered, especially after analyzing a sample
containing high analyte concentrations. A thorough col-
umn rinsing sequence following elution of the analytes is
necessary to minimize such interferences.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible
for maintaining a current awareness file of OSH A regula-
tions regarding the safe handling of the chemicals speci-
fied in this method.3"6 A reference file of material data
handling sheets should also be made available to all
personnel involved in the chemical analysis. Specifically,
concentrated nitric and hydrochloric acids present vari-
ous hazards and are moderately toxic and extremely
irritating to skin and mucus membranes. Use these
reagents in a fume hood whenever possible and if eye or
skin contact occurs, flush with large volumes of water.
Always wear safety glasses or a shield for eye protection,
protective clothing and observe proper mixing when
working with these reagents.
5.2 Acidification of samples containing reactive mate-
rials may result in release of toxic gases, such as cya-
nides or sulfides. Samples should be acidified in a fume
hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 The graphite tube during atomization emits in-
tense U V radiation. Suitable precautions should be taken
to protect personnel from such a hazard.
5.5 The use of the argon/hydrogen gas mixture during
the dry and char steps may evolve a considerable amount
of HCI gas. Therefore, adequate ventilation is required.
5.6 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Equipment and Supplies
6.1 Graphite Furnace A tomic Absorption
Spectrometer
6.1.1 The GFAA spectrometer must be capable of pro-
grammed heating of the graphite tube and the associated
delayed atomization device. The instrument should be
equipped with an adequate background correction de-
vice capable of removing undesirable non-specific ab-
sorbance over the spectral region of interest. The capa-
bility to record relatively fast (< 1 sec) transient signals
and evaluate data on a peak area basis is preferred. In
addition, a recirculating refrigeration unit is recommended
for improved reproducibility of furnace temperatures.
The data shown in the tables were obtained using the
stabilized temperature platform and Zeernan background
correction.
6.1.2 Single element hollow cathode lamps or single
element electrodeless discharge lamps along with the
associated power supplies.
6.1.3 Argon gas supply (high-purity grade, 99.99%).
6.1.4 A 5% hydrogen in argon gas mix and the neces-
sary hardware to use this gas mixture during specific
furnace cycles.
6.1.5 Autosampler— Although not specifically required,
the use of an autosampler is highly recommended.
6.1.6 Graphite Furnace Operating Conditions—A guide
to experimental conditions for the applicable elements is
provided in Table 1
6.2 Preconcentration System - System containing
no metal parts in the analyte flow path, configured as
shown with a sample loop in Figure 1 and without a
sample loop in Figure 2. .
6.2.1 Column — Macroporous iminodiacetate chelating
resin (Dionex Metpac CC-1 or equivalent).
6.2.2 Control valves — Inert double stack, pneumati-
cally operated four-way slider valves with connectors.
6.2.2.1 Argon gas supply regulated at 80-100 psi.
6.2.3 Solution reservoirs — Inert containers, e.g., high
density polyethylene (HOPE), for holding eluent and
carrier reagents.
6.2.4 Tubing— High pressure, narrow bore, inert tubing
such as Tefzel ETFE (ethylene tetra-fluoroethylene) or
equivalent for interconnection of pumps/valve assem-
blies and a minimum length for connection of the
preconcentration system with the sample collection ves-
sel.
6.2.5 Eluent pumping system (Gradient Pump) — Pro-
grammable flow, high-pressure pumping system, ca-
pable of delivering either one of three eluents at a
pressure up to 2000 psi and a flow rate of 1-5 mUmin.
6.2.6 System setup, including sample loop (See Figure
1).
6.2.6.1 Sample loop — 10-mL loop constructed from
narrow bore, high-pressure inert tubing, Tefzel ETFE or
equivalent.
6.2.6.2 Auxiliary pumps — On-line buffer pump, piston
pump (Dionex QIC pump or equivalent) for delivering 2M
ammonium acetate buffer solution; carrier pump, peri-
staltic pump (Gilson Minipuls or equivalent) for delivering
1 % nitric acid carrier solution; sample pump, peristaltic
pump for loading sample loop.
6.2.7 System setup without sample loop (See Figure 2).
6.2.7.1 Auxiliary Pumps — Sample pump (Dionex QIC
Pump or equivalent) for loading sample on the column.
200.13-4
Revision 1.0 November 1992
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Carrier pump (Dionex QIC Pump or equivalent) used to
flush collection line between samples.
6.3 Labware — For determination of trace elements,
contamination and loss are of critical consideration.
Potential contamination sources include improperly
cleaned laboratory apparatus and general contamina-
tion within the laboratory environment. A clean laboratory
work area, designated for trace element sample handling
must be used. Sample.containers can introduce positive
and negative errors in determination of trace elements by
(1) contributing contaminants through surface desorp-
tion or leaching and (2) depleting element concentrations
through adsorption processes. For these reasons, boro-
silicate glass is nol recommended for use with this
method. All labware in contact with the sample should be
cleaned prior to use. Labware may be soaked overnight
and thoroughly washed with laboratory-grade detergent
and water, rinsed with water, and soaked for 4 h in a
mixture of dilute nitric and hydrochloric acids, followed by
rinsing with ASTM type I water and oven drying.
6.3.7 Griffin beakers, 250 mL, polytetrafluoroethylene
(PTFE) or quartz.
6.3.2 Storage bottles — Narrow mouth bottles, Teflon
FEP (fluorinated ethylene propylene), or HOPE, 125-mL
and 250-mL capacities.
6.4 Sample Processing Equipment
6.4.1 Air displacement pipetter — Digital pipet system
capable of delivering volumes from 100 to 2500 nL with
an assortment of metal-free, disposable pipet tips.
6.4.2 Balances — Analytical balance, capable of accu-
rately weighing to ± 0.1 mg; top pan balance, accurate to
± 0.01 g.
6.4.3 Hotplate— Corning PC100 or equivalent.
6.4.4 Centrifuge—Steel cabinet with guard bowl, elec-
tric timer and brake.
6.4.5 Drying oven— Gravity convection oven with ther-
mostatic control capable of maintaining 105°C ± 5°C.
6.4.6 pH meter— Bench mounted or hand-held elec-
trode system with a resolution of ± 0.1 pH units.
6.4.7 Class 100 hoods are recommended for all sample
handling.
7.0 Reagents and Standards
7.1 Reagents may contain elemental impurities which
might affect analytical data. Only high-purity reagents
that conform to the American Chemical Society specifi-
cations7 should be used whenever possible. If the purity
of a reagent is in question, analyze for contamination. All
acids used for this method must be of ultra high-purity
grade or equivalent. Suitable acids are available from a
number of manufacturers. Redistilled acids prepared by
sub-boiling distillation are acceptable.
7.1.1 Nitric acid, concentrated (sp.gr. 1.41).
7.1.1.1 Nitric acid 0.75M—Dilute 47.7 mL (67.3 g) cone.
nitric acid to 1000 mL with ASTM type I water.
7.1.1.2 Nitric acid (1 +1)—Dilute 500 mL cone, nitric acid
to 1000 mL with ASTM type I water.
7.1.1.3 Nitricacid(1+9)—Dilute 100 mLconc. nitricacid
to 1000 mL with ASTM type I water.
7.1.2 Matrix Modifier, dissolve 300 mg Palladium (Pd)
powder in a minimum amount of concentrated HNO, (1
mL of HNO, adding concentrated HCI only if necessary)
Dissolve 200 mg of MgJNO^-eH O in ASTM type 1
water. Pour the two solutions together and dilute to 100
mL with ASTM type I water.
Note: It is recommended that the matrix modifier be
analyzed separately in orderto assess the contribution of
the modifier to the overall laboratory blank.
7.1.3 Acetic acid, glacial (sp.gr. 1.05). High purity acetic
acid is recommended.
7.1.4 Ammonium hydroxide (20%). High purity ammo-
nium hydroxide is recommended.
7.1.5 Ammonium acetate buffer 1M, pH 5.5— Add 58
mL (60.5 g) of glacial acetic acid to 600 mL of ASTM type
I water. Add 65 mL (60 g) of 20% ammonium hydroxide
and mix. Check the pH of the resulting solution by
withdrawing a small aliquot and testing with a calibrated
pH meter, adjusting the solution to pH 5.5 ± 0.1 with small
volumes of acetic acid or ammonium hydroxide as nec-
essary. Cool and dilute to 1 L with ASTM type I water.
7.1.6 Ammonium acetate buffer 2M, pH 5.5— Prepare
as for Section 7.1.5 using 116 mL (121 g) glacial acetic
acid and 130 mL (120 g) 20% ammonium hydroxide,
' diluted to 1000 mL with ASTM type I water.
Note: If the system is configured as shown in Figure 1,
the ammonium acetate buffer solutions may be further
purified by passing them through the chelating column at
a flow rate of 5.0 mL/min. Collect the purified solution in
a container. Following this, elute the collected contami-
nants from the column using 0.75M nitric acid for 5 min at
a flow rate of 4.0 mL/min. If the system is configured as
shown in Figure 2, the majority of the buffer is being
purified in an on-line configuration via the clean-up col-
umn.
7.1.7 Oxalic acid dihydrate (CASRN 6153-56-6), 0.2M -
Dissolve 25.2 g reagent grade C2H O4'2H2O in 250 mL
ASTM type I water and dilute to 1000 mL with ASTM type
I water. CAUTION - Oxalic acid is toxic; handle with care.
7.2 Water— For all sample preparation and dilutions,
ASTM type I water (ASTM D1193) is required.
7.3 Standard Stock Solutions — May be purchased
from a reputable commercial source or prepared from
ultra high-purity grade chemicals or metals (99.99 -
99.999% pure). All salts should be dried for one hour at
105°C, unless otherwise specified. (CAUTION - Many
metal salts are extremely toxic if inhaled or swallowed.
Wash hands thoroughly after handling.) Stock solutions
200.13-5
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should be stored in plastic bottles. The following proce-
dures may be used for preparing standard stock solu-
tions:
Note: Some metals, particularly those which form sur-
face oxides require cleaning prior to being weighed. This
may be achieved by pickling the surface of the metal in
acid. An amount in excess of the desired weight should
bepickledrepeatedly.rinsedwithwater.driedandweighed
until the desired weight is achieved.
7.3.1 Cadmium s'olution, stock 1 mL = 1000 jig Cd —
Pickle cadmium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5 mL (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100 mL with
ASTM type I water.
7.3.2 Cobalt solution, stock 1 mL=1000 ng Co—Pickle
cobalt metal in (1 +9) nitric acid to an exact weight of 0.100
g. Dissolve in 5 mL (1+1) nitric acid, heating to effect
solution. Cool and dilute to 100 mL with ASTM type I
water.
7.3.3 Coppersolution, stock 1 mL= 1000 ngCu—Pickle
copper metal in (1+9) nitric acid l° an ex.act we'9ht of
0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 mL with ASTM type
I water.
7.3.4 Lead solution, stock! mL=1000 ngPb—Dissolve
0.1599 g PbNO3 in 5 mL (1 +1) nitric acid. Dilute to 100 mL
with ASTM type I water.
7.3.5 Nickel solution, stock 1 mL = 1000 jig Ni —
Dissolve 0.100 g nickel powder in 5 mL cone, nitric acid,
heating to effect solution. Cool and dilute to 100 mL with
ASTM type I water.
7.4 Multielement Stock Standard Solution—Care
must be taken in the preparation of multielement stock
standards that the elements are compatible and stable.
Originating element stocks should be checked for the
presence of impurities which might influence the accu-
racy of the standard. Freshly prepared standards should
be transferred to acid cleaned, new FEP or HOPE bottles
for storage and monitored periodically for stability. A
multielement stock standard solution containing cad-
mium, cobalt, copper, lead, and nickel may be prepared
by diluting an appropriate aliquot of each single element
stock in the list to 100 mL with ASTM type I water
containing 1% (v/v) nitric acid.
7.4.7 Preparation of calibration standards — Fresh
multielement calibration standards should be prepared
weekly. Dilute the stock multielement standard solution
in 1 % (v/v) nitric acid to levels appropriate to the required
operating range. The element concentrations in the stan-
dards should be sufficiently high to produce good mea-
surement precision and to accurately define the slope of
the response curve.
7.5 Blanks— Fourtypesofblanksarerequiredforthis
method. A calibration blank is used to establish the
analytical calibration curve, the laboratory reagent blank
(LRB) is used to assess possible contamination from the
sample preparation procedure and to assess spectral
background. The laboratory fortified blank is used to
assess routine laboratory performance, and a rinse blank
is used to flush the instrument autosampler uptake sys-
tem. All diluent acids should be made from concentrated
acids (Section 7.1) and ASTM Type I water.
7.5.7 The calibration blank consists of the appropriate
acid diluent in ASTM Type I water. The calibration blank
should be stored in a FEP bottle.
7.5.2 The laboratory reagent blanks must contain all the
reagents in the same volumes as used in processing the
samples. The preparation blank must be carried through
the entire sample digestion and preparation scheme.
7.5.3 The laboratory fortified blank (LFB) is prepared by
fortifying an aliquot of the laboratory reagent blank with
all analytes to provide a final concentration which will
produce an absorbance of approximately 0.1 for each
analyte. The LFB must be carried through the complete
procedure as used for the samples.
7.5.4 The rinse blank is prepared as needed by adding
1.0 mL of cone. HNO3 and 1.0 mL cone. HCI to 1 L of
ASTM Type I water and stored in a convenient manner.
7.6 Instrument Performance Check (IPC) Solution
- The IPC solution is used to periodically verify instru-
ment performance during analysis. The IPC solution
should be a fortified seawater prepared in the same acid
mixture as the calibration standards and should contain
method analytes such that the resulting absorbances are
near the midpoint of the calibration curve. The IPC
solution should be prepared from the same standard
stock solutions used to prepare the calibration standards
and stored in a FEP bottle. Agency programs may specify
or request that additional instrument performance check
solutions be prepared at specified concentrations in
order to meet particular program needs.
7.7 Quality Control Sample (QCS) — A quality con-
trol sample having certified concentrations of the analytes
of interest should be obtained from a source outside the
laboratory. Dilute the QCS if necessary with 1% nitric
acid, such that the analyte concentrations fall within the
proposed instrument calibration range.
8.0 Sample Collection, Preservation and
Storage
8.1 Prior to collection of an aqueous sample, consider-
ation should be given to the type of data required, so that
appropriate preservation and pretreatrnent steps can be
taken. Acid preservation, etc., should be performed at the
time of sample collection or as soon thereafter as prac-
tically possible. The pH of all aqueous samples must be
tested immediately prior to aliquoting for analysis to
ensure the sample has been properly preserved. If prop-
erly acid-preserved, the sample can be held up to 6
months before analysis.
8.2 For determination of total recoverable elements in
aqueous samples, acidify with (1 +1) nitric acid at the time
of collection to pH < 2. Normally 3 ml. of (1+1) acid per
200.13-6
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liter of sample is sufficient. The sample should not be
filtered prior to analysis.
Note: Samples that cannot be acid-preserved at the time
of collection because of sampling limitations or transport
restrictions, or have pH > 2 because of high alkalinity
should be acidified with nitric acid to pH <2 upon receipt
in the laboratory. Following acidification, the sample
should be held for 16 h and the pH verified to be <2 before
withdrawing an aliquot for sample processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container type and acid as used in sample
collection.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability and periodic
analysis of laboratory reagent blanks, fortified blanks and
other laboratory solutions as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of
linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) prior to samples being analyzed
by this method.
9.2.2 Linear dynamic range (LDR) — The upper limit of
the LDR must be established for the wavelength utilized
for each analyte by determining the signal responses
from a minimum of 6 different concentration standards
across the range, two of which are close to the upper limit
of the LDR. Determined LDRs must be documented and
kept on file. The linear calibration range which may be
used for analysis of samples should be judged by the
analyst from the resulting data. The upper LDR limit
should be an observed signal no more than 10% below
the level extrapolated from the four lower standards.
New LDRs should be determined whenever there is a
significant change in instrument response, a change in
instrument analytical hardware or operating conditions.
Note: Multiple cleanout furnace cycles may be neces-
sary in order to fully define or utilize the LDR for certain
elements such as nickel. For this reason, the upper limit
of the linear calibration range may not correspond to the
upper LDR limit.
Measured sample analyte concentrations that exceed
the upper limit of the linear calibration range must either
be diluted and reanalyzed with concern for memory
effects (Section 4.4) or analyzed by another approved
method.
9.2.3 Quality control sample (QCS) — When beginning
the use of this method, on a quarterly basis or as required
to meet data-quality needs, verify the calibration stan-
dards and acceptable instrument performance with the
preparation and analyses of a QCS (Section 7.7). If the
determined concentrations are not within ± 10% of the
stated values, performance of the determinative step of
the method is unacceptable. The source of the problem
must be identified and corrected before either proceed-
ing on with the initial determination of method detection
limits or continuing with ongoing analyses.
9.2.4 Method detection limit (MDL) — MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.8To determine MDL
values, take.seven replicate aliquots of the fortified
reagent water and process through the entire analytical
method. Perform all calculations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t =' 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
Note: If the relative standard deviation (RSD) from the
analyses of the seven aliquots is < 15%, the concentra-
tion used to determine the analyte MDL may have been
inappropriately high forthie determination. If so, this could
result in the calculation of an unrealistically low MDL. If
additional confirmation of the MDL is desired, reanalyze
the seven replicate aliquots on two more nonconsecutive
days and again calculate the MDL values for each day.
An average of the three MDL values for each analyte may
provide for a more appropriate MDL estimate. Determi-
nation of MDL in reagent water represents a best case
situation and does not reflect possible matrix effects of
real world samples. However, successful analyses of
LFMs (Section 9.4) can give confidence to the MDL value
determined in reagent water. Typical single laboratory
MDL values using this method are given in Table 1 MDLs
should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory reagent blank (LRB) — the laboratory
must analyze at least one LRB (Section 7.5.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and
the samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
200.13-7
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9.3.2 Laboratory fortified blank (LFB) — the laboratory
must analyze at least one LFB (Section 7.5.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that ana-
lyte is judged out of control, and the source of the problem
should be identified and resolved before continuing analy-
ses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance againstthe required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
Internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and.the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each 5-10
new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
establish an ongoing precision statement for the level of
concentrations included in the LFB. These data must be
kept on file and be available for review.
9.3.4 Instrument Performance Check (IPC) Solution—
For all determinations the laboratory must analyze the
IPC solution (Section 7.6) and a calibration blank imme-
diatelyfollowingeachcalibration.aftereverytenthsample
(or more frequently, if required) and at the end of the
sample run. The IPC solution should be afortified seawa-
ter matrix. Analysis of the IPC solution and calibration
blank Immediately following calibration must verify that
the instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must be within ± 10% of
calibration. If the calibration cannot be verified within the
specified limits, reanalyze the IPC solution. If the second
analysis of the IPC solution confirms calibration to be
outside the limits, sample analysis must be discontinued,
the cause determined and/or in the case of drift the
Instrument recalibrated. All samples following the last
acceptable IPC solution must be reanalyzed. The analy-
sis data of the calibration blank and I PC solution must be
kept on file with the sample analyses data.
9.3.5 The overall sensitivity and precision of this method
are strongly influenced by a laboratory's ability to control
the method blank. Therefore, it is recommended that the
calibration blank response be recorded for each-set of
samples. This record will aid the laboratory in assessing
both its long and short term ability to control the method
blank.
9.4 Assessing Analyte Recovery and Data
Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and data
quality. Taking separate aliquots from the sample for
replicate and fortified analyses can, in some cases,
assess these effects. Unless otherwise specified by the
data user, laboratory or program, the following laboratory
fortified matrix (LFM) procedure (Sect 9.4.2) is required.
9.4.2 The laboratory must add a known amount of each
analyte to a minimum of 10% of routine samples. In each
case, the LFM aliquot must be a duplicate of the aliquot
used for sample analysis and for total recoverable deter-
minations added prior to sample preparation. For water
samples, the added analyte concentration must be the
same as that used in the laboratory fortified blank (Sec-
tion 7.5.3). Over time, samples from all routine sample
sources should be fortified.
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is <25% of the
unfortified sample concentration. Percent recovery may
be calculated in units appropriate to the matrix, using the
following equation:
R = CS-C x100
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte
added to sample.
9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range (but is still within the
range of calibration and the background absorbance is <
1 abs.) and the laboratory performance for that analyte is
shown to be in control (Section 9.3), the recovery prob-
lem encountered with the LFM is judged to be either
matrix or solution related, not system related. This situa-
tion should be rare given the matrix elimination
preconcentration step prior to analysis. If a low recovery
is found, check the pH of the sample plus the buffer
mixture. The resulting pH should be about 5.5. The pH of
' the sample strongly influences the column's ability to
preconcentrate the metals; therefore, a low recovery
may be caused by a low pH. If the pH for the LFM/buffer
mixture is about 5.5, the analyst is advised to make an in
furnace analyte addition to the LFM using the
preconcentrated standard solution. If recovery of the in
furnace analyte addition is shown to be out of control, a
matrix interference is confirmed and the sample must be
analyzed by MSA.
9.5 Utilizing Reference Materials
9.5.1 It is recommended that a reference material such
as NASS-3 (from the Research Council of Canada) be
fortified and used as an Instrument Performance Check
Solution.
200.13-8
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10.0 Calibration and Standardization
10.1 The preconcentration system can be configured
utilizing a sample loop to define the sample volume
(Figure 1) or the system can be configured such that a
sample pump rate and a pumping time defines the
sample volume (Figure 2). The system illustrated in
Figure 1 is recommended for sample sizes of < 10 ml. A
thorough rinsing of the sample loop between samples
with HNO3 is required. This rinsing will minimize the
cross-contamination which may be caused by the sample
loop. The system in Figure 2 should be used for sample
volumes of > 10 ml. The sample pump used in Figure 2
must be calibrated to assure that a reproducible/defined
volume is being delivered.
10.2 Specific wavelengths and instrument operating
conditions are listed in Table 1. However, because of
differences among makes and models of spectropho-
tometers and electrothermal furnace devices, the actual
instrument conditions selected may vary from those
listed.
10.3 Prior to the use of this method, instrument operat-
ing conditions must be optimized. The analyst should
follow the instructions provided by the manufacturer
while using the conditions listed in Table 1 as a guide. Of
particular importance is the determination of the charring
temperature limit for each analyte. This limit is the fur-
nace temperature setting where a loss in analyte will
occur prior to atomization. This limit should be deter-
mined by conducting char temperature profiles for each
analyte and when necessary, in the matrix of question.
The charring temperature selected should minimize back-
ground absorbance while providing some furnace tem-
perature variation without loss of analyte. For routine
analytical operation the charring temperature is usually
set at least 100°C below this limit. The optimum condi-
tions selected should provide the lowest reliable MDLs
and be similar to those listed in Table 1. Once the
optimum operating conditions are determined, they should
be recorded and available for daily reference.
10.4 Prior to an initial calibration, the linear dynamic
range of the analyte must be determined (Sect 9.2.2)
using the optimized instrument operating conditions. For
all determinations allow an instrument and hollow cath-
ode lamp warm-up period of not less than 15 min. If an
EDL is to be used, allow 30 min for warm-up.
10.5 Before using the procedure (Section 11.0) to ana-
lyze samples, data must be available to document initial
demonstration of performance. The required data and
procedure are described in Section 9.2. This data must
be generated using the same instrument operating con-
ditions and calibration routine (Section 11.4) to be used
for sample analysis. These documented data must be
kept on file and be available for review by the data user.
11.0 Procedure
11.1 Sample Preparation - Total Recoverable
Elements
11.1.1 Add 2 ml_ (1 +1) nitric acid to the beaker contain-
ing 100 mL of sample. Place the beaker on the hot plate
200
for solution evaporation. The hot plate should be located
in a fume hood and previously adjusted to provide evapo-
ration at a temperature of approximately but no higher
than 85°C. (See the following note.) The beaker should
be covered with an elevated (ribbed) watch glass or other
necessary steps should be taken to prevent sample
contamination from the fume hood environment.
Note: For proper heating adjust the temperature control
of the hot plate such that an uncovered Griffin beaker
containing 50 mL of water placed in the center of the hot
plate can be maintained at a temperature approximately
but no higher than 85°C. (Once the beaker is covered
with a watch glass the temperature of the water will rise
to approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20 mL by gentle heating at 85°C. DO NOT BOIL.
This step takes about 2 h for a 100-mL aliquot with the
rate of evaporation rapidly increasing as the sample
volume approaches 20 mL. (A spare beaker containing
20 mL of water can be used as a gauge.)
11.1.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the sample
for 30 min. Slight boiling may occur, but vigorous boiling
must be avoided.
11.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
11.1.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight the
sample contains suspended solids that would clog or
affect the sample introduction system, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination
from filtration.) The sample is now ready for analysis.
Because the effects of various matrices on the stability of
diluted samples cannot be characterized, all analyses
should be performed as soon as possible after the
completed preparation.
11.2 Prior to first use, the preconcentration system
should be thoroughly cleaned and decontaminated using
0.2M oxalic acid.
11.2.1 Precleaning the Preconcentration System
11.2.1.1 Place approximately 500 mL 0.2M oxalic acid
in each of the sample/eluent containers. Flush the entire
system by running the program used for sample analysis
. 3 times.
11.2.1.2 Rinse the containers with ASTM Type I water
and repeat the sequence described in Section 11.2.1.1
using,0.75M nitric acid and again using ASTM type I
water in place of the 0.2M oxalic acid.
11.2.1.3 Rinse the containers thoroughly with ASTM
type I water, fill them with their designated reagents and
run through the program used for sample analysis in
order to prime the pump and all eluent lines with the
correct reagents.
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11.2.2 Peak Profile Determination
11.2.2.1 The peak elution time or the collection window
should be determined using an ICP-AES (Inductively
Coupled Plasma Atomic Emission Spectrometer) or Flame
AA. Figure 3 is a plot of time vs. emission intensity for Cd,
Pb, Ni.and Cu. The collection window is marked in Figure
3 and should provide about 30 sec buffer on either side
of the peak. If an ICP-AES is not available, it is recom-
mended that the peak profile be determined by collecting
200-jiL samples during the elution part of the
preconcentration cycle and then reconstructing the peak
profile from the analysis of the 200-nL samples.
11.3 Sample Preconcentration
11.3.1 Preconcentration utilizing a sample loop.
11.3.1.1 Loading Sample Loop—With valve 1 in the off
position and valve 2 in the on position, load sample
through the sample loop to waste using the sample pump
for 4 min at 4 mL/min. Switch on the carrier pump and
pump 1% nitric acid to flush the sample collection line.
11.3.1.2 Column Loading — With valve 1 in the on
position, load sample from the loop onto the column
using 1M ammonium acetate for 4.5 min at 4.0 mL/min.
Switch on the buffer pump, and pump 2M ammonium
acetate at a flow rate of 1 mL/min. The analytes are
retained on the column, while the majority of the matrix is
passed through to waste.
11.3.1.3 Elution Matrix—With valve 1 in the on position
the gradient pump is allowed to elute the matrix using the
1M ammonium acetate. During which time the carrier,
buffer and the sample pumps are all off.
11.3.1.4 Elute Analytes — Turn off valve 1 and begin
eluting the analytes by pumping 0.75M nitric acid through
the column and turn off valve 2 and pump the eluted
analytes into the collection flask. The analytes should be
eluted into a 2-mL sample volume.
11.3.1.5 Column Reconditioning — Turn on valve 2 to
direct column effluent to waste, and pump 0.75M nitric
acid, 1M ammonium acetate, 0.75M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min. Each solvent should be pumped through the
column for 2 min. During this process, the next sample
can be loaded into the sample loop using the sample
pump.
11.3.1.6 Preconcentration of the sample may be
achieved by running through an eluent pump program.
The exact timing of this sequence should be modified
according to the internal volume of the connecting tubing
and the specific hardware configuration used.
11.3.2 Preconcentration utilizing an auxiliary pump to
determine sample volume.
11.3.2.1 Sample Loading—With the valves 1 and 2 on
and the sample pump on, load the sample on the column
buffering the sample utilizing the gradient pUmp and the
2M buffer. The actual sample volume is determined by
knowing the sample pump rate and the time. While, the
sample is being loaded the carrier pump can be used to
flush the collection line.
11.3.2.2 Elution Matrix—With valve 1 in the off position
the gradient pump is allowed to elute the matrix using the
1M ammonium acetate. During which time the carrier,
buffer and the sample pumps are all off.
11.3.2.3 Elution of Analytes—with valves 1 and 2 inthe
off position the gradient pump is switched to 0.75M HNO3
and the analytes are eluted into the collection vessel. The
analytes should be eluted into a 2 mL sample volume.
11.3.2.4 Column Reconditioning — Turn on valve 2 to.
direct column effluent to waste, and pump 0.75M nitric
acid, 1M ammonium acetate, 0.75M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min.
Note: When switching the gradient pump from nitric acid
back to the ammonium acetate it is necessary to flush the
line connecting the gradient pump to valve 2 with the
ammonium acetate priorto switching the valve. If the line
contains nitric acid it will elute the metals from the clean-
up column.
11.3.2.5 Preconcentration of the sample may be
achieved by running through an eluent pump program.
The exact timing of this sequence should be modified
according to the internal volume of the connecting tubing
and the specific hardware configuration used.
11.4 Repeat the sequence described in Section 11.3.1
or 1.1.3.2 for each sample to be analyzed. At the end of
the analytical run leave the column filled with 1M ammo-
nium acetate buffer until it is next used.
11.5 Samples having concentrations higher than the
established linear dynamic range should be diluted into
range and reanalyzed.
11.6 Sample Analysis
11.6.1 Prior to daily instrument calibration, inspect the
graphite furnace, the sample uptake system and
autosampler injector for any change that would affect
instrument performance. Clean the system and replace
the graphite tube and/or platform when needed or on a
daily basis. A cotton swab dipped in a 50/50 mixture of
isopropyl alcohol (I PA) and H2O (such that it is damp but
not dripping) can be used to remove the majority of the
salt buildup. A second cotton swab is dipped in IPA and
the contact rings are wiped down to assure they are
clean. The rings are then allowed to thoroughly dry and
then a new tube is placed in the furnace and conditioned
according to instrument manufacturers specifications.
11.6.2 Configure the instrument system to the selected
optimized operating conditions as determined in Sec-
tions 10.1 and 10.2.
11.6.3 Before beginning daily calibration the instrument
should be reconfigured to the optimized conditions. Ini-
tiate data system and allow a period of not less than 15
min for instrument and hollow cathode lamp warm-up. If
an EDL is to be used, allow 30 min for warm-up.
200.13-10
Revision 1.0 November 1992
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11.6.4 After the warm-up period but before calibration,
instrument stability must be demonstrated by analyzing
a standard solution with a concentration 20 times the IDL
a minimum of five times. The resulting relative standard
deviation of absorbance signals must be < 5%. If the
relative standard deviation is > 5%, determine and cor-
rect the cause before calibrating the instrument.
11.6.5 For initial and daily operation calibrate the instru-
ment according to the instrument manufacturer's recom-
mended procedures using the calibration blank (Section
7.5.1) and calibration standards (Section 7.4) prepared
at three or more concentrations within the usable linear
dynamic range of the analyte (Sections 4.4 & 9.2.2).
11.6.6 An autosampler must be used to introduce all
solutions into the graphite furnace. Once the standard,
sample or QC solution plus the matrix modifier is injected,
the furnace controller completes furnace cycles and
cleanout period as programmed. Analyte signals must be
integrated and collected as peak area measurements.
Background absorbances, background corrected analyte
signals, and determined analyte concentrations on all
solutions must be able to be displayed on a CRT for
immediate review by the analyst and be available as hard
copy for documentation to be kept on file. Flush the
autosampler solution uptake system with the rinse blank
(Section 7.5.4) between each solution injected.
11.6.7 After completion of the initial requirements of this
method (Section 9.2), samples should be analyzed in the
same operational manner used in the calibration routine.
11.6.8 During sample analyses, the laboratory must
comply with the required quality control described in
Sections 9.3 and 9.4.
11.6.9 Determined sample analyte concentrations that
are > 90% of the upper limit of calibration must either be
diluted with acidified reagent water and reanalyzed with
concern for memory effects (Section 4.4), or determined
by another approved test procedure that is less sensitive.
Samples with a background absorbance > 1.0 must be
appropriately diluted with acidified reagent water and
reanalyzed (Section 9.4.6). If the method of standard
additions is required, follow the instructions described in
Section 11.5.
11.6.10 Report data as directed in Section 12.
11.7 Standard Additions — If the method of standard
addition is required, the following procedure is recom-
mended:
11.7.1 The standard addition technique9 involves pre-
paring new standards in the sample matrix by adding
known amounts of standard to one or more aliquots of the
processed sample solution. This technique compen-
sates for a sample constituent that enhances or de-
presses the analyte signal, thus producing a different
slope from that of the calibration standards. It will not
correct for additive interference, which causes a baseline
shift. The simplest version of this technique is the single-
addition method. The procedure is as follows: Two iden-
tical aliquots of the sample solution, each of volume Vx,
are taken. To the first (labeled A) is added a small volume
Vs of a standard analyte solution of concentration Cs. To
the second (labeled B) is added the same volume Vs of
the solvent. The analytical signals of A and B are mea-
sured and corrected for nonanalyte signals. The un-
known sample concentration Cx is calculated:
cx= SBVSCS
. (SA-SB)VX
where, SA and SB are the analytical signals (corrected for
the blank) of solutions A and B, respectively. Vs and Cs
should be chosen so that SA is roughly twice S on the
average. It is best if Vs is made much less than V , and
thus Cs is much greater than Cx, to avoid excess dilution
of the sample matrix. If a separation or concentration step
is used, the additions are best made first and carried.
through the entire procedure. For the results from this
technique to be valid, the following limitations must be
taken into consideration:
1. The analytical curve must be linear.
2. The chemical form of the analyte added must re-
spond in the same manner as the analyte in the
sample.
3. The interference effect must be constant over the
working range of concern.
4. The signal must be corrected for any additive inter-
ference.
12.0 Data Analysis and Calculations
12.1 Sample data should be reported in units of jxg/L for
aqueous samples.
12.2 For total recoverable aqueous analytes (Section
11.1), when 100-mL aliquot is used to produce the 100
mL final solution, round the data to the tenths place and
report the data in jig/L up to three significant figures. If an
aliquot volume other than 100 mL is used for sample
preparation, adjust the dilution factor accordingly. Also,
account for any additional dilution of the prepared sample
solution needed to complete the determination of analytes
exceeding the upper limit of the calibration curve. Do not
report data below the determined analyte MDL concen-
tration or below an adjusted detection limit reflecting
smaller sample aliquots used in processing or additional
dilutions required to complete the analysis.
12.3 The QC data obtained during the analyses provide
an indication of the quality of the sample data and should
be provided with the sample results.
13.0 Method Performance
13.1 Experimental conditions used for single laboratory
testing of the method are summarized in Table 1.
13.2 Table 2 contains precision and recovery data ob-
tained from a single laboratory analysis of a fortified and
a non-fortified sample of NASS-3. The samples were
prepared using the procedure described in Sect. 11.1.
Four replicates of the non-fortified samples were ana-
200.13-11
Revision 1.0 November 1992
-------
lyzed and the average of the replicates was used for
determining the sample analyte concentration. The forti-
fied samples of NASS-3 were also analyzed and the
average percent recovery and the percent relative stan-
dard deviation is reported. The reference material certi-
fied values are also listed for comparison.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation (e.g., Section 7.8). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 115516th Street N.W., Wash-
ington D.C. 20036, (202)872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section14.2.
16.0 References
1. A. Siraraks, H.M. Kingston and J.M. Riviello,
Anal Chem. §2 1185 (1990).
2. E.M. Heithmar, T.A. Hinners, J.T. Rowan and J.M.
Riviello, Anal Chem. £2 857 (1990).
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
4. Carcinogens - Working With Carcinogens,
Department of Health, Education, and Welfare, Public
Health Service, Centers for Disease Control, National
Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
5. Proposed OSHA Safety and Health Standards,
Laboratories, Occupational Safety and Health
Administration, Federal Register, July 24,1986.
6. Safety in Academic Chemistry Laboratories,
American Chemical Society Publication, Committee
on Chemical Safety, 3rd Edition, 1979.
7. Rohrbough, W.G. et al. Reagent Chemicals, American
Chemical Society Specifications, 7th edition.
American Chemical Society, Washington, DC, 1986.
8. Code of Federal Regulations 40, Ch. 1, Pt. 136
Appendix B.
9. Winefordner, J.D., Trace Analysis: Spectroscopic
Methods for Elements, Chemical Analysis, Vol. 46,
pp. 41-42, 1976.
200.13-12
Revision 1.0 November 1992
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Method Detection Limits for Total Recoverable Analytes in Reagent Water1
Element
Cadmium
Cobalt
Copper
Lead
Nickel
Slit,
nm
0.7
0.2
0.7
0.7
0.2
Recommended
Analytical
Wavelengths, nm
228.8
242.5
324.8
283.3
232.4
Char
Temp, °C
800
1400
1300
1250
1400
Atomization
Temp, °C
1600
2500
2600-
2000
2500
MDL2,
MQ/L
0.016
-
0.36
0.28
-
1 MDLs were calculated using NASS-3 as the matrix.
2 MDLs were calculated based on a 10-mL sample loop.
* MDL was not calculated because the concentration in the matrix exceeds the MDL spike level.
- Not Determined.
Table 2. Precision and Recovery Data for NASS-3 using System Illustrated in Figure 1
Analyte
Cd
Co
Cu
Pb
Ni
Certified
Value,
ng/L3
0.029 ± 0.004
0.004 ± 0.001
0.109 + 0.011
0.039 ± 0.006
0.257 ± 0.027
Sample
Cone.,
W3/L3
0.026 + 0.012
-
<0.36
<0.28
0.260 ± 0.04
Fortified
Cone.,
ug/L
0.25
5.0
5.0
5.0
Avg.
Recovery, %
93
_
87
90
117
% RSD
3.3
1.4
3.7
8.3
1 Data collected using 10-mL sample loop.
2 Matrix modifier is Pd/MgfNCg./H,,
3 Uncertainties based on 95% confidence limits.
- Not determined.
200.13-13
Revision 1.0 November 1992
-------
Sample Loop
Loading
Column
Loading
Elulion of
Matrix
Elulion of
Analytes
Column
Recondition
1
Off
On
On
Off
Off
Valves
2
On
On
On
Off
On
Buffer
Pump
Off
On
Off
Off
Off
Carrier
Pump
On
Off
Off
Off
Off
Sample
Pump
On
Off
Off
Off
Off
Waste
On ' r
-•*" \
i 'lug < L i ^ —
Waste ^S-,1**
.. I ,..„., Mixing Tee
..•V. || J
Sample --* - ^v /">>
Pump \..-" S-^
1 , , fix I Column [^ —
J . 1
1 1 i f^
a> .'^"L 1 1 ^••J^s iQJ
Ttt »" 4 ^*^. II •" * ^^, ^r^f »•
to t 1 .5 ~* ^~~~ C. i .' " .»'
> \. -• 'V^ -• r^Qm.iln r*
-• ^Sm.- ^*k •* Sample i — 'C
^ J" Loop 1 \
™9 ^ Waste
01
<3
T Carrier
Grad enl Pump 1 ' —
1
1 M 0.75 M
NH4OAc HNO3
Buffer
Pump
2 >— ,
.'*
Col.
VesssI
Figure 1. Sample Loop Configuration.
Revision 1.0 November 1992
200.13-14
-------
Event
Sample
Loading
Elution of
Matrix
Elution of
Analytes
Column
Recondition
1
On
Off
Off
Off
Valves
2
On
On
Off
On
Carrier
Pump
On
Off
Off
Off
Sample
Pump
On
Off
Off
On
.Off
.On
Waste
**
X1
X
.*
«•
Sample
Pump
Mixing Tee
Figure 2. System Diagram without Sample Loop.
200.13 -15
Revision 1.0 November 1992
-------
80
Time (sec)
Figure 3. Peak Collection Window from ICP-AES. ,
200.13-16
Revision 1.0 November 1992
-------
Method 353.4
Determination of Nitrite + Nitrate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis
Adapted by
Carl F. Zimmermann
Carolyn W. Keefe
University of Maryland System
Center for Environmental Estuarine Studies
' Chesapeake Biological Laboratory
Solomns, MD 20688-0038
Revision 1.3
November 1992
Edited by
Elizabeth J. Arar
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
353.4 -1
Revision 1.3 November 1992
-------
Method 353.4
Determination of Nitrite + Nitrate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for the determi-
nation of low level nitrite + nitrate concentrations nor-
mally found in estuarine and/or coastal waters using the
cadmium (Cd) reduction technique.1 Nitrate concentra-
tions are obtained by subtracting nitrite values, which
have been previously determined by this method without
the Cd reduction procedure, from the nitrite + nitrate
values.
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Nitrite
Nitrate
14797-65-0
14797-55-8
1.2 A statistically determined method detection limit
(MDL) of 0.001 mg N/L in 3 parts per thousand (ppt)
saline water has been determined by one laboratory.2
The method is linearto 0.42 mg N/L using an AutoAnalyzer
II System (Bran & Luebbe, Buffalo Grove, IL).
1.3 Approximately 40 samples can be analyzed in an
hour.
1.4 This method should be used by analysts experi-
enced in the use of automated colorimetric analyses and
familiarwith matrix interferences and procedures fortheir
correction. A minimum of 6 months experience under
experienced supervision is recommended.
15 This method was tested by 11 laboratories using
deionized distilled water, high salinity sea water (36 ppt)
and three estuarine waters of 8,12, and 18 ppt salinity.
When nitritewasdetermined (sample not passed through
Cd reduction column), precision and accuracy were high
and there were no statistically significant matrix effects.
However, when nitrate was determined (sample passed
through the Cd reduction column), 50% of the laborato-
ries reported unacceptable data. Precision and accuracy
decreased as salinity increased and nitrate concentra-
tion decreased. The user of this method is, therefore,
cautioned as to its lack of ruggedness and accuracy
when determining nitrate, and the user is admonished to
carefully check and maintain the Cd reduction column
required for the determination of nitrate.
2.0 Summary of Method
2.1 An automated colorimetric method for the analysis
of low level nitrite + nitrate concentrations is described.
Filtered samples are passed through a copperized cad-
mium column to reduce nitrate to nitrite. The nitrite
originally present and the reduced nitrate are then deter-
mined by diazotizing with sulfanilamide and coupling with
N-1-naphthylethylenediamine dihydrochloride to form a
colored azo dye. The color produced is proportional to
the nitrite + nitrate concentration present in the sample.
Nitrate is obtained by subtracting nitrite values, which
have been previously determined without the cadmium
reduction step, from the nitrite + nitrate values.
3.0 Definitions
3.1 Calibration Standard (CAL) — A solution pre-
pared from the primary dilution standard solution or stock
standard solution containing the internal standards and
surrogate analytes. The CAL solutions are used to cali-
brate the instrument response with respect to analyte
concentration.
3.2 Dissolved Analyte (DA) —- The concentration of
analyte in an aqueous sample that passes through a 0.45
jim membrane filter assembly prior to sample acidifica-
tion or other processing.
3.3 Laboratory Fortified Blank (LFB)—An aliquot of
reagent water or other blank matrix to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly as a sample, and
its purpose is to determine whether the method is in
control and whether the laboratory is capable of making
accurate and precise measurements.
3.4 Laboratory Reagent Blank (LRB) — An aliquot
of reagent water or other blank matrix that is treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, re-
agents, or apparatus.
3.5 Linear Dynamic Range (LDR)—The concentra-
tion range over which the analytical working curve re-
mains linear.
3.6 Method Detection Limit (MDL) — The minimum
concentration of an analyte that can be identified, mea.-
sured and reported with 99% confidence that the analyte
concentration is greater than zero.2
3.7 Reagent Water— Type 1 reagent grade water
equal to or exceeding standards established by Ameri-
can Society for Testing and Materials (ASTM). Reverse
353.4 - 2
Revision 1.3 November 1992
-------
osmosis systems or distilling units which produce 18
megohm water are two examples of acceptable water
sources.
3.8 Refractive Index (Rl) — The ratio of the velocity
of light in a vacuum to that in a given medium. The relative
refractive index is the ratio of the velocity of light in two
different media, such as sea or estuarine water versus
reagent water. The correction for this difference is re-
ferred to as the refractive index correction in this method.
3.9 Stock Standard Solution (SSS) — A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
4.0 Interferences
4.1 Concentrations of iron, copper, or other metals
above several mg/L alter reduction efficiency.3 The
presence of large concentrations of sulfide and/or sulfate
will cause a loss of sensitivity of nitrate to the copperized
cadmium column.4'5
4.2 Suspended solids restrict sample flow through the
column. Sample turbidity should be removed by filtration
prior to analysis.
4.3 This method corrects for refractive index and "salt
error" interferences (Sections 12.2 and 12.3) which occur
if calibration standards and samples are not matched in
salinity.
5.0 Safety
5.1 Water samples collected from estuarine and/or
ocean environments are generally not hazardous. How-
ever, the individual who collects samples should use
proper techniques to insure safety.
5.2 Good laboratory technique should be followed
when preparing reagents. A lab coat, safety goggles and
gloves should be worn when preparing the reagents,
particularly the copper sulfate solution, and color re-
agent.
6.0 Equipment and Supplies
6.1 Continuous Flow Automated Analytical
System Consisting of:
6.1.1 Sampler.
6.1.2 Manifold or analytical aartridge equipped with
copper/cadmium reduction column (prepared according
to specifications in Section 7.2.1).
6.1.3 Proportioning pump.
6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular
flow cell and 550 nm filter.
6.1.5 Phototube sensitive to 550 nm light.
6.1.6 Recorder or computer based data system.
6.2 Nitrogen-Free Glassware — All glassware used
in the determination must be low in residual nitrate to
avoid sample/reagent contamination. Washing with 10%
HCI and thoroughly rinsing with reagent water have been
found to be effective.
7.0 Reagents and Standards
7.1 Stock Reagent Solutions
7.1.1 Ammonium Chloride Reagent— Dissolve 10.0 g
of ammonium chloride (NH4CL)(CASRN 12125-02-9) in
1 Lof reagent water. Adjust to pH 8.5 by adding 3-4 NaOH
(CASRN1310-73-2) pellets as necessary. Add 5 drops of
2% copper sulfate solution (Section 7.1.3). No addition of
EDTA is necessary. This reagent is stable for 1 week
when kept refrigerated.
7.1.2 Color Reagent— Combine 1500 ml_ reagent wa-
ter, 200.0 ml concentrated phosphoric acid (H3PO4,
CASRN 7664-38-2), 20.0 g sulfanilamide (CASRN 63-
74-1), and 1.0 g N-1-napthylethylenediamine di-
hydrochloride (CASRN 1465-25-4). Dilute to 2000 ml
with reagent water. Add 2.0 mL BRIJ-35 (Bran & Luebbe,
Buffalo Grove, IL). Store at 4°C in the dark. This reagent
should be prepared every 6 weeks.
7.1.3 Copper Sulfate Solution — Dissolve 2.0 g of cop-
per sulfate (CuSO4.5H2O) (CASRN 7758-98-7) in 90 mL
of reagent water. Dilute to 100 mL with reagent water.
7.1.4 Refractive Reagent— Combine 100 mL of con-
centrated phosphoric acid (H3PO4) to 800 mL reagent
water. Add 1.0 mL BRIJ-35. Dilute to 1000 mL with
reagent water.
7.1.5 Stock Nitrate Solution — Dissolve 0.721 g of pre-
dried (60°C for 1 h) potassium nitrate (KNO3)(CASRN
7758-09-0) in reagent water and dilute to 1000 mL. 1.0
mL = 0.100 mg N. The stability of this stock standard is
approximately 3 months when kept refrigerated.
7.1.6 Stock Nitrite Solution — Dissolve 0.493 g of pre-
dried (60°C for 1 hr) sodium nitrite (NaNO2) (CASRN
7632-00-0) in reagent water and dilute to 1000 mL. 1.0
mL = 0.100 mg N. The stability of this stock standard is
approximately 3 months, when kept refrigerated.
7.1.7 Low Nutrient Seawater—Obtain natural low nutri-
ent seawater (36 ppt salinity; <0.0002 mg N/L) or prepare'
synthetic seawater by dissolving 31 g analytical reagent
grade sodium chloride, NaCI, (CASRN 7647-14-5); 10 g
analytical reagent grade magnesium sulfate, MgSO4
CASRN 10034-99-8); and 0.05 g analytical reagent grade
sodium bicarbonate, NaHCO3 (CASRN 144-55-8), in 1 L
of reagent water.
7.2 Cadmium Preparation — Use good quality cad-
mium (CASRN 7440-43-9) filings. Depending on the
reductor column shape and size, cadmium filings should
generally be <0.5 mm but >0.3 mm for glass columns and
353.4 - 3
Revision 1,3 November 1992
-------
in the 25-60 mesh size (0.25 mm to 0.71 mm) range for
columns prepared by using flexible tubing.
New cadmium filings should be rinsed with diethyl ether
to remove dirt and grease.
Approximately 10 g of this cadmium is treated with 50 ml
of 6N HCI in a 150-mL beaker. Swirl very carefully for 1
min. Carefully decant the HCI and thoroughly rinse the
cadmium (at least 10 times) with reagent water. Decant
the reagent water and add a 50-mL portion of 2% (w/v)
copper sulfate solution (Section 7.1.3). While swirling,
brown flakes of colloidal copper will appear and the blue
color of thesolution will fade. Decant and repeat sequen-
tial washing with reagent water and copper sulfate solu-
tion until blue color does not fade. Avoid exposure of
treated copper-cadmium to air.
Wash the filings thoroughly with reagent water until all
blue color is gone and the supernatant is free of panicu-
late matter. Usually a minimum of 10 rinses is necessary.
The filings are now ready to be packed into the column.
7.2.1 Column Preparation — The column should be
prepared from flexible plastic tubing or glass. The follow-
ing column dimensions are acceptable.
Glass tube: U-shaped, 35 cm (13.78 in.) in length
with 2 mm (0.079 in.) ID.
Flexible plastic tube: 22 cm (8.66 in.) in length with
2.8 mm (0.11 in.) ID.
Plug one end of the column with glass wool. Fill the
reductor column with ammonium chloride reagent (Sec-
tion 7.1.1) and transfer the prepared cadmium filings to
the column using a Pasteur pipette or some other method
that prevents contact of the Cd filings with air. Pack the
entire column uniformly with filings such that, visually, the
packed filings have separation gaps no greater than
approximately 1 mrn. If the column is too densely packed,
sample flow is restricted. Insert another glass wool plug
at the top of the column and with reagents pumping
through the system, attach the column to the valve
assembly. Remember to have no air bubbles in the valve
(Rgure 1) and to attach the column to the intake side of
the valve first.
Check for good flow characteristics (regular bubble pat-
tern) after the addition of air bubbles beyond the column.
If the column is packed too tightly, an inconsistent flow
pattern will be evident.
Prior to sample analysis, condition the column by pump-
ing through the sample line approximately 1 mg N (ni-
trate)/L (Section 7.2.2) for 5 min.
7.2.2 Secondary Nitrate Solution — Dilute 1.0 ml_ of
stock nitrate solution (Section 7.1.5) to 100 ml_ with
reagent water. 1.0 mL of this solution = 0.001 mg N.
Refrigerate and prepare fresh weekly.
7.2.3 Prepare a series of calibration standards (CAL) by
diluting suitable volumes of Secondary Nitrate Solution
(Section 7.2.2) to 100 mL with reagent water. Prepare
these standards daily. When working with samples of
known salinity, it is recommended that the CAL solutions
be prepared in Low Nutrient Seawater (Section 7.1.7)
diluted to the salinity of the samples, and the Sampler
Wash Solution also be Low Nutrient Seawater (Section
7.1.7) diluted to that salinity. If this procedure is per-
formed, it is unnecessary to perform the salt error and
refractive index correction outlined in Sections 12.2 and
12.3. When analyzing samples of varying salinities, it is
recommended that the standard curve be prepared in
reagent water and refractive index corrections be made
to the sample concentrations (Section 12.2). The follow-
ing solutions, diluted to 100 mL with reagent water, are
suggested.
Volume (mL) of secondary
nitrate solution (7.2.2)
diluted to 100mL
Cone.,
mgN/L
0.5
1.0
2.0
4.0
6.0
0.005
0.010
0.020
0.040
0.060
7.2.4 Saline Nitrate Standards — If CAL solutions will
not be prepared to match salinity, then they must be
prepared in a series of salinities in order to quantify the
"salt error," the increase or decrease in the colorimetric
response of nitrate due to the change in the ionic strength
of the solution. The following dilutions prepared in 100-
mL volumetric flasks, diluted to volume with reagent
water, are suggested.
Salinity
(PPt)
0
g
18
27
34
Volume (mL) of
low nutrient
seawater (7.1.7)
0
25
50
75
94
Volume (mL) of
secondary nitrate
solution (7.2.2)
6.0
6.0
6.0
6.0
6.0
Cone.,
mgN/L
0.060
0.060
0.060
0.060
0.060
7.2.5 Secondary Nitrite Solution — Dilute 1.0 mL of
stock nitrite solution (Section 7.1.6) to 100 mL with
reagent water. 1.0 mL of this solution = 0.001 mg N.
Refrigerate and prepare fresh weekly.
7.2.6 Working Nitrite Solution — Prepare one working
standard to act as a check on the reduction capability of
the cadmium column. Dilute 6.0 mL of (Section 7.2.5) to
100 mL to yield a concentration of 0.060 mg N/L. Store at
4°C and prepare fresh every 2 to 3 days.
8.0 Sample Collection, Preservation and
Storage
8.1 Sample Collection—Samples collected for nutri-
ent analyses from estuarine and coastal waters are
normally collected using one of two methods, hydrocast
or submersible pump systems. Filtration of the sample
353.4 - 4
Revision 1.3 November 1992
-------
through a 0.45-|im membrane or glass fiber filter imme-
diately after collection is recommended.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Nansen, Go-Flo or equivalent) which are at-
tached at fixed intervals to a hydro wire. These bottles are
sent through the water column open and are closed
either electronically or via a mechanical "messenger
when the bottles have reached the desired depth,
8.1.2 When a submersible pump system is used, a
weighted hose is sent to the desired depth in the water
column and water is pumped to the deck of the ship for
processing.
8.1.3 Another method used to collect surface samples
involves the use of a plastic bucket or large plastic bottle.
While not the most ideal method, it is commonly used in
citizen monitoring programs.
8.2 Sample Preservation — After collection and fil-
tration, samples should be analyzed as quickly as pos-
sible. Jf the samples will be analyzed within 24 h of
collection, then refrigeration at 4°C is acceptable.
8.3 Sample Storage — Long-term storage of frozen
samples should be in clearly labeled polyethylene bottles
or polystyrene cups compatible with the analytical
system's automatic sampler. If the samples cannot be
analyzed within 24 h, then freezing at -20°C for a maxi-
mum period of 2 months is acceptable.6-8
9.0 Quality Control
9.1 A formal quality control (QC) program is required.
The minimum requirements of this program consists of
an initial demonstration of laboratory capability (Section
9.2), and the continued analysis of laboratory reagent
blanks, laboratory duplicates, arid laboratory fortified
blanks with each set of samples as a continuing check on
performance.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (MDLs and linear
dynamic range) and laboratory performance (analysis of
QC samples) prior to analysis of samples using this
method.
9.2.2 MDLs should be established for all analytes, using
a low level estuarine water sample containing, or fortified
at, approximately 5 times the estimated detection limit.
To determine MDL values, analyze 7 replicate aliquots of
water which have been processed through the entire
analytical method. Perform all calculations defined in the
method and report concentration in the appropriate units.
Calculate the MDL as follows:
MDL = (t)(S)
where, S = Standard deviation of the
replicate analyses.
t = Student's t value for n-1 degrees
of freedom at the 99% confidence
limit; t = 3.143 for 6 degrees of free-
dom.
MDLs should be determined every six months or when-
ever a significant change in background or instrument
response occurs or when a new matrix is encountered.
9.2.3 Linear Dynamic Range (LDR) — The LDR should
be determined by analyzing a minimum of 5 calibration
standards ranging from 0.005 mg N/L to 0.30 mg N/L
across all sensitivity settings of the autoanalyzer. Nor-
malize responses by dividing the response by the sensi-
tivity setting multiplier. Perform the linear regression of
normalized response vs. concentration and obtain the
constants m and b, where m is the slope arid b is the y-
intercept. Incrementally analyze standards of higher con-
centration until the measured absorbance response, R,
of a standard no longer yields a calculated concentration,
Cc, that is ± 10% of the known concentration, C, where
Cc= (R - b)/m. That concentration defines the upper limit
of the LDR for your instrument. Should samples be
encountered that have a concentration that is >90% of
the upper limit of the LDR then these samples must be
diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) — A laboratory
should analyze at least one reagent blank (Section 3.4)
with each set of samples. Reagent blank data are used
to assess contamination from the laboratory environ-
ment. Should an analyte value in the reagent blank
exceed the MDL, then laboratory or reagent contamina-
tion should be suspected and corrective actions must be
taken before continuing analyses.
9.3.2 Laboratory Fortified Blank (LFB) — A laboratory
should analyze at least one fortified blank (Section 3.3)
with each set of samples. Calculate accuracy as percent
recovery. If the recovery of an analyte is not within 90-
110%, then the source of the problem should be identi-
fied and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 90-110% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20 to 30 analyses), optional control limits can
be developed from the percent mean recovery (x) and the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
353.4 - 5
Revision 1.3 November 1992
-------
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery — Laboratory
Fortified Sample Matrix
9.4.1 A laboratory should add aknown amount of analyte
to a minimum of 5% of the routine samples or one sample
per sample set, whichever is greater. The analyte con-
centration should be 2 to 4 times the ambient level and
should be at least four times greater than the MDL
9.4.2 Calculate the percent recovery of the analyte,
corrected for background concentrations measured in
the unfortified sample, and compare these values with
the values obtained from the LFB's. Percent recoveries
may be calculated using the following equation:
where, R » percent recovery
Ca s determined fortified sample concen-
tration (background + addition in mg
N/L)
C « Sample background concentration
(mg N/L)
S - Concentration in mg N/L added to the
environmental sample.
9.4.3 If the recovery of an analyte falls outside the
designated range of 85-115% but the laboratory perfor-
mance for that analyte is in control, the fortified sample
should be prepared again and reanalyzed. If the result is
the same after reanalysis, the recovery problem encoun-
tered with the fortified sample is judged to be matrix
related and the sample data should be flagged.
10.0 Calibration and Standardization
10.1 Calibration (Refer to Section 12.1).
10.2 Standardization (Refer to Sections 12.2, and
12.3).
11.0 Procedure
11.1 If samples are frozen, thaw the samples to room
temperature.
11.2 Set up the manifold as shown in Figure 2. The
tubing, flow rates, sample:wash ratio, sample rate, etc.
are based on the Technicon AutoAnaiyzer II System.
Specifications for other segmented flow analyzers vary,
so slight adjustments may be necessary.
11.3 Allow both colorimeter and recorder to warm up for
30 min. Obtain a steady baseline with reagent water
pumping through the system. Add reagents to the sample
stream and after the baseline has equilibrated; note the
rise (reagent baseline), and adjust the baseline.
For analysis of samples with a narrow salinity range, it is
advisable to use low nutrient seawater as wash water in
the sampler in place of reagent water. For samples with
a large salinity range, it is suggested that reagent water
and procedures in Sections 12.2 and 12.3 be employed.
11.4 A good sampling rate is approximately 40 samp-
les/h with a 9:1 sample to wash ratio.
11.5 Place CAL solutions (Section 7.2.3) and saline
standards (Section 7.2.4) (optional) and the working
nitrite standard (Section 7.2.6) in sampler in order of
decreasing concentration. Complete filling the sampler
tray with samples, laboratory reagent blanks, laboratory
fortified blanks, laboratory fortified matrices, and QC
samples.
11.6 Commence Analysis
11.6.1 If the peak height of the 0.060 mg N/L nitrate
standard prepared in reagent water (Section 7.2.3) is
<90% of the peak height of the 0.060 mg. N/L nitrite
standard (Section 7.2.6), halt analyses and prepare a
new cadmium reduction column (Section 7.2).
11.6.2 If a low concentration sample peak follows a high
concentration sample peak, a certain amount of carry-
over can be expected. It is recommended that if there is
not a clearly resolved low concentration peak, the sample
be reanalyzed at the end of the sample set.
11.6.3 Obtain a second set of peak heights for all
samples and standards with refractive reagent (Section
7.1.4) being pumped through the system in place of color
reagent (Section 7.1.2). The peak heights obtained from
these analyses must be subtracted from the peak heights
of samples analyzed with color reagent to eliminate
positive bias due to color of the water sample.
12.0 Data Analysis and Calculations
12.1 Concentrations of nitrite + nitrate are calculated
from the linear regression obtained from the standard
curve in which the concentrations of the standards are
entered as the independent variable and their .corre-
sponding peak heights are the dependent variable.
Note: If the standards are prepared in low nutrient sea-
water of same salinity as the samples, there is no need
to apply the correction factor for "salt error."
12.2 Refractive Index Correction for Estuarine/
Coastal Systems
12.2.1 The absorbance peak obtained by an automated
system for nitrate in a seawater sample (when compared
to a reagent water baseline) represents the sum of
absorbances from at least four sources: (1) the light
changes due to the differences in the index of refraction
of the seawater and reagent water; (2) reaction products
(e.g., precipitates) of BRIJ-35 and the seawater; (3) the
absorbance of colored substances dissolved in the
sample; and (4) reaction products of the nitrite and the
nitrate (reduced to nitrite by the cadmium column) in the
sample with the color reagent.9
353.4 - 6
Revision 1.3 November 1992
-------
72.2.2 Obtain a second set of peak heights for all
samples and standards with refractive reagent (Section
7.1.4) being pumped through the system in place of color
reagent (Section 7.1.2). All other reagents remain the
same. Peak heights for the refractive index correction
must be obtained at the same standard calibration setting
and on the same colorimeter as the corresponding
samples and standards.10
72.2.3 Subtract the refractive index peak heights from
the heights obtained for the nitrate determination.
72.2.4 When a large data set has been amassed in
which each sample's salinity is known, a regression for
the refractive index correction on a particular colorimeter
can be calculated. First analyze a set of nitrate standards
(Section 7.2.3) with color reagent (Section 7.1.2) and
obtain a linear regression from the standard curve (Sec-
tion 12.1). For each sample, the apparent nitrate concen-
tration due to refractive index is then calculated from its
peak height obtained with refractive reagent (Section
7.1.4) and the regression of nitrate standards obtained
with color reagent (Section 7.1.2) for each sample. Salin-
ity is entered as the independent variable and the appar-
ent nitrate due to refractive index in that colorimeter is
entered as the dependent variable. The resulting regres-
sion allows the operator to subtract an apparent nitrate
concentration when the salinity is known, as long as other
matrix effects (Sections 12.2.1-2) remain unchanged.
Thus, the operator would not be required to obtain
refractive index peak heights for all samples after a large
data set has been found to yield consistent apparent
nitrate concentrations due to salinity. An example of
typical results from one laboratory follows:
Salinity (ppt)
Apparent nitrate cone, due
to refractive index (mg N/L)
1
6
10
22
0.0001
0.0004
0.0007
0.0015
72.2.5 An example of a typical equation is:
mg N/L apparent NO3 = 0.000069 X Salinity (ppt)
where 0.000069 is the slope of the line
12.3 Correction for Salt Error in Estuarine/Coastal
Samples
12.3.1 When calculating concentrations of samples of
varying salinities from standards prepared in reagent
water, it is necessary to first correct for refractive index
errors (Section 12.2), then correct for the alteration in
color development due to the ionic strength of the samples
("salt error").
72.3.2 Plot the salinity of the saline standards as the
independent variable and the apparent concentration of
nitrate (mg N/L) from the peak height corrected for
refractive index (Section 12.2) calculated from the re-
gression of standards in reagent water (Sections 7.2.3
and 12.1) as the dependent variable for all 0.060 mg N/
L standards. The resulting regression equation allows
the operator to correct the concentrations of the samples
of known salinity for the color enhancement due to "Salt
Error". An example of typical results from one laboratory
follows:
'•
Salinity
(ppt)
0
9
18
27
34
Peak height of
0.060 mg N/L
standard after
correction for
refractive index
85
87
89
92
94
Uncorrected
mg N/L calculated
from regression
of standards
in reagent water
0.0600
0.0614
0.0628
0.0649
0.0663
72.3.3 An example of a typical equation to correct for
"salt error" is:
Corrected mg N/L =
y
Uncorrected
X 0.0600
(Salinity X 0.0001 87) + 0.060
where 0.0600 is the concentration of nitrate standard
(Section 7.2.4) present in each saline standard; salinity of
the sample is in parts per thousand; 0.0001 87 is the slope
of the regression equation (Section 12.3.1); and 0.060 is
the y-intercept of the regression equation (Section 1 2.3. 1 ).
1 2.4 Results of sample analyses should be reported in
mg N/L or in ng N/L.
mg N/L = ppm (parts per million)
Mg N/L = ppb (parts per billion)
13.0 Method Performance
13.1 Single-Analyst Precision
13. 1. 1 A single laboratory analyzed three samples col-
lected from the Chesapeake Bay, MD, and East Bay, FL.
Seven replicates of each sample were processed and
analyzed, randomly throughout a group of 75 samples
with salinities ranging from 3 to 36 ppt. The results were
as follows:
Sample
1
2
3
Salinity
(PPt)
36
18
3
Concentration
(mg N/L)
0.0165
0.0251
0.0040
% Relative
standard deviation
5.2
0.7
4
13.2 Pooled Precision and A ccuracy
No data are available at this time. In a collaborative
validation study of the method, precision and accuracy
decreased as salinity increased and concentration de-
creased.
353.4 - 7
Revision 1.3 November 1992
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14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the
next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society, Department of Government Rela-
tions and Science Policy, 115516th Street N.W., Wash-
ington D.C. 20036, (202)872-4477.
15.0 Waste Management
15.1 The U.S. Environmental Protection Agency re-
quires that laboratory waste management practices be
conducted consistent with all applicable rules and regu-
lations. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section 14.2.
16.0 References
1. Wood, E.D., F.A.G. Armstrong, and F.A. Richards.
1967. Determination of Nitrate in Seawater by
Cadmium-Copper Reduction to Nitrite. J. Mar. Biol.
Assoc.U,K.47:23.
2. 40 CFR, Part 136, Appendix B. Definition and
Procedure for the Determination of the Method
Detection Limit. Revision 1.11.
3. American Public Health Association, American Water
Works Association, Water Pollution Control
Federation, 1989. Standard Methods for the
Examination of Water and Wastewater. 17th Edition.
American Public Health Association, Washington,
DC 20005.
4. Methods for Chemical Analysis of Water and Wastes,
1983. Methods Development and Quality Assurance
Research Laboratory (EPA/600/4-79/020). U.S.
Environmental Protection Agency, Cincinnati, OH
45268.
5. Grasshoff, K., M. Ehrhardt and K. Kremling. 1983.
Methods of Seawater Analysis. Verlag Chemie,
Federal Republic of Germany. 419 pp.
6. MacDonald, R.W. and F.A. McLaughlin. 1982. The
Effect of Storage by Freezing on Dissolved Inorganic
Phosphate, Nitrate and Reactive Silicate for Samples
from Coastal and Estuarine Waters. Water Research,
76:95-104.
7. Thayer, G.W. 1970. Comparison of Two Storage
Methods for the Analysis of Nitrogen and Phosphorus
Fractions in Estuarine Water. Ches. Sci. 11:3,155-
158.
8. Salley, B.A., J.G. Bradshaw and B.J. Neilson. 1986.
Results of Comparative Studies of Preservation
Techniques for Nutrient Analysis on Water Samples.
Virginia Institute of Marine Science, Gloucester Point,
VA., 23062. 32pp.
9. Loder, T.C. and P.M. Gilbert. 1977. Blank and Salinity
Corrections for Automated Nutrient Analysis of
Estuarine and Seawaters. 7th Technicon International
Congress: 48-56, Tarrytown, N.Y.
10. Froelich, P.N. and M.E.Q, Pilson. 1978. Systematic
Absorbance Errors with Technicon AutoAnalyzer II
Colorimeters. Water Research 12: 599-603.
Additional Bibliography
1. Klingamann, E.D. and D.W. Nelson, 1976. Evaluation
of Methods for Preserving the Levels of Soluble
Inorganic Phosphorus and Nitrogen in Unfiltered
Water Samples. J. Environ. Qua/. 5:1 42-46.
353.4 - 8
Revision 1.3 November 1992
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Reductor column
Valve assembly
(For column dimensions see Section 7.2.1)
Figure 1. Reductor column valve assembly.
mL/min
. 22 turns
00000 3
sample WE
Debut
I
jsh receptacle
r
r
c
Deb
)admium
eduction
olumn
bier
Colorimeter
55 nm filters
Jbbler
Stums
JQCQQCL
2.0
0.32
0.6
0.32
1.2
' 0.32
0.32
1.0
Pump
Water (GRN/GRN)
Air (BLK/BLK)
Debubbler (WHT/WHT)
Air (BLK/BLK)
NH,CL (YEL/YEL)
SE
A
Sample (BLK/BLK)
ampler
0/hr.
9:1
Color reagent (BLK/BLK)
Waste from F/C (GRY/GRY)
F/C to waste
50x1.5 mm ID F/C
199-B021-01
Figure 2. Manifold configuration for nitrite + nitrate.
353.4-9
Revision 1.3 November 1992
-------
-------
Method 365.5
Determination of Orthophosphate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis
Adapted by
Carl F. Zimmermann
Carolyn W. Keefe
University of Maryland System
Center for Environmental and Estuarine Studies
Chesapeake Biological Laboratory
Solomons, MD 20688-0036
Revision 1.4
November 1992
Edited by
Elizabeth J. Arar
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
365.5-1
Revision 1.4 November 1992
-------
Method 365.5
Determination of Orthophosphate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for the determi-
nation of low-level Orthophosphate concentrations nor-
mally found in estuarine and/or coastal waters. It is based
upon the method of Murphy and Riley1 adapted for
automated segmented flow analysis2 in which the two
reagent solutions are added separately for greater re-
agent stability and facility of sample separation.
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Phosphate
14265-44-2
- •:
1.2 A statistically determined method detection limit
(MDL) of 0.0007 mg P/L has been determined by one
laboratory in 3 parts perthousand (ppt) saline water.3 The
method is linear to 0.39 mg P/L using a Technicon
AutoAnalyzer II system (Bran & Luebbe, Buffalo Grove,
IL).
1.3 Approximately 40 samples per hour can be ana-
lyzed.
1.4 This method should be used by analysts experi-
enced in the use of automated colorimetric analyses, and
familiar with matrix interferences and procedures fortheir
correction. A minimum of 6-months experience under
experienced supervision is recommended.
2.0 Summary of Method
2.1 An automated colorimetric method for the analysis
of low-level Orthophosphate concentrations is described.
Ammonium molybdate and antimony potassium tartrate
react in an acidic medium with dilute solutions of phos-
phate to form an antimony-phospho-molybdate com-
plex. This complex is reduced to an intensely blue-
colored complex by ascorbic acid. The color produced is
proportional to the phosphate concentration present in
the sample. Positive bias caused by differences in the
refractive index of seawater and reagent water is cor-
rected for prior to data reporting.
3.0 Definitions
3.1 Calibration Standard (CAL) — A solution pre-
pared from the stock standard solution that is used to
calibrate the instrument response with respect to analyte
concentration. One of the standards in the standard
curve.
3.2 Dissolved Analyte (DA) — The concentration of
analyte in an aqueous sample that will pass through a
0.45-nm membrane filter assembly prior to sample acidi-
fication or other processing.
3.3 Laboratory Fortified Blank (LFB)—An aliquot of
reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is ana-
lyzed exactly like a sample, and its purpose is to deter-
mine; whether method performance is within acceptable
control limits. This is basically a standard prepared in
reagent water that is analyzed as a sample.
3.4 Laboratory Fortified Sample Matrix (LFM)—An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory: The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background
concentrations.
3.5 Laboratory Reagent Blank (LRB)—An aliquot of
reagent water that is treated exactly as a sample includ-
ing exposure to all glassware, equipment, and reagents
that are used with other samples. The LRB is used to
determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or
apparatus.
3.6 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instru-
ment;response to an analyte is linear.
3.7 Method Detection Limit (MDL} — The minimum
concentration of an analyte that can be identified, mea-
sured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.8 Reagent Water (RW) — Type 1 reagent grade
water equal to or exceeding standards; established by
American Society of Testing Materials (ASTM). Reverse
osmosis systems or distilling units that produce 18
megohm water are two examples of acceptable water
sources.
3.9 Refractive Index (Rl)—The ratio of the velocity of
light in.a vacuum to that in a given medium. The relative
refractjve index is the ratio of the velocity of light in two
different media, such as sea or estuarine water versus
reagent water. The correction for this difference is re-
ferred to as the refractive index correction in this method.
3.10 Stock Standard Solution (SSS) — A concen-
trated solution of method analyte prepared in the labora-
365.5 - 2
Revision 1.4 November 1992
-------
tory using assayed reference compounds or purchased
from a reputable commercial source.
4.0 Interferences
4.1 Interferences caused by copper, arsenate and
silicate are minimal relative to the orthophosphate deter-
mination because of the extremely low concentrations
normally found in estuarine or coastal waters. High iron
concentrations can cause precipitation of and subse-
quent loss of phosphate from the dissolved phase. Hy-
drogen sulfide effects, such as occur in samples col-
lected from deep anoxic basins, can be treated by simple
dilution of the sample since high sulfide concentrations
are most often associated with high phosphate values.4
4.2 Sample turbidity is removed by filtration prior to
analysis.
4.3 Refractive Index interferences are corrected for
estuarine/coastal samples (Section 12.2).
5.0 Safety
5.1 Water samples collected from the estuarine and/or
ocean environment are generally not hazardous. How-
ever, the individual who collects samples should use
proper technique.
5.2 Good laboratory technique should be used when
preparing reagents. A lab coat, safety goggles, and
gloves should be worn when preparing the sulfuric acid
reagent.
6.0 Equipment and Supplies
6.1 Continuous Flow Automated Analytical Sys-
tem Consisting of:
6.1.1 Sampler.
6.1.2 Manifold or Analytical Cartridge equipped with
37°C heating bath.
6.1.3 Proportioning pump.
6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular
flow cell and a 880 nm filter.
6.1.5 Phototube that can be used for 600-900 nm range.
6.1.6 Strip chart recorder or computer based data sys-
tem.
6.2 Phosphate-Free Glassware and Polyethylene
Bottles
6.2.1 All labware used in the determination must be low
in residual phosphate to avoid sample or reagent con-
tamination. Washing with 10% HCI (v/v) and thoroughly
rinsing with distilled, deionized water was found to be
effective.
6.2.2 Membrane or glass fiber filters, 0.45 nm nominal
pore size.
7.0 Reagents and Standards
7.1 Stock Reagent Solutions
7.1.1 Ammonium Molybdate Solution (40 g/L) — Dis-
solve 20.0 g of ammonium molybdate tetrahydrate
((NH )6Mo7O24'4H2O, CASRN 12027-67-7) in approxi-
mately 400 ml of reagent water and dilute to 500 ml.
Store in a plastic bottle out of direct sunlight. This reagent
is stable for approximately three months.
7.1.2 Antimony Potassium Tartrate Solution (3.0 g/L)—
Dissolve 0.3 g of antimony potassium tartrate
[(K(SbO)C4H406-1/2 H20, CASRN 11071-15-1] in ap-
proximately 90 mL of reagent water and dilute to 100 ml_.
This reagent is stable for approximately three months.
7.1.3 Ascorbic Acid Solution (18.0 g/L)—Dissolve 18.0
g of ascorbic acid (C6H6O6, CASRN 50-81-7) in approxi-
mately 800 mL of reagent water and dilute to 1 L.
Dispense approximately 75 mL into clean polyethylene
bottles and freeze. The stability of the frozen ascorbic
acid is approximately three months. Thaw overnight in
the refrigerator before use. The stability of the thawed,
refrigerated reagent is less than 10 days.
7.7.4 Sodium Lauryl Sulfate Solution (30.0 g/L) — So-
dium dodecyl sulfate (CH3(CH2)11OSO3Na, CASRN 151-
21-3). Dissolve 3.0 g of sodium lauryl sulfate (SLS) in
approximately 80 mL of reagent water and dilute to 100
mL. This solution is the wetting agent and its stability is
approximately three weeks.
7.7.5 Sulfuric Acid Solution (4.9 N)—Slowlyadd 136mL
of concentrated sulfuric acid (H2SO4, CASRN 7664-93-9)
to approximately 800 mL of reagent water. After the
solution is cooled, dilute to 1 L with reagent water.
7.1.6 Stock Phosphorus Solution— Dissolve 0.439 g of
pre-dried (105°C for 1 h) monobasic potassium phos-
phate (KH PO4, CASRN 7778-77-0) in reagent water and
dilute to 1000 mL (1.0 mL = 0.100 mg P.) The stability of
this stock standard is approximately three months when
kept refrigerated.
7.1.7 Low Nutrient Seawater—Obtain natural low nutri-
ent seawater (36 ppt salinity; <0.0003 mg P/L) or dissolve
31 g analytical reagent grade sodium chloride, (NaCI,
CASRN 7647-14-5); 10 g analytical grade magnesium
sulfate, (MgSO4, CASRN 10034-99-8); and 0.05 g ana-
lytical reagent grade sodium bicarbonate, (NaHCO ,
CASRN 144-55-8), in 1 L of reagent water.
7.2 Working Reagents
7.2.1 Reagent A — Mix the following reagents in the
following proportions for 142 mL of Reagent A: 100 mL of
4.9N H2SO4 (Section 7.1.5), 30 mL of ammonium molyb-
date solution (Section 7.1.1), 10 mL of antimony potas-
sium tartrate solution (Section 7.1.2), and 2.0 mL of SLS
solution (Section 7.1.4). Prepare fresh daily.
7.2.2 ReagentB—Add approximately 0.5 mLof the SLS
solution (Section 7.1.4) to the 75 mL of ascorbic acid
365.5 - 3
Revision 1.4 November 1992
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solution (Section 7.1.3). Stability is approximately 10
days when kept refrigerated.
7.2.3 Refractive Reagent A—Add 50 mLof 4.9 N H2SO
(Section 7.1.5) to 20 mL of reagent water. Add 1 ml_ of
SLS (Section 7.1.4) to this solution. Prepare fresh every
few days.
7.2.4 Secondary Phosphorus Solution—Take 1.0 mLof
Stock Phosphorus Solution (Section 7.1.6) and dilute to
100 mL with reagent water. (1.0 mL = 0.0010 mg P.)
Refrigerate and prepare fresh every 10 days.
7.2.5 Prepare a series of standards by diluting suitable
volumes of standard solutions (Section 7.2.4) to 100 mL
with reagentwater. Prepare these standards daily. When
working with samples of known salinity, it is recom-
mended that the standard curve concentrations be pre-
pared in low-level natural seawater (Section 7.1.7) di-
luted to match the salinity of the samples. Doing so
obviates the need to perform the refractive index correc-
tion outlined in Section 12.2. When analyzing samples of
varying salinities, it is recommended that the standard
curve be prepared in reagent water and refractive index
corrections be made to the sample concentrations (Sec-
tion 12.2). The following dilutions are suggested.
mL of Secondary
Phosphorus Solution (7.2.4)
0.1
0.2
0.5
1.0
2.0
4.0
5.0
Cone.
mgP/L
0.0010
0.0020
0.0050
0.0100
0.0200
0.0400
0.0500
8.0 Sample Collection, Preservation and
Storage
8.1 Sample Collection—Samples collected for nutri-
ent analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems. Filtration of the sample
through a 0.45-jim membrane or glass fiber filter imme-
diately after collection is required.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Nansen, Go-Flo or equivalent) that are attached
at fixed intervals to a hydro wire. These bottles are sent
through the water column open and are closed either
electronically or via a mechanical "messenger" when the
bottles have reached the desired depth.
8.1.2 When a submersible pump system is used, a
weighted hose is sent to the desired depth in the water
column and water is pumped from that depth to the deck
of the ship for processing.
8.1.3 Another method used to collect surface samples
involves the use of a plastic bucket or large plastic bottle.
While not the most ideal method, it is commonly used in
citizen monitoring programs.
•Or
8.2 Sample Preservation — After collection and
filtration, samples should be analyzed as quickly as
possible. If the samples are to be analyzed within 24 h of
collection, then refrigeration at 4°C is acceptable.
8.3 Sample Storage — Long-term storage of frozen
samples should be in clearly labelled polyethylene bottles
or polystyrene cups compatible with the analytical
system's automatic sampler (Section 6.1.1). If samples
cannot be analyzed within 24 h, then freezing at -20°C for
a maximum period of two months is acceptable.5"8
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of. an
initial demonstration of laboratory capability, the contin-
ued analysis of LRBs, laboratory duplicates, and LFBs as
a continuing check on performance.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (MDLs and linear
dynamic range) and laboratory performance (analysis of
QC samples) prior to analyses of samples using this
method.
9.2.2 MDLs should be established using a low-level
estuarine water sample fortified to approximately five
times the estimated detection limit.3 To determine MDL
values, analyze seven replicate aliquots of water and
process through the entire analytical method. Perform all
calculations defined in the method and report the con-
centration values in the appropriate units. Calculate the
MDL as follows:
MDL = (t)(S)
where, S = the standard deviation of the replicate
analyses.
t *= the Student's t value for n-1 degrees of
freedom at the 99% confidence limit, t =
3.143 for six degrees of freedom.
MDLs should be determined every six months or when-
ever a significant change in background or instrument
response occurs or when a new matrix: is encountered.
9.2.3 Linear Dynamic Range (LDR) — The LDR should
be determined by analyzing a minimum of five calibration
standards ranging in concentration from 0.001 mgP/Lto
0.20. mg P/L across all sensitivity settings of the
autoanalyzer. Normalize responses by dividing the re-
sponse by the sensitivity setting multiplier. Perform the
linear regression of normalized response vs. concentra-
365.5 - 4
Revision 1.4 November 1992
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tion and obtain the constants m and b, where m is the
slope and b is the y-intercept. Incrementally analyze
standards of higher concentration until the measured
absorbance response, R, of a standard no longer yields
a calculated concentration C^ that is ± 10% of the known
concentration, C, where CC=(R- b)/m. That concentra-
tion defines the upper limit of the LDR for your instrument.
. Should samples be encountered that have a concentra-
tion that is >90% of the upper limit of the LDR, then these
samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) — A laboratory
should analyze at least one LRB (Section. 3.5) with each
set of samples. LRB data are used to assess contamina-
tion from the laboratory environment. Should an analyte
value in the LRB exceed the MDL, then laboratory or
reagent contamination should be suspected. When LRB
values constitute 10% or more of the analyte level deter-
mined for a sample, fresh samples or field duplicates of
the samples must be prepared and analyzed again after
the source of contamination has been corrected and
acceptable LRB values have been obtained.
9.3.2 Laboratory Fortified Blank (LFB) — A laboratory
should analyze at least one LFB (Section 3.3) with each
batch of samples. Calculate accuracy as percent recov-
ery. If the recovery of the analyte falls outside the re-
quired control limits of 90 -110%, the analyte is judged
put of control and the source of the problem should be
identified and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB data to assess labo-
ratory performance against the required control limits of
90 -110% (Section 9.3.2). When sufficient internal per-
formance data become available (usually a minimum of
20 to 30 analyses), optional control limits can be devel-
oped from the percent mean recovery (x) and the stan-
dard deviation (S) of the mean recovery. These data can
be used to establish the upper and lower control limits as
follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90 -110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also, the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery - Laboratory
Fortified Sample Matrix
9.4.1 A laboratory should add a known amount of analyte
to a minimum of 5% of the routine samples or one sample
per sample set, whichever is greater. The analyte con-
centration should be two to four times the ambient
concentration and should be at least four times the MDL.
9.4.2 Calculate the percent recovery of the analyte,
corrected for background concentrations measured in
the unfortified sample, and compare these values with
the values obtained from the LFBs.
Percent recoveries may be calculated using the following
equation:
R=(cs-c)x100
S
where, R = percent recovery
Cs= measured fortified sample concentration
(background + concentrated addition in
mg P/L)
C = sample background concentration
(mg P/L)
S = concentration in mg P/L added to the
environmental sample.
9.4.3 If the recovery of the analyte falls outside the
designated range of 90-110% recovery, but the labora-
tory performance for that analyte is in control, the fortified
sample should be prepared again and analyzed. If the
result is the same after reanalysis, the recovery problem
encountered with the fortified sample is judged to be
matrix related, not system related.
10.0 Calibration and Standardization
10.1 Calibration (Refer to Sections 11.5 and 12.0).
10.2 Standardization (Refer to Section 12.2).
11.0 Procedure
11.1 If samples are frozen, thaw the samples to room
temperature.
11.2 Set-up manifold as shown in Figure 1. The tubing,
flow rates, sample:wash ratio, sample rate, etc., are
based on a Technicon AutoAnalyzer II system. Specifi-
cations for similar segmented flow analyzers vary, so
slight adjustments may be necessary.
11.3 Allow both colorimeter and recorder to warm up for
30 min. Obtain a steady baseline with reagent water
pumping through the system, add reagents to the sample
stream and after the reagent water baseline has equili-
brated, note that rise (reagent water baseline), and adjust
baseline.
For analysis of samples with a narrow salinity range, it is
advisable to use low nutrient seawater matched to sample
salinity as wash water in the sampler in place of reagent
water. For samples with a large salinity range, it is
suggested that reagent wash water and procedure (Sec-
tion 12.2) be employed.
365.5 - 5
Revision 1.4 November 1992
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11.4 Agoodsampling rate is approximately 40 samples/
h with a 9:1, sample:wash ratio.
1,1.5 Place standards (Section 7.2.5) in sampler in order
of decreasing concentration. Complete filling the sam-
pler tray with samples, LRBs, LFBs, and LFMs.
11.6 Commence analysis.
11.7 Obtain a second set of peak heights for all samples
and standards with Refractive Reagent A (Section 7.2.3)
being pumped through the system in place of Reagent A
(Section 7.2.1). This "apparent" concentration due to
coloration of the water should be subtracted from con-
centrations obtained with Reagent A pumping through
the system.
12.0 Data Analysis and Calculations
12.1 Concentrations of orthophosphate are calculated
from the linear regression obtained from the standard
curve in which the concentrations of the calibration
standards are entered as the independent variable and
the corresponding peak height is the dependent variable.
12.2 Refractive Index Correction for Estuarine/
Coastal Systems
12.2.1 Obtain a second set of peak heights for all
samples and standards with Refractive Reagent A (Sec-
tion 7.2.3) being pumped through the system in place of
Reagent A (Section 7.2.1). Reagent B (Section 7.2.2)
remains the same and is also pumped through the
system. Peak heights for the refractive index correction
must be obtained at the same Standard Calibration
Setting and on the same colorimeter as the correspond-
ing samples and standards.9
12.2,2 Subtract the refractive index peak heights from
the heights obtained for the orthophosphate determina-
tion. Calculate the regression equation using the cor-
rected standard peak heights. Calculate the concentra-
tion of samples from the regression equation using the
corrected sample peak heights.
12.2.3 When a large data set has been amassed in
which each sample's salinity is known, a regression for
the refractive index correction on a particular colorimeter
can be calculated. For each sample, the apparent or-
thophosphate concentration due to refractive index is
calculated from its peak height obtained with Refractive
Reagent A (Section 7.2.3) and Reagent B (Section 7.2.2)
and the regression of orthophosphate standards ob-
tained with orthophosphate Reagent A (Section 7.2.1)
and Reagent B (Section 7.2.2) for each sample. Its
salinity is entered as the independent variable and its
apparent orthophosphate concentration due to its refrac-
tive index in that colorimeter is entered as the dependent
variable. The resulting regression equation allows the
operatorto subtract an apparent orthophosphate concen-
tration when the salinity is known, as long as other matrix
effects are not present. Thus, the operator would not be
required to obtain the refractive index peak heights for all
samples after a large data set has been found to yield
consistent apparent orthophosphate concentrations due
to salinity. An example follows:
Apparent orthophosphate
cone, due to refractive
Salinity (ppt)
1,
5
10
20
index (mg P/L)
0.0002
0.0006
0.0009
0.0017
12.2.4 An example of a typical equation is:
mg P/L apparent PO43' = 0.000087 X Salinity (ppt)
where, 0.000087 is the slope of the line.
12.3 Results should be reported in mg PO43'- P/L or ^g
P043-- P/L.
mg PO43" - P/L = ppm (parts per million)
ng PO43' - P/L = ppb (parts per billion)
13.0 Method Performance
13.1 Single Analyst Precision — A single laboratory
analyzed three samples collected from Chesapeake
Bay, Maryland, and East Bay, Florida. Seven replicates
of each sample were processed and analyzed randomly
throughout a group of 75 samples with salinities ranging
from 3 to 36 ppt. The results were as follows:
Sample
1
2
3
Salinity
(ppt)
36
18
3
Concentration
(mg P/L)
0.0040
0.0024
0.0007
Percent Relative
Standard Deviation
6.5
10
24
13.2 Pooled Precision and Accuracy
13.2.1 This method was tested by nine laboratories
using reagent water, high salinity seawater from the
Sargasso Sea (36 ppt) and three different salinity waters
from Chesapeake Bay, Maryland (8.3 ppt, 12.6 ppt, and
19.5 ppt). The reagent water and the Sargasso Sea water
were fortified at four Youden pair concentrations ranging
from0.0012mg P/Lto0.1000mg P/L10 The Chesapeake
Bay waters were fortified at three Youden pair concentra-
tions ranging from 0.0050 mg P/L to 0.0959 mg P/L with
the highest salinity waters containing the lowest Youden
pair and the lowest salinity waters containing the highest
Youden pair. Analysis of variance (ANOVA) at the 95%
365.5 - 6
Revision 1.4 November 1992
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confidence level found no statistical differences between
water types indicating that the refractive index correction
for different salinity waters is an acceptable procedure.
Table 1 contains the linear equations that describe the
single-analyst standard deviation, overall standard de-
viation, and mean recovery of orthophosphate from each
water type.
13.2.2 Pooled Method Detection Limit (p-MDL) — The
p-MDL is derived from the pooled precision obtained by
single laboratories for the lowest analyte concentration
level used in the multilaboratory study. The p-MDLs
using reagent water and Sargasso Sea water were
0.00128 and 0.00093 mg P/L, respectively.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity ortoxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the
next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 115516th Street N.W., Wash-
ington, D.C. 20036, (202)872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions., The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
pus waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management, consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. Murphy, J. and J.P. Riley. 1962. A Modified Single
Solution Method for the Determination of Phos-
phate in Natural Waters. Analytica Chim. Acta 27,
31-36.0.
2. Technicon Industrial Systems. 1973. Orthophos-
phate in Water and Seawater. Industrial Method
155-71W. Technicon Industrial Systems,
Tarrytown, NY 10591.
3. 40CFR, 136 Appendix B. Definition and
Procedure for the Determination of the Method
Detection Limit. Revision 1.11.
4. Grasshoff, K., M. Ehrhardt, and K. Kremling. 1983.
Methods of Seawater Analysis. Verlag Chemie,
Federal Republic of Germany, 419 pages.
5. Klingamann, E.D. and D.W. Nelson. 1976. Evalua-
tion of Methods for Preserving the Levels of
Soluble Inorganic Phosphorus and Nitrogen in
Unfiltered Water Samples. J. Environ. Qua/., 5:1,
42-46.
6. MacDonald, R.W. and F.A. McLaughlin. 1982.
The Effect of Storage by Freezing on Dissolved
Inorganic Phosphate, Nitrate, and Reactive Sili-
cate for Samples from Coastal and Estuarine Wa-
ters. Water Research, 16:95-104.
7. Thayer, G.W. 1979. Comparison of Two Storage
Methods for the Analysis of Nitrogen and Phospho-
rus Fractions in Estuarine Water. Ches. Sci., 11:3,
155-158.
8. Salley, B.A., J.G, Bradshaw, and B.J. Neilson .
1986. Results of Comparative Studies of Preser-
vation Techniques for Nutrient Analysis on Water
Samples. VIMS, Gloucester Point, VA 23062. 32
PP-
9. Froelich, P.N. and M.E.Q. Pilson. 1978. System-
atic Absorbance Errors with Technicon
AutoAnalyzer II Colorimeters. Water Research 12:
599-603.
10. Edgell, K.W., E.J. Erb, and J.E. Longbottom, "De-
termination of Orthophosphate in Estuarine and
Coastal Waters by Automated Colorimetric Analy-
sis: Collaborative Study," submitted in November
1992 for publication in Marine Chemistry.
365.5 - 7
Revision 1.4 November 1992
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Single-Analyst Precision, Overall Precision and Recovery from
Muftilaboratory Study _^__
Reagent Water
(0.0012-O.IOOmg P/L)
Mean Recovery
Overall Standard Deviation
Single-Analyst Standard Deviation
X =0.9720-0.000018
SR = 0.033X + 0.000505
Sr =0.002X +0.000448
Sargasso Sea Water
(0.0012- 0.100 mgP/L)
Mean Recovery
Overall Standard Deviation
Single-Analyst Standard Deviation
Chesapeake Bay Water
(0.005-0.100 mgP/L)
Mean Recovery
Overall Standard Deviation
Single-Analyst Standard Deviation
X =0.9710-0.000002
SR = 0.021 X + 0.000550^
Sr =0.01 OX+ 0.000249
X =1.0190-0.000871
So «= 0.066X + 0.000068
.Sr =0.030X + 0.000165
C True value or spike concentration, mg P/L.
X Mean concentration found, mg P/L, exclusive of outliers.
So Overall standard deviation, mg P/L, exclusive of outliers.
S" Single-analyst standard deviation, mg P/L, exclusive of outliers.
To Sample Wash Re<
37°C S Turns
Heating
Bath
oonm
Debubbler
I
Colorimeter
880 nm Filters
5 Turns
nmnn
F/C to Waste
mL/min
2.0
0.32
1.2
0.23
0.10
0.42
Pump
Water (GRN/GRN)
Air,(BIWBIk)
Sample (YEL/YEL)
Sampler
40/hr
9:1
Reagent A (ORN/WHT)
Reagent B (ORN/GRN)
Waste from F/C (ORN/ORN)
50x1.5 mm ID F/C
199-B021-04 Phototube
Figure 1. Manifold Configuration for Orthophosphate.
365.5 - 8
Revision 1.4 November 1992
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Method 440.0
Determination of Carbon and Nitrogen in Sediments and Particulates of
Estuarine/Coastal Waters Using Elemental Analysis
Adapted by
Carl F. Zimmermann
Carolyn W. Keefe
University of Maryland System
Center for Environmental Estuarine Studies
Chesapeake Biological Laboratory
Solomns, MD 20688-0038
and
Jerry Bashe
Technology Applications, Inc.
26 W. Martin Luther King Drive
Cincinnati, OH 45219
Revision 1.4
November 1992
Work Assignment Manager
Elizabeth J. Arar
Inorganic Chemistry Branch
Chemistry Research Division
Environmental Monitoring Systems Laboratory
Office of Research and Devlopment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
440.0 -1
Revision 1.4 November 1992
-------
Method 440.0
Determination of Carbon and Nitrogen in Sediments and Participates
of Estuarine/Coastal Waters Using Elemental Analysis
1.0 Scope and Application
1.1 Elemental analysis is used to determine particu-
late carbon (PC) and paniculate nitrogen (PN) inrestua-
rine and coastal waters and sediment. The method
measures the total carbon and nitrogen irrespective of
source (inorganic or organic).
Analylo
Chemical Abstracts Service
Registry Numbers (CASRN)
Carbon
Nitrogen
7440-44-0
1333-74-0
1.2 The need to qualitatively or quantitatively deter-
mine the paniculate organic fraction from the total par-
ticulate carbon and nitrogen depends on the data-quality
objectives of the study. Section 11.4 outlines procedures
to ascertain the organic/inorganic paniculate ratio. The
method performance presented in the method was ob-
tained on paniculate samples with greater than 80%
organic content. Performance on samples with a greater
proportion of paniculate inorganic versus organic carbon
and nitrogen has not been investigated.
1.3 Method detection limits (MDLs)1 of 10.5 ng/L and
62.3 ng/Lfor PN and PC, respectively, were obtained for
a 200-mL sample volume. Sediment MDLs of PN and PC
are 84 mg/kg and 1300 mg/kg, respectively, for a sedi-
ment sample weight of 10.00 mg. The method has been
determined to be linear to 4800 jig of C and 700 ng of N
in a sample.
1.4 This method should be used by analysts experi-
encedinthetheoryandapplication of elemental analysis.
A minimum of 6 months experience with an elemental
analyzer is recommended.
1.5 Users of the method data should set the data-
quality objectives prior to analysis. Users of the method
must document and have on file the required initial
demonstration of performance data described in Section
9.2 prior to using the method for analysis.
2.0 Summary of Method
2.1 An accurately measured amount of paniculate
matter from an estuarine water sample or an accurately
weighed dried sediment sample is combusted at 975°C
using an elemental analyzer. The combustion products
are passed over a copper reduction tube to convert the
oxides of N into molecular N. Carbon dioxide, water
vapor and N are homogeneously mixed at a known
volume, temperature and pressure. The mixture is re-
leased to a series of thermal conductivity detectors/traps,
measuring in turn by difference, hydrogen (as water
vapor), C (as carbon dioxide) and N (as N2). Inorganic
and organic C may be determined by two methods which
are also presented.
3.0 Definitions
3.1 Sediment Sample — A fluvial,, sand, or humic
sample matrix exposed to a marine, brackish or fresh
water environment. It is limited to that portion which may
be passed through a number 10 sieve or a 2-mm mesh
sieve.
3.2 Material Safety Data Sheet (MSDS) — Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
3.3 Instrument Detection Limit (IDl) — The mini-
mum quantity of analyte or the concentration equivalent
which gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance
line, etc.
3.4 Method Detection Limit (MDL) — The minimum,
concentration of an analyte that can be identified, mea-
sured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.5 Linear Dynamic Range (LDP) — The absolute
quantity overwhichthe instrument response to an analyte
is linear.
3.6 Calibration Standard (CAL) — An accurately
weighed amount of a certified chemical used to calibrate
the instrument response with respect to analyte mass.
3.7 Conditioner — A standard chemical which is not
necessarily accurately weighed that is used to coat the
surfaces of the instrument with the analytes (water
vapor, carbon dioxide, and nitrogen).
3.8 External Standards (ES) — A pure analyte(s)
that is measured in an experiment separate from the
experiment used to measure the analyte(s) in the sample.
The signal observed for a known quantity of the pure
external standard(s) is used to calibrate the instrument
response for the corresponding analyte(s). The instru-
Revlslon 1.4 November 1992
440.0 - 2
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ment response is used to calculate the concentrations of
the analyte(s) in the sample.
3.9 Response Factor (RF) — The ratio of the re-
sponse of the instrument to a known amount of analyte.
3.10 Laboratory Reagent Blank (LRB) — A blank
matrix (i.e., a precombusted filter or sediment capsule)
that is treated exactly as a sample including exposure to
all glassware, equipment, solvents, and reagents that
are used with other samples. The LRB is used to deter-
mine if method analytes or other interferences are present
in the laboratory environment, the reagents, or the appa-
ratus.
3.11 Field Reagent Blank (FRB) — An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sam-
pling site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.12 Laboratory Duplicates (LD1 and LD2) — Two
aliquots of the same sample taken in the laboratory and
analyzed separately with identical procedures. Analyses
of LD1 and LD2 indicate precision associated with labo-
ratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.13 Field Duplicates (FD1 andFD2) —Two separate
samples cojlected at the same time and place under
identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of
FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well
as with laboratory procedures.
3.14 Laboratory Fortified Blank (LFB) — An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the method is in
control, and whether the laboratory is capable of making
accurate and precise measurements.
3.15 Laboratory Fortified Sample Matrix (LFM) —
An aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background
concentrations.
3.16 Standard Reference Material (SRM) — Material
which has been certified for specific analytes by a variety
of analytical techniques and/or by numerous laboratories
using similar analytical techniques. These may consist of
pure chemicals, buffers or compositional standards. These
materials are used as an indication of the accuracy of a
specific analytical technique.
3.17 Quality Control Sample (QCS) — A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
4.0 Interferences
4.1 There are no known interferences for estuarine/
coastal water or sediment samples. The presence of C
and N compounds on laboratory surfaces, on fingers, in
detergents and in dust necessitates the'utilization of
careful techniques (i.e., the use of forceps and gloves) to
avoid contamination in every portion of this procedure.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible
for maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals speci-
fied in this method.2-5 A reference file of material safety
data sheets (MSDS) should also be made available to all
personnel involved in the chemical analysis.
5.2 The acidification of samples containing reactive
materials may result in the release of toxic gases, such as
cyanides or sulfides. Acidification of samples should be
done in a fume hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 Although most instruments are adequately
shielded, it should be remembered that the oven tem-
peratures are extremely high and that care should be
taken when working near the instrument to prevent
possible burns.
5.5 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Apparatus and Equipment
6.1 Elemental Analyzer
6.1.1 An elemental analyzer capable of maintaining a
combustion temperature of 975°C and analyzing particu-
late samples and sediment samples for elemental C and
N. The Leeman Labs Model 240 XA Elemental Analyzer
was used to produce the data presented in this method.
440.0 - 3
Revision 1.4 November 1992
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6.2 A gravity convection drying oven. Capable of main-
taining 103-105°C for extended periods of time.
6.3 Muffle furnace. Capable of maintaining 875°C +
15°C.
6.4 Ultra-micro balance. Capable of accurately weigh-
ing to 0.1 ng. Desiccant should be kept in the weighing
chamber to prevent hygroscopic effects.
6.5 Vacuum pump orsource capable of maintaining up
to 10 in. Hg of vacuum.
6.6 Mortar and pestle.
6.7 Desiccator, glass.
6.8 Freezer, capable of maintaining -20°C + 5°C.
6.9 47-mmor25-mmvacuumfilterapparatusmadeup
of a glass filter tower, fritted glass disk base and 2-L
vacuum flask.
6.10 13-mmSwinlok filter holder.
6.11 Teflon-tipped, flat blade forceps.
6.12 Labware— All reusable labware (glass, quartz,
polyethylene, PTFE, FEP, etc.) should be sufficiently
clean for the task objectives. Several procedures found
to provide clean labware include washing with a deter-
gent solution, rinsing with tap water, soaking for 4 h or
more in 20% (v/v) HCI, rinsing with reagent water and
storing clean. All traces of organic material must be
removed to prevent C-N contamination.
6.12.1 Glassware — Volumetric flasks, graduated cyl-
inders, vials and beakers.
6.12.2 Vacuum filter flasks — 250 mL and 2 L, glass.
6.12.3 Funnel, 6.4 mm i.d., polyethylene.
6.12.4 Syringes, 60-mL, glass.
7.0 Reagents and Standards
7.1 Reagents may contain elemental impurities which
affect analytical data. High-purity reagents that conform
to the American Chemical Society specifications6 should
be used whenever possible. If the purity of a reagent is in
question, analyze for contamination. The acid used for
this method must be of reagent grade purity or equiva-
lent. A suitable acid is available from a number of manu-
facturers.
7.2 Hydrochloric acid, concentrated (sp. gr. 1.19)-HCI.
7.3 Acetanilide, 99.9% + purity, C8H19NO (CASRN
103-84-4).
7.4 Blanks — Three blanks are used for the analysis.
Two blanks are instrument related. The instrument zero
response (ZN) is the background response of the instru-
ment without sample holding devices such as capsules
and sleeves. The instrument blank response (BN) is the
response of the instrument when the sample capsule,
sleeve and ladle are inserted for analysis without stan-
dard or sample. The BN is also the laboratory reagent
blank (LRB) for sediment samples. The LRB for water
samples includes the capsule, sleeve, ladle and a
precombusted filter without standard or sample. These
blanks are subtracted from the uncorrected instrument
response used to calculate concentration in Sections
12.3 and 12.4.
7.4.1 Laboratory fortified blank (LFB) — The third blank
is the laboratory fortified blank. For sediment analysis,
add a weighed amount of acetanilide in an aluminum
capsule and analyze for PC and PN (Section 9.3.2). For
aqueous samples, place a weighed amount of acetanil-
ide on a glass fiber filter the same size as used for the
sample filtration. Analyze the fortified filter for PC and PN
(Section 9.3.2)
7.5 Quality Control Sample (QCS)—Forthis method,
the QCS can be, any assayed and certified sediment or
particulate sample which is obtained from an external
source. The Canadian Reference Material, BCSS-1, is
just such a material and was used in this capacity for the
data presented in this method. The percent PC has been
certified in this material but percent PN has not.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection — Samples collected
for PC and PN analyses from estuarine/coastal waters
are normally collected from a ship using one of two
methods; hydrocast or submersible pump systems. Fol-
low the recommended sampling protocols associated
with the method used. Whenever possible, immediately
filter the samples as described in Section 11.1.1. Store
the filtered sample pads by freezing at -20°C or storing in
a desiccator after drying at 103-105° C for 24 hr. No
significant difference has been noted in comparing the
two storage procedures for a time period of up to 100
days. If storage of the water sample is necessary, place
the sample into a clean amber bottle and store at 4°C until
filtration is done.
8.1.1 The volume of water sample collected will vary with
the type of sample being analyzed. Table 1 provides a
guide for a number of matrices of interest. If the matrix
cannot be classified by this guide, collect 2 x 1L of water
from each site. A minimum filtration volume of 200 mL is
recommended.
8.2 SedimentSample Collection — Estuarine/coastal
sediment samples are collected with benthic samplers.
The type of sampler used will depend on the type of
sample needed by the data-quality objectives.7 Store the
wet sediment in a clean jar and freeze at -20°C until ready
for analysis.
8.2.1 The amount of sediment collected will depend on
the sample matrix and the elemental analyzer used. A
minimum of 10 g is recommended.
440.0 - 4
Revision 1.4 November 1992
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9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability and the
continued analysis of laboratory reagent blanks, labora-
tory duplicates, field duplicates and calibration standards
analyzed as samples as a continuing check on perfor-
mance. The laboratory is required to maintain perfor-
mance records that define the quality of data thus gener-
ated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (MDLs, linear dy-
namic range) and laboratory performance (analysis of
QC samples) prior to the analyses conducted by this
method.
9.2.2 Linear dynamic range (LDP) — The upper limit of
the LDR must be established by determining the signal
responses from a minimum of three different concentra-
tion standards across the range, one of which is close to
the upper limit of the LDR. Determined LDRs must be
documented and kept on file. The LDR which may be
used for the analysis of samples should be judged by the
analyst from the resulting data. The upper LDR limit
should be an observed signal no more than 10% below
the level extrapolated from the lower standards. Deter-
mined sample analyte concentrations that are 90% and
above the upper LDR must be reduced in mass and
reanalyzed. New LDRs should be determined whenever
there is a significant change in instrument response and
for those analytes that periodically approach the upper
LDR limit, every 6 months or whenever there is a change
in instrument analytical hardware or operating condi-
tions.
9.2.3 Quality control sample (QCS) (Section 7.5) —
When beginning the use of this method, on a quarterly
basis or as required to meet data quality needs, verify the
calibration standards and acceptable instrument perfor-
mance with the analyses of a QCS. If the determined
concentrations are not within ± 5% of the stated values,
performance of the determinative step of the method is
unacceptable. The source of the problem must be iden-
tified and corrected before either proceeding with the
initial determination of MDLs or continuing with analyses.
9.2.4 Method detection limits (MDLs) — MDLs should
be established for PC and PN using a low level estuarine
water sample, typically three to five times higher than the
estimated MDL. The same procedure should be followed
for sediments. To determine MDL values, analyze seven
replicate aliquots of water or sediment and process
through the entire analytical procedure (Section 11).
These replicates should be randomly distributed through-
out a group of typical analyses. Perform all calculations
defined in the method (Section 12) and report the con-
centration values in the appropriate units. Calculate the
MDL as follows:1
MDL = (t) X (S)
where, S = Standard deviation of the repli-
cate analyses.
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees of
freedom.
MDLs should be determined whenever a significant
change in instrumental response, change of operator, or
a new matrix is encountered.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory reagent blank (LRB) —The laboratory
must analyze at least one LRB (Section 3.10) with each
batch of 20 or fewer samples of the same matrix. LRB
data are used to assess contamination from the labora-
tory environment. LRB values that exceed the MDL
indicate laboratory or reagent contamination. When LRB
values constitute 10% or more of the analyte level deter-
mined for a sample, fresh samples or field duplicates of
the samples must be prepared and analyzed again after
the source of contamination has been corrected and
acceptable LRB values have been obtained. For aque-
ous samples the LRB is a precombusted filter of the same
type and size used for samples.-
9.3.2 Laboratory fortified blank (LFB) — The laboratory
must analyze at least one LFB (Section 7.4.1) with each
batch of samples. Calculate accuracy as percent recov-
ery. If the recovery of any analyte falls outside the
required control limits of 85-115%, that analyte is judged
put of control, and the source of the problem should be
identified and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most recent 20-30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
440.0 - 5
Revision 1.4 November 1992
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9.4 Assessing Analyte Recovery and Data Quality
9.4.1 Percent recoveries cannot be readily obtained
from particulate samples. Consequently, accuracy can
only be assessed by analyzing check standards as
samples and quality control samples (QCS). The use of
laboratory fortified matrix samples has not been as-
sessed.
10.0 Calibration and Standardization
10.1 Calibration—After following manufacturer's instal-
lation and temperature stabilization procedures, daily
calibration procedures must be performed and evaluated
before sample analysis may begin. Single point or stan-
dard curve calibrations are possible, depending on in-
strumentation.
10.1.1 Establish single response factors (RF) for each
element (C,H, and N) by analyzing three weighed por-
tions of calibration standard (acetanilide). The mass of
calibration standard should provide a response within
20% of the response expected for the samples being
analyzed. Calculate the (RF) for each element using the
following formula:
Response factor (nv/jig) =
RN - ZN - BN
WTN
Average instrument response to
standard
where, RN =
ZN » Instrument zero response (M.V)
BN - Instrument blank response (pv)
and, WTN - (M)(Na)(AW/MW)
where, M - The mass of standard material in
PO
Na « Number of atoms of C, N or H, in a
molecule of standard material
AW = AtomicweightofC(12.01),N(14.01)
or H (1.01)
MW = Molecular weight of standard mate-
rial (135.2 for acetanilide)
If instrument response is in units other than jiv, then
change the formula accordingly. '
10.1.2 For standard curve preparation, the range of
calibration standard masses used should be such that
the low concentration approaches but is above the MDL
and the high concentration is above the level of the
highest sample, but no more than 90% of the linear
dynamic range. A minimum of three concentrations should
be used in constructing the curve. Measure response
versus mass of element in the standard and perform a
regression on the data to obtain the calibration curve.
11.0 Procedure
11.1 Aqueous Sample Preparation
11.1.1 Water Sample Filtration — Precombust GF/F
glass fiber filters at 500°C for 1.5 h. The diameter of filter
used will depend on the sample composition and instru-
ment capabilities (Section 8.1.1). Store filters covered if
not immediately used. Place a precombusted filter on
fritted filter base of the filtration apparatus and attach the
filtration tower. Thoroughly shake the sample container
to suspend the particulate matter. Measure and record
the required sample volume using a graduated cylinder.
Pour the measured sample into the filtration tower, no
more than 50 mL at a time. Filter the sample using a
vacuum no greater than 10 in. of Hg. Vacuum levels
greater than 10 in. of Hg can cause filter rupture. If less
than the measured volume of sample can be practically
filtered due to clogging, measure and record the actual
volume filtered. Do not rinse the filter following filtration.
It has been demonstrated that sample loss occurs when
the filter is rinsed with an isotonic solution or the filtrate.8
Air dry the filter after the sample has passed through by
continuing the vacuum for 30 sec. Using Teflon-coated
flat-tipped forceps, fold the filters in half while still on the
fritted glass base of the filter apparatus. Store filters as
described in Section 8.
11.1.2 If the sample has been stored frozen, place the
sample in a drying oven at 103-105° C for 24 h before
analysis and dry to a constant weight. Precombust one
nickel sleeve at 875° C for 1 h for each sample.
11.1.3 Remove the filter pads containing the particulate
material from the drying oven and insert into a
precombusted nickel sleeve using Teflon-coated flat-
tipped forceps. Tap the filter pad using a stainless steel
rod. The sample is ready for analysis,.
11.2 Sediment Samples Preparation
11.2.1 Thaw the frozen sediment sample in a 102-
105°C drying oven for at least 24 h before analysis and
dry to a constant weight. After drying, homogenize the
dry sediment with a mortar and pestle. Store in a desic-
cator until analysis. Precombust aluminum capsules at
550°C in a muffle furnace for 1.5 h for each sediment
sample being analyzed. Precombust one nickel sleeve at
875°C for 1 h for each sediment sample.
11.2.2 Weigh 10 mg of the homogenized sediment to
the nearest 0.001 mg with an ultra-micro balance into a
precombusted aluminum capsule. Crimp the top of the
aluminum capsule with the Teflon-coated flat-tipped for-
ceps and place into a precombusted nickel sleeve. The
sample is ready for analysis.
11.3 Sample Analysis
11.3.1 Measure instrument zero response (Section 7.4)
and instrument blank response (Section 7.4) and record
Revision 1.4 November 1992
440.0 - 6
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values. Condition the instrument by analyzing a condi-
tioner. Calibrate the instrument according to Section 10
and analyze all preliminary QC samples as required by
Section 9. When satisfactory control has been estab-
lished, analyze samples according to the instrument
manufacturer's recommendations. Record all response
data.
11.3.2 Report data as directed in Section 12.
11.4 Determination of Paniculate Organic and
Inorganic Carbon
11.4.1 Method 1: Thermal Partitioning — The differ-
ence found between replicate samples, one of which has
been analyzed for total PC and PN and the other which
was muffled at 550°C and analyzed is the particulate
organic component of that sample. This method of ther-
mally partitioning organic and inorganic PC may under-
estimate slightly the carbonate minerals' contribution in
the inorganic fraction since some carbonate minerals
decompose below 500°C, although CaCO3 does not.9
11.4.2 Method2: Fuming HCI — Allow samples to dry
overnight at 103-105°C and then place in a desiccator
containing concentrated HCI, cover and fume for 24 h in
a hood. The fuming HCI converts inorganic carbonate in
the samples to water vapor, CO2 and calcium chloride.
Analyze the samples for particulate C. The resultant data
are particulate organic carbon.10
12.0 Data Analysis and Calculations
12.1 Sample data should be reported in units of jig/L for
aqueous samples and mg/kg dry weight for sediment
samples.
12.2 Report analyte concentrations up to three signifi-
cant figures for both aqueous and sediment samples.
12.3 For aqueous samples, calculate the sample con-
centration using the following formula:
Corrected
sample response (|iv)
Sample volume (L) x RF
Concentration (ng/L) =
where, RF = Response Factor (Section 10.1.1)
Corrected Sample Response (Section 7.4)
12.4 For sediment samples, calculate the sample con-
centration using the following formula:
Concentration (mg/kg) =
Corrected
sample response (jxv)
Sample weight (g) x RF (p.v/|ig)
where, RF = Response Factor (Section 10.1.1)
Corrected Sample Response (Section 7.4)
Note: Units of ng/g = mg/kg
12.5 The QC data obtained during the analyses provide
an indication of the quality of the sample data and should
be provided with the sample results..
13.0 Method Performance
13.1 Single-laboratory performance data for aqueous
samples from the Chesapeake Bay are provided in Table
2.
13.2 Single-laboratory precision and accuracy data for
the marine sediment reference material, BCSS-1, are
listed in Table 3.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the
•next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 115516th Street N.W., Wash-
ington D.C. 20036, (202) 872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. 40 CFR, Part 136, Appendix B. Definition and
Procedure for the Determination of the Method
Detection Limit. Revision 1.11.
2. Carcinogens - Working With Carcinogens,
Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control,
440.0 - 7
Revision 1.4 November 1992
-------
National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
4. Safely in Academic Chemistry Laboratories,
American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. Proposed OSHA Safety and Health Standards,
Laboratories, Occupational Safety and Health
Administration, Federal Register, July 24,1986.
6. Rohrdough, W.G. et al. Reagent Chemicals,
American Chemical Society Specifications, 7th
Edition. American Chemical Society,
Washington, DC, 1986.
7. Holme, N.A. and A.D. Mclntyre (eds). 1971.
Methods for the Study of Marine Benthos.
International Biome Program. IBP Handbook #16.
F.A. Davis Co., Philadelphia, PA.
8. Hurd, D.C. and D.W. Spencer (eds). 1991. Marine
Particles: Analysis and Characterization.
Geophysical Monograph: 63, American
Geophysical Union, Washington, DC 472p.
9. Hirota, J. and J.P. Szyper. 1975. Separation of
total particulate carbon into inorganic and organic
components. Limnol. and Oceanogr. 20: 896-900.
10. Grasshoff, K., M. Ehrhardt and K. Kremling (eds).
1983. Methods of Seawater Analysis. Verlag
Chemie.
17.0 Tables, Diagrams, Flowcharts, and
Validation Data
Table 1. Filter Diameter Selection Guide
Filter diameter
Sample matrix 47mm 25mrn 13mm
Sample matrix volume
Open ocean
Coastal
Estuarine
(low particulate)
Estuarine
(high particulate)
2000 ml
1000ml
500-700 ml
100-400 ml
500mL 100ml
400-600 ml 100ml
250-400 ml 50 ml
75-200 ml
25ml
Table 2. Performance Data—Chesapeake Bay
Aqueous Samples
Sample
1
2
3
4
Measured
nitrogen
concentration
(W3/L)
147
148
379
122
S.D.A
(M9/L)
±4
+ 11
±51
±9
Measured
carbon
concentration
(ug/D
1210
1240
3950
1010
S.D.A
(H9/L)
± 49
±179
±269
± 63
* Standard deviation based on 7 replicates.
Table 3. Precision and Accuracy Data - Canadian
Sediment Reference Material BCSS-1
Element
Carbon
Nitrogen
T.V.A
2.19%
0.195%
Mean
measured
value (%)
2.18
0.194
%RSDB
±3.3
±3.9
%Recoveryc
99:5
99.5
A True value. Carbon value is certified; nitrogen valus is listed but not
certified.
" Percent relative standard deviation based on 10 replicates.
c As calculated from T.V.
Revision 1.4 November 1992
440.0 - 8
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Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytin a in
Marine and Freshwater Phytoplankton by Fluorescence
Adapted by
Elizabeth J. Arar
and
Gary B. Collins
Revision 1.1
November 1992
Environmental Monitoring Systems Laboratory
Office of Research and Devlopment
U.S. Environmental Protection Agency
Cincinnati, OH 45268
445.0 -1
Revision 1.1 November 1992
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Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytiia a im
Marine and Freshwater Phytoplankton by Fluorescence
1.0 Scope and Application
1.1 This method provides a procedure for the low level
determination of chlorophyll a (chl a) and its magnesium-
free derivative, pheophytin a, (pheo a) in marine and
freshwater phytoplankton using fluorescence detection.1'2
Phaeophorbides present in the sample are determined
collectively as pheo a.
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Chl a
479-61-8
1.2 Instrumental detection limits of 0.05 ng chl a/L and
0.06 jig pheo a/L in a solution of 90% acetone were
determined by this laboratory. Method detection limits
using mixed assemblages of algae provide little informa-
tion because of interferences from other pigments in the
fluorescence of chl a and pheo a.3 An estimated detec-
tion limit for chl a was determined to be 0.11 ng/L in 10
mL of final extraction solution. The upper limit of the linear
dynamic range forthe instrumentation used in this method
evaluation was 250 ng chl a/L.
1.3 This method uses 90% acetone as the extraction
solvent because of its efficiency for most types of algae.
There is evidence that certain chlorophylls and caro-
tenoids are more thoroughly extracted with methanol ^
or dimethyl sulfoxide.7 Bowles, et al.6 found that for chl
a, however, 90% acetone was an effective extractant
when the extraction period was optimized for the domi-
nant species present in the sample.
1.4 Depending on the type of algae under investiga-
tion, this method can have uncorrectable interferences
(Section 4.0). In cases where taxpmonic classification is
unavailable, a spectrophotometric or high performance
liquid chromatographic (HPLC) method may provide
more accurate data for chl a and pheo a.
1.5 This method is for use by analysts experienced in
the handling of photosynthetic pigments and in the op-
eration of fluorescence detectors or by analysts under
the close supervision of such qualified persons.
2.0 Summary of Method
2,1 Chlorophyll-containing phytoplankton in a mea-
sured volume of sample water are concentrated by
filtering at low vacuum through a glass fiber filter. The
pigments are extracted from the phytoplankton in 90%
acetone with the aid of a mechanical tissue grinder and
allowed to steep for a minimum of 2 h, lout not to exceed
24 h, to ensure thorough extraction of the chl a. The filter
slurry is centrifuged at 675 g for 15 miri (or at 1000 g for
5 min) to clarify the solution. An aliquot of the supernatant
is transferred to a glass cuvette and fluorescence is
measured before and after acidification to 0.003 N HCI
with 0.1 N HCI. Sensitivity calibration factors, which have
been previously determined on solutions of pure chl a of
known concentration, are used to calculate the concen-
tration of chl a and pheo a in the sample extract. The
concentration in the natural water sample is reported in
ng/L.
3.0 Definitions
3.1 Estimated Detection Limit (EDL) — The mini-
mum concentration of an analyte that yields a fluores-
cence 3X the fluorescence of blank filters which have
been extracted according to this method.
3.2 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instru-
ment response to an analyte is linear.
3.3 Instrument Detection Limit (IDL) — The mini-
mum quantity of analyte or the concentration equivalent
that is detectable by the f luorometer. For this method the
background is a solution of 90% acetone.
3.4 Stock Standard Solution (SSS) — A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
3.5 Primary Dilution Standard Solution (PDS) — A
solution of the analytes prepared in the laboratory from
stock standard solutions and diluted as needed to pre-
pare calibration solutions and other needed analyte
solutions.
3.6 Calibration Standard (CAL) — A solution pre-
pared from the primary dilution standard solution or stock
standard solutions containing the internal standards and
surrogate analytes. The CAL solutions are used to cali-
brate the instrument response with respect to analyte
concentration.
3.7 Response Factor (RF) — The ratio of the re-
sponse of the instrument to a known amount of analyte.
3.8 Laboratory Reagent Blank (LRB) — An aliquot
of reagent water or other blank matrix that is treated
exactly as a sample including exposure to all glassware,
445.0 - 2
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equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, re-
agents, or apparatus.
3.9 Field Duplicates (FD1 andFD2)—Two separate
samples collected at the same time and place under
identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of
FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well
as with laboratory procedures.
3.10 Quality Control Sample (QCS) — A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.11 Material Safety Data Sheet (MSDS) — Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
4.0 Interferences
4.1 Any substance extracted from the filter or acquired
from laboratory contamination that fluoresces in the red
region of the spectrum may interfere in the accurate
measurement of both chl a and pheo a.
4.2 The relative amounts of chl a, b, and c vary with
the taxonomic composition of the phytoplankton. Chl b
and cmay significantly interfere with chl a measurements
depending on the amount present. Due to the spectral
overlap of chl & with phep a and chl a, underestimation of
chl a occurs accompanied by overestimation of pheo a
when chl b is present in the sample. The degree of
interference depends upon the ratio of a:b. This labora-
tory found that at a ratio of 5:1, using the acidification
procedure to correct for pheo a, chl a was underesti-
mated by approximately 5%. Loftis and Carpenter8 re-
ported an underestimation of 16% when the a:b ratio was
2.5:1. A ratio of 2:1 is the highest ratio likely to occur in
nature. They also reported overestimation of chl a in the
presence of chl c of as much as 10% when the a: c ratio
was 1:1 (the theoretical maximum likely to occur in
nature). The presence of chl c also causes the under-
estimation of pheo a. The effect of chl cis not as severe
as the effect of chl b on the measurement of chl a and
pheo a. Knowledge of the taxonomy of the algae under
consideration will aid in determining if the spectrophoto-
metric method using trichromatic equations to determine
chl a, b, and c or an HPLC method would be more
appropriate. <"*
4.3 Quenching effects are observed in highly concen-
trated solutions or in the presence of high concentrations
of other chlorophylls or carotenoids. Minimum sensitivity
settings on the fluorometer should be avoided; samples
should be diluted instead.
4.4 Fluorescenceistemperaturedependentwithhigher
sensitivity occurring at lower temperatures. Samples,
standards, LRBs and QCSs must be at the same tem-
perature to prevent errors and/or poor precision. Analy-
ses of samples at ambient temperature is recommended
in this method. Ambient temperature should not fluctuate
more than ± 3°C between calibrations or recalibration of
the fluorometer will be necessary.
4.5 Samples must be clarified by centrifugation prior to
analysis.
4.6 All photosynthetic pigments are light and tempera-
ture sensitive. Work must be performed in subdued light
and all standards, QC materials and filter samples must
be stored in the dark at -20°C to prevent degradation.
5.0 Safety
5.1 The toxicity or carcinogenicity of the chemicals
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and handled with caution and respect. Each laboratory is
responsible for maintaining a current awareness file of
Occupational Safety and Health Administration (OSHA)
regulations regarding the safe handling of the chemicals
specified in this method.1!M8 A file of MSDS should also
be made available to all personnel involved in the chemi-
cal analysis.
5.2 The grinding of filters during the extraction step of
this method should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.
6.0 Apparatus and Equipment
6.1 Fluorometer — Equipped with a high intensity
F4T5 blue lamp, red-sensitive photomultiplier, and filters
for excitation (CS-5-60) and emission (CS-2-64). (The
F4T5D daylight white lamp is an acceptable substitute for
the F4T5 blue lamp.) A Turner Designs Model 10 Series
fluorometer was used in the evaluation of this method.
6.2 Centrifuge, capable of 675 g.
6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)
with grooves in the tip with 1/4" stainless steel rod long
enough to chuck onto a suitable drive motor and 30-mL
capacity glass grinding tube.
6.4 Precombusted filters, glass fiber, 47-mm, nominal
pore size of 0.45 or 0.7 nm. Whatman GF/F filters were
used in this work.
6.5 Petri dishes, plastic, 50 X 9-mm, or some other
solid container for transporting and storing sampled
filters.
6.6 Aluminum foil.
6.7 Laboratory tissues.
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6.8 Tweezers or flat-tipped forceps.
6.9 Vacuum pump or source capable of maintaining a
vacuum up to 6 in. Hg.
6.10 Room thermometer.
6.11 Labware— All reusable labware (glass, polyeth-
ylene, Teflon, etc.) that comes in contact with chlorophyll
solutions should be clean and acid free. An acceptable
cleaning procedure is soaking for 4 h in laboratory grade
detergent and water, rinsing with tap water, distilled
deionized water and acetone.
6.11.1 Assorted Class A calibrated pipets.
6. /1.2 Graduated cylinders, 500-mL and 1 -L.
6.11.3 Volumetric flasks, Class A calibrated, 25-mL,
50-mL, 100-mL and 1-L capacity.
6.11.4 Glass rods.
6.11.5 Pasteur Type pipet or medicine dropper.
6.11.6 Disposable glass cuvettes for the fluorometer.
6.11.7 Filtration apparatus consisting of 1 or 2-L filtra-
tion flask, 47-mm fritted glass disk base and a glass filter
tower.
6.11.8 Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.11.9 Polyethylene squirt bottles.
7.0 Reagents and Standards
7.1 Acetone, HPLC grade, (CASRN 67-64-1).
7.2 Hydrochloricacid (HCI), concentrated (sp. gr. 1.19),
(CASRN 7647-01-0).
7.3 Magnesium carbonate (MgCO3), light powder
(CASRN 39409-82-0).
7.4 Chi a free of chl b. May be obtained from a
commercial supplier such as Sigma Chemical (St. Louis,
MO).
7.5 Water— ASTM Type I water (ASTM D1193) is
required. Suitable water may be obtained by passing
distilled water through a mixed bed of anion and cation
exchange resins.
7.6 0.1 N HCI Solution—Md 8.5 rhL of concentrated
HCI to approximately 500 ml_ water and dilute to 1 L.
7.7 Saturated Magnesium Carbonate Solution —
Add 10 g MgCO3 powder to a 1-L flask and dilute to
volume with water (Section 7.5). Cap the flask and invert
it several times. Let the suspended powder settle before
using the solution in subsequent work.
7.8 Aqueous Acetone Solution — 90% acetone/
10% saturated magnesium carbonate solution. Carefully
measure 100 mLof the saturated magnesium carbonate
solution into the 1-L graduated cylinder. Transfer to a 1-
L flask or storage bottle. Measure 900 mL of acetone into
the graduated cylinder and transfer to the flask or bottle
containing the saturated magnesium carbonate solution.
Mix, label and store.
7.9 Chl Stock Standard Solution (SSS) — Chl a
from a commercial supplier will be shipped in an amber
glass ampoule which has been flame sealed. This dry
standard should be stored at -20°C in the dark and the
SSS prepared just prior to use. Tap the ampoule until all
the dried chl is in the bottom of the ampoule. In subdued
light, carefully break the tip off the ampoule. Weigh the
ampoule and its contents to the nearest .1 mg. Transfer
the entire contents of the ampoule into a 50-mL volumet-
ric flask and reweigh the empty ampoule. Determine by
difference the mass of chl a added to the flask. Dilute to
volume with 90% acetone, determine the concentration
in mg/L (1 mg in 50 mL = 20 mg/L), label the flask and
wrap with aluminum foil to protect from light. The concen-
tration of the solution must be confirmed spectrophoto-
metrically using a multiwavelength spectrophotometer.9
When stored at -20°C, the SSS is stable for months.
However, confirmation of the chl a concentration spec-
trophotometrically is required each time dilutions are
made from the SSS.
7.10 Laboratory Reagent Blank (LRB)—A blankfilter
which is extracted and analyzed just as a sample filter.
The LRB should be the last filter extracted of a sample
set. It is used to assess possible contamination of the
reagents or apparatus.
7.11 Chl a Primary Dilution Standard Solution (PDS)
— Add 1 mL of the SSS (Section 7.9) to a clean 100-mL
flask and dilute to volume with the eiqueous acetone
solution (Section 7.8). If exactly 1 mg of pure chl a was
used to prepare the SSS, the concentration of the PDS is
200 \ig/L. Prepare fresh just prior to use.
7.12 Quality Control Sample (QCS) — Chl a QCSs
can be obtained from the Quality Assurance Research
Division, Environmental Monitoring Systems Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH
45268. QCSs are supplied with a calibration solution.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection — Water may be ob-
tained by a pump orgrab sampler. Data quality objectives
will determine the depth at which samples are taken.
Healthy phytoplankton, however, are generally obtained
from the photic zone (depth at which the illumination level
is 1% of surface illumination). Enough water should be
collected to concentrate phytoplankton on at least three
filters. Filtration volume size will depend on the particu-
late load of the water. Four liters may be required for open
ocean water where phytoplankton density is usually low,
whereas 1 L or less is generally sufficient for lake, bay or
estuary water. All apparatus should be clean and acicl-
445.0 - 4
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free. Filtering should be performed in subdued light as
soon as possible after sampling. Aboard-ship filtration is
highly recommended.
Assemble the filtration apparatus and attach the vacuum
source wifh vacuum gauge and regulator. Vacuum filtra-
tion should not exceed 6 in. Hg (20 kPa). Higher filtration
pressures may damage cells and result in loss of chloro-
phyll.
Prior to drawing a subsample from the water sample
container, thoroughly shake the containerto suspend the
particulates. Pour the subsample into a graduated cylin-
der and accurately measure the volume. Pour the
subsample into the filter tower of the filtration apparatus
and apply a vacuum (not to exceed 20 kPa). A sufficient
volume has been filtered when a visible green or brown
color is apparent on the filter. Do not suck the filter dry
with the vacuum; instead slowly release the vacuum as
the final volume approaches the level of the filter and
completely release the vacuum as the last bit of water is
pulled through the filter. Remove the filter from the fritted
base with tweezers, fold once with the paniculate matter
inside, lightly blot the filter with a tissue to remove excess
moisture and place it in the petri dish or other suitable
container. If the filter will not be immediately extracted,
then wrap the container with aluminum foil to protect the
phytoplankton from light and store the filter at-20°C.
Short term storage (2 to 4 h) on ice is acceptable, but
samples should be stored at -20°C as soon as possible.
8.2 Preservation—Sampled filters should be stored
frozen (-20°C or -70°C) in the dark until extraction.
8.3 Holding Time — Filters can be stored frozen for
as long as 3-112. weeks without significant loss of chl a.19
9.0 Quality Control
9.1 Each Laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability and the
continued analysis of laboratory reagent blanks, field
duplicates and quality control samples as a continuing
check on performance. The laboratory is required to
maintain performance records that define the quality of
the data thus generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (instrumental de-
tection limits, linear dynamic range and EDLs) and labo-
ratory performance (analyses of QCSs) prior to sample
analyses.
9.2.2 Linear Dynamic Range (LDR)—The LDR should
be determined by analyzing a minimum of 5 calibration
standards ranging in concentration from 0.2 fig/L to 200
ng chl alL across all sensitivity settings of the fluorom-
eter. Normalize responses by dividing the response by
the sensitivity setting multiplier. Perform the linear re-
gression of normalized response vs. concentration and
obtain the constants m and b, where m is the slope and
b is the y-intercept. Incrementally analyze standards of
higher concentration until the measured fluorescence
response, R, of a standard no longer yields a calculated
concentration, Cc, that is ± 10% of the known concen-
tration, C, where Cc = (R - b)/m. That concentration
defines the upper limit of the LDR for your instrument.
Should samples be encountered that have a concentra-
tion which is 90% of the upper limit of the LDR, these
samples must be diluted and reanalyzed.
9.2.3 Instrumental Detection Limit (IDL) — Zero the
fluorometer with a solution of 90% acetone on the maxi-
mum sensitivity setting. Pure chl a in 90% acetone should
be serially diluted until it is no longer detected by the
fluorometer on a maximum sensitivity setting.
9.2.4 Estimated Detection Limit (EDL) — Several blank
filters should be extracted according to the procedure in
Section 11, using clean glassware and apparatus, and
the fluorescence measured. A solution of pure chl a in
90% acetone should be serially diluted until it yields a
response which is 3X the average response of the blank
filters.
9.2.5 Quality Control Sample (QCS)—When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify the calibration standards
and acceptable instrument performance with the analy-
sis of aQCS (Section 7.12). If the determined value is not
within the confidence interval provided with the reference
value, then the determinative step of this method is
unacceptable. The source of the problem must be iden-
tified and corrected before continuing analyses.
9.2.6 Extraction Proficiency — Personnel performing
this method for the first time should demonstrate profi-
ciency in the extraction of sampled filters (Section 11.1).
Twenty to thirty natural samples should be obtained
using the procedure outlined in Section 8.1 of this method.
Sets of 10 samples or more should be extracted and
analyzed according to Section 11.2. The percent relative
standard deviation (%RSD) of uncorrected values of chl a
should not exceed 15% for samples that are approxi-
mately 10X the IDL. RSD for pheo a might typically range
from 10 to 50%.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) — The labora-
tory must analyze at least one blank filter with each
sample batch. The LRB should be the last filter extracted.
LRB data are used to assess contamination from the
laboratory environment. LRB values that exceed the IDL
indicate contamination from the laboratory environment.
When LRB values constitute 10% or more of the analyte
level determined for a sample, fresh samples or field
duplicates must be analyzed after the contamination has
been corrected and acceptable LRB values have been
obtained.
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10.0 Calibration and Standardization
10,1 Calibration — Calibration should be performed
bimonthly or when there has been an adjustment made
to the instrument, such as replacement of lamp, filters or
photomultiplier. Prepare 0.2,2,5,20 and 200 jig chl a/L
calibration standards from the PDS (Section 7.11). Alter-
nately, a calibration solution can be obtained from the
address listed in Section 7.12. Allow the instrument to
warm up for at least 15 min. Measure the fluorescence of
each standard at sensitivity settings that provide midscale
readings. Obtain response factors for chl a for each
sensitivity setting as follows:
where,
Fs«response factor for sensitivity setting, S.
Rs s fluorometer reading for sensitivity setting, S.
C. ^ concentration of chl a.
Avoid using the minimum sensitivity setting dueto quench-
ing effects.
if pheo a determinations will be made then it will be
necessarytoobtainbefore-to-afteracidification response
ratios of the chl a calibration standards as follows: (1)
measure the fluorescence of the standard, (2) remove
the cuvette from the fluorometer, (3) acidify the solution
to 0.003 N HCI4 with the 0.1 N HCI solution, (4) wait 90
sec. and measure the fluorescence of the standard
solution again. Addition of the acid may be made using a
medicine dropper. It will be necessary to know how many
drops are equal to 1 ml_ of acid. For a cuvette that holds
5 mL of extraction solution, it will be necessary to add
0.15 mL of 0.1 N HCI to reach a final acid concentration
of 0.003N in the 5 mL Calculate the ratio, r, as follows:
where,
Rb ^ fluorescence of pure chl a standard solution
before acidification.
Ra = fluorescence of pure chl a standard solution
after acidification.
11.0 Procedure
11.1 Extraction of Filter Samples
11.1.1 If sampled filters have been frozen, remove them
from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand tissues and squirt
bottles containing water and acetone. Workspace light-
ing should be the minimum that is necessary to read
instructions and operate instrumentation. Remove a filter
from its container and place it in the glass grinding tube.
Push it to the bottom of the tube with a glass rod. With a
volumetric pipet, add 4 mL of the aqueous acetone
solution (Section 7.8) to the grinding tube. After the filter
has been converted to a slurry, grind the filter for approxi-
mately 1 min at 500 rpm. Pour the slurry into a 15-mL
screw-cap centrifuge tube and, using a 6-mL volumetric
pipet, rinse the pestle and the grinding tube with 90%
acetone. Add the rinse to the centrifuge tube containing
the filter slurry. Cap the tube and shake it vigorously.
Place it in the dark before proceeding to the next filter
extraction. Before placing another filter in the grinding
tube, use the acetone and water squirt bottles to thor-
oughly rinse the pestle, grinding tube and glass rod. The
last rinse should be with acetone. Use a clean tissue to
remove any filter residue that adheres to the pestle or to
the steel rod of the pestle. Proceed to the next filter and
repeat the steps above. The entire extraction with trans-
ferring and rinsing steps takes 5 min. Approximately 500
mL of acetone and water waste are generated per 20
samples from the rinsing of glassware and apparatus.
11.1.2 Shake each tube vigorously before placing them
to steep in the dark at 4°C. Samples should be allowed to
steep for a minimum of 2 h but not to exceed 24 h. Tubes
should be shaken at least once during the steeping
period or placed horizontally to allow the extraction
solution to have maximum contact with the filter slurry.
11.1.3 After steeping is complete, centrifuge samples
for 15 min. at 675 g or for 5 min. at 1000 g. Samples
should be allowed to come to ambient temperature
before analysis. This can be done by placing the tubes in
a constant temperature water bath or by letting them
stand at room temperature for 30 min., Recalibrate the
fluorometer iftheroomtemperaturefluctuated±3°Cfrom
the last calibration date.
11.2 Sample Analysis
11.2.1 After the fluorometer has warmed up for at least
15 min, use the 90% acetone solution to zero the instru-
ment on the sensitivity setting that will be used for sample
analysis.
11.2.2 Pour or pipet the supernatant of the extracted
sample into a sample cuvette. The volume of sample
required in your instrument's cuvette should be known so
that the correct amount of acid can be added in the pheo a
determinative step. For a cuvette that holds 5 mL of.
extraction solution, 0.15 mL of the 0.1 N HCI solution is
required to achieve 0.003 N HCI. Choose a sensitivity
setting that yields a midscale reading when possible and
avoid the minimum sensitivity setting. If the concentra-
tion of chl a in the sample is >90% of the upper limit of the
LDR, then dilute the sample with the 90% acetone
solution and reanalyze. Record the fluorescence mea-
surement and sensitivity setting used for the sample.
Remove the cuvette from the fluorometer and acidify the
extract to a final concentration of 0.003 N HCI using the
0.1 N HCI solution. Wait 90 sec. before measuring
445.0 - 6
Revision 1.1 November 1992
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fluorescence again. Twenty-five to thirty-five samples
can be extracted and analyzed in one 8-h day.
12.0 Data Analysis and Calculations
12.1 "Uncorrected" chl a may be determined in a sample
extract by multiplying the fluorescence response of the
sample by the appropriate response factors determined
in Section 10.1. Determine the "corrected" chl a concen-
tration in the sample extract and the pheo a concentration
in ng/L as follows:
Chla,ng/l_=Fs(r/r-1)(Rb-Ra)
Pheo a, jig/L = Fs(r/r-1) (rRa - Rb)
where,
Fs = response factor for the sensitivity setting
used.
Rb = fluorescence of sample extract before acidi-
fication.
Ra = fluorescence of sample extract after acidifi-
cation.
r = thebefore-to-afteracidificationratioofapure
chl a solution (Section 10.1).
12.2 The concentration of chl a and pheo a in the natural
water sample is calculated by multiplying the results
obtained in Section 12.1 by 10 ml (the extraction vol-
ume) and dividing by the volume (ml) of natural water
sample that was filtered. Any other dilution or concentra-
tion factors should be incorporated accordingly.
12.3 LRB and QCS data should be reported with each
sample data set.
13.0 Method Performance
13.1 The IDL for the instrument used in the evaluation
of this method was 0.05 ng/L for chl a and 0.06 u.g/L
pheoa. An EDL of 0.11 jig chl a/L was determined. ,
13.2 The precision (%RSD) for chl a in mostly blue-
green and green phytoplankton natural samples which
were steeped for 2 h vs. 24 h is reported in Table 1.
Although the means were the same, precision was better
for samples which were allowed to steep for 24 h prior to
analysis. Since pheo a was found in the samples, the
chla values are "corrected" (Section 12.1). Table 2 con-
tains precision data for pheo a. A statistical analysis of the
pheo a data indicated a significant difference at the 0.05
significance level in the mean values obtain. The cause
of the lower pheo a values in samples extracted for 24 h
is not known.
13.3 Three QCS ampoules obtained from the USEPA
were analyzed and compared to the reported confidence
limits in Table 3. The reference values for QCS obtained
from the USEPA are periodically updated and new con-
fidence limits established.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation (e.g., Section 11.1.1). When wastes cannot
be feasibly reduced at the source, the Agency recom-
mends recycling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 115516th Street N.W., Wash-
ington DC 20036, (202)872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section 14.2.
16.0 References
1. Yentsch, C.S. and D.W. Menzel, "A method for the
determination of phytoplankton chlorophyll and
pheophytin by fluorescence," Deep Sea Res 10
(1963), pp. 221-231.
2. Strickland, J.D.H. and T.R. Parsons, A Practical
Handbook of Seawater Analysis, Bull. Fish Res
Board Can., 1972, No.167, p. 201.
3. Trees, C.C., M.C. Kennicutt, andJ.M. Brooks, "Errors
associated with the standard fluorometric determin-
ation of chlorophylls and pheopigments," Mar. Chem
17 (1985) pp. 1-12.
4. Holm-Hansen.O., "Chlorophyll a determination-
improvements in methodology," OIKOS, 30 (1978)
pp. 438-447.
5. Wright, S.W. and J.D. Shearer, "Rapid extraction
and HPLC of chlorophylls and carotenoids from
marine phytoplankton," J. Chrom., 294 (1984) pp
281-295. HH'
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6. Bowies, N.D., H.W. Paerl, and J. Tucker, "Effective
solvents and extraction periods employed in
phytoplankton carotenoid and chlorophyll
determination," Can. J. Fish. Aquat. Sci., 42 (1985)
pp. 1127-1131.
7. Shoaf, W.T. and B.W. Lium, "Improved extraction of
chlorophyll a and b from algae using dimethyl
sulfoxide," Limnol. and Oceanogr., 21 (6), (1976) pp.
926-928.
8. Loftis, M.E. and J.H. Carpenter, "A fluorometric
method for determining chlorophylls a, b, and c,1" J.
Mar. Res., 29 (1971) pp.319-338.
9. Standard Methods for the Analysis of Water and
Wastes, 17th Ed., 1989,10200H, Chlorophyll.
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phytoplankton," paper submitted for publication in
1991.
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waters by reverse-phase high performance liquid
chromatography/Mna/. Chim.Acta., 151 (1983) pp.
297-314.
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pressure liquid chromatography," Can. J. Fish. Aquat.
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"Rapid determination of chlorophylls and their
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445.0 - 8
Revision 1.1 November 1992
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Comparison of Precision of Two Extraction
Periods
Corrected Chi a
Sample A1
2h3 24 h3
Sample B2
2h3 24 h3
Mean 49.6
concentation
(ng/L)
52.9
78.6
78.8
Standard
deviation
Relative
standard
deviation (%)
4.89
9.9
2.64
5.0
6.21
7.9
2.77
3.5
1 Values reported are the mean measured concentrations (n=6) of
chl a in the natural water based on a 100 mL filtration volume.
2 Values reported are the mean measured concentrations (n=9) in
the extraction solution. Sample filtration volume was 300 mL
3 The length of time that the filters steeped after they were ground.
Table 3. Analyses of USEPA QC Samples
Analyte Reference value Confidence limits
Chl a
Pheoa
Reference value
2.1 u.g/L
0.3 u,g/L
0.5 to 3.7 u,g/L
-0.2 to 0.8 u,g/L
Analyte
Chl a
Pheo a
% Relative Standard
Mean Measured Value deviation
2.8 jig/L
0.3 jig/L
1.5
33
Table 2. Comparison of Precision of Two Extractions
Periods for Pheo a
Pheo a
Sample A1
2 h3 24 h3
Sample B2
2 h3 24 h3
Mean
concentation
(ng/U
Standard
deviation
(W3/L)
Relative
standard
deviation
9.22
2.36
25.6
8.19
3.55
43.2
13.10 10.61
3.86 2.29
29.5 21.6
1 Values reported are the mean measured concentrations (n=6) of
chl a in the natural water based on a 100 mL filtration volume.
2 Values reported are the mean measured concentrations (n=9) in
the extraction solution. Sample filtration volume was 300 mL.
3 The length of time that the filters steeped after they were ground.
445.0-9
Revision 1.1 November 1992
•&U.S. GOVERNMENT PRINTING OFFICE: 1993-750-002/60177
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