UnitedStates National Training EPA-430/1 -80-011
Environmental Protection and Operational September 1980
Aaencv Technology Center
Cincinnati OH 45258
Water
Organic Analyses in
Water Quality
Control Programs
Training Manual
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TECHNICAL REPORT DATA
(Please read Inductions on the reverse before completing)
1. REPORT NO.
EPA-430/1/80-011
2,
3. RECIPIENT'S ACCESS I or* NO.
M81 12441«
4. TITLE AND SUBTITLE
ORGANIC ANALYSES IN WATER QUALITY CONTROL PROGRAMS -
TRAINING MANUAL
5. REPORT DATE
November 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Charles Feldmann
8, PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
U.S. Environmental Protection Agency
National Training & Operational Technology Center
26 West St. Clair Street
Cincinnati, Ohio 45268
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO,
12. SPONSORING AGENCY NAME AND ADDRESS
N/A
13, TYPE OF REPORT AND PERIOD COVERED
14, SPONSORING AGENCY CODE
15, SUPPLEMENTARY NOTES
Supercedes PB297-713/AS
16, ABSTRACT
A lecture/laboratory manual dealing with the analysis of selected organic
pollutants. Intended for use by those having little or no experience in
the field, but having one year (or equivalent) of college organic chemistry,
and having basic laboratory skills (volumetric glassware, titration assem-
blies, analytical and trip balances). Topics include dissolved oxygen,
biochemical oxygen demand, ammonia, nitrates, nitrites, carbon analysis
chemical oxygen demand, surfactants, oil and grease phenolics, gas
chromatography, and polychlorinated biphenyls.
117.
KEY WORDS AND DOCUMENT ANALYSIS
L DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group ]
| Chemical analysis, analysis, instru-
I mentation, pollutant identification,
water analysis
N/A
07/C
18, DISTRIBUTION STATEMENT
Released unlimited t
19. SECURITY CLASS (This Report)
None
21. NO. OF PAGES
20. SECURITY CLASS (This page)
None
22. PRICE
EPA Form 2220-1 (9-73)
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EPA-430/ 1-80-011
September 1980
Organic Analyses in Water Quality Control Programs
This course is for chemists and technicians with little
or no experience in organic analyses commonly required
for NPDES and NIPDWR regulations and for other water
quality programs. Applicants should have a fundamental
knowledge of organic chemistry and quantitative analyses,
and should have basic laboratory skills including the
use of volumetric glassware and titration assemblies.
They should be actively engaged in a water quality
control program.
After successfully completing the course, the student
will know how to perform analyses for selected organic
pollutants listed as approved in the Federal Register.
The training is a five-day course which includes classroom
instruction, student performance of laboratory procedures,
and discussion of each laboratory assignment and reported
results.
Course topics include NPDES methodology, laboratory
safety, dissolved oxygen (Winkler), five-day biochemical
oxygen demand, total organic carbon, oil and grease
(separatory funnel extraction), surfactants (MBAS),
total Kjeldahl and organic nitrogen (Nesslerization),
phenol (4-AAP), polychlorinated biphenyls, pesticides,
and control of analytical performance. Emphasis is
on laboratory operations.
U. 5. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Program Operations
National Training and Operational Technology Center
ii
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FOREWORD
These manuals are prepared for reference use of students enrolled in scheduled
training courses of the Office of Water Program Operations, U. S. Environmental
Protection Agency.
Due to the limited availability of the manuals it is not appropriate
to cite them as technical references in bibliographies or other
forms of publication.
References to products and manufacturers are for illustration only;
such references do not imply product endorsement by the Office of
Water Program Operations, U. S. Environmental Protection Agency.
The reference outlines in this manual have been selected and developed with a
goal of providing the student with a fund of the best available current information
pertinent to the subject matter of the course. Individual instructors may provide
additional material to cover special aspects of their own presentations.
This manual will be useful to anyone who has need for information on the subjects
covered. However, it should be understood that the manual will have its greatest
value as an adjunct to classroom presentations. The inherent advantages of class-
room presentation is in the give-and-take discussions and exchange of information
between and among students and the instructional staff.
Constructive suggestions for improvement in the coverage, content, and format
of the manual are solicited and will be given full consideration.
ill
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CONTENTS
Title or Description Outline Number
Laboratory Safety Practices 1
Sample Handling - Field Through Laboratory 2
I
Methodology For Chemical Analysis Of Water And Wastewater 3
Statistics For Chemists 4
Accuracy-Precision-Error 5
Elements Of A Quality Assurance Program 6
Use Of A Spectrophotometer 7
Dissolved Oxygen - Factors Affecting DO Concentration In Water 8
Dissolved Oxygen - Determination by the Winkler Iodometric Titration - 9
Azide Modification
Laboratory Procedure For Dissolved Oxygen Winkler Method - 10
Azide Modification
Dissolved Oxygen-Determination By Electronic Measurement 11
Biochemical Oxygen Demand Test Procedures 12
Biochemical Oxygen Demand Test Dilution Technique 13
Sources And Analysis Of Organic Nitrogen 14
Ammonia, Nitrites And Nitrates 15
Determination Of Kjeldahl Nitrogen (Micro Apparatus-Nesslerization) 16
Total Carbon Analysis 17
Chemical Oxygen Demand And COD/BOD Relationships 18
Laboratory For Routine Level Chemical Oxygen Demand Determination 19
Determination Of Surfactants 20
Laboratory Determination Of Surfactants (Methylene Blue Active Substances, 21
MBAS)
Oil And Grease 22
Laboratory Determination Of Oil And Grease 23
Determination Of Phenolic.s 24
Laboratory Determination Of Phenol Direct Photometric Method 25
Introduction To Gas-Liquid Chromatography 26
Quantitation In Gas Chromatography 27
Poly chlorinated Biphenyls 28
Method For Poly chlorinated Biphenyls (PCB's) In Industrial Effluents 29
Method For Organochlorine Pesticides In Industrial Effluents 30
| y/
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LABORATORY SAFETY PRACTICES
I INTRODUCTION
A Safe Use,- Handling and Storage of Chemicals
1 Chemicals in any farm can he safely
stored, handled, and used il' their
hazardous physical and chemical
properties are fully understood and the
necessary precautions, including the
use of proper safeguards and personal
protective equipment are observed.
2 The management of every unit within a
manufacturing establishment must give
wholehearted support to a well integrated
safety policy,
B General Rules for Laboratory Safety
1 Supervisory personnel should think
"safety. " Their attitude toward fire
and safety standard practices is reflected
in the behavior of their entire staff.
2 A safety program is only as strong as
the worker's will to do the correct
things at the right time.
3 The fundamental weakness of most
safety programs lies in too much lip
service to safety rules and not enough
action in putting them into practice.
4 Safety practices should be practical and
enforceable.
5 Accident prevention is based on certain
common standards of education, training
of personnel and provision of safeguards
against accidents.
II LABORATORY DESIGN AND EQUIPMENT
A Type of Construction
1 Fire-resistant or noncombustible
2 Multiple story buildings should have
adequate means of exit.
3 Stairways enclosed with brick or
concrete walls
4 Laboratories should have adequate exit
doors to permit quick, safe escape in
an emergency and to protect the
occupants from fires or accidents in
adjoining rooms. Fach room should be
checked to make sure there is no
chance of a person being trapped by
fire, explosions, or release of dangerous
gases.
5 Laboratory rooms in which most of the
work is carried out with flammable
liquids or gases should be provided
with explosion-venting windows.
B Arrangement of Furniture and Equipment
1 Furniture should be arranged for
maximum utilization of available space
and should provide working conditions
that arc efficient and safe.
2 Aisles between benches should be at
least 4 feet wide to provide adequate
room for passage of personnel and
equipment.
3 Desks should be isolated from benches
or adequately protected.
4 Every laboratory should have an eye-
wash station and a safety shower.
C Hoods and Ventilation
1 Adequate hood facilities should be
installed where work with highly toxic
or highly flammable materials are used.
2 Hoods should be ventilated separately
and the exhaust should be terminated
at a safe distance from the building.
3 Make-up air should be supplied to
rooms or to hoods to replace the
quantity of air exhausted through the
hoods.
PC. SA. lab. 1. 11.77
1-1
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Laboratory Safety Practices
4 Hood ventilation systems are best
designed to have an air flow of not less
than 60 linear feet per minute across
.the face of the hood, with all doors open
and 1-50, if toxic materials are involved,
5 Exhaust fans should be spark-proof if
exhausting flammable vapors and
corrosive resistant if handling corrosive
fumes.
6 Controls for all services should be
located at the front of the hood and
should be operable when the hood door
is closed.
7 All laboratory rooms should have the
air changed continuously at a rate
depending on the materials being
handled.
D Electrical Services
1 Electrical outlets should be placed
outside of hoods to afford easy access
and thus protect them from spills and
corrosion by gases.
2 Noninterchangeable plugs should be
provided for multiple electrical services.
3 Adequate outlets should be provided and
should be of the three-pole type to
provide for adequate grounding.
E Storage
1 Laboratories should provide for adequate
storage space for mechanical equipment
and glassware which will be used
regularly.
2 Flammable solvents should not be stored
in glass bottles over one liter in size.
Large quantities should be stored in
metal safety cans. Quantities requiring
containers larger than one gallon should
be stored outside the laboratory.
3 Explosion proof refrigerators should be
used for the storage of highly volatile
and flammable solvents.
4 Cylinders of compressed or liquified
gases should not be stored in the
laboratory.
F Housekeeping
1 Housekeeping plays an important role
in reducing the frequency of laboratory
accidents. Rooms should be kept in a
neat orderly condition. Floors, shelves,
and tables should be kept free from
dirt and from all apparatus and chemi-
cals not in use.
2 A cluttered laboratory is a dangerous
place to work. Maintenance of a clean
and orderly work space is indicative of
interest, personal pride, and safety-
mindedness.
3 Passageways should be kept clear to all
building exits and stairways.
4 Metal containers should be provided for
the disposal of broken glassware and
should be properly labeled.
5 Separate approved waste disposal cans,
should be provided for the disposal of
waste chemicals.
6 Flammable liquids not miscible with
water and corrosive materials, or
compounds which are likely to give off
toxic vapors should never be poured
into the sink.
G Fire Protection
1 Laboratory personnel should be
adequately trained regarding pertinent
fire hazards associated with their work.
2 Personnel should know rules of fire
prevention and methods of combating
fires.
3 Fire extinguishers (CO^ type) should
be provided at convenient locations and
personnel should be instructed in their
use.
4 Automatic sprinkler systems are
effective for the control of fires in
chemical laboratories.
1-2
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Laboratory Safety Practices
H Alarms
1 An approved fire alarm system should
be provided.
2 Wherever a hazard of accidental release
of toxic gases exists, a gas alarm
system to warn occupants to evacuate
the building should be provided.
3 Gas masks of oxygen or compressed air
type should be located near exits and
selected personnel trained to use them.
Ill HANDLING GLASSWARE
A Receiving, Inspection and Storage
1 Packages containing glassware should
be opened and inspected for cracked or
nicked pieces, pieces with flaws that
may become cracked in use, and badly
shaped pieces.
2 ' Glassware should be stored on well-
lighted stockroom shelves designed and
having a coping of sufficient height
around the edges to prevent the pieces
from falling off.
B Laboratory Practice
1 Select glassware that is designed for the
type of work planned.
2 To cut glass tubing or a rod, make a
straight clean cut with a cutter or file
at the point where the piece is to be
severed. Place a towel over the piece
to protect the hands and fingers, then
break away from the body.
3 Large size tubing is cut by means of a
heated nichrome wire looped around the
piece at the point of severance.
4 When it is necessary to insert a piece
of glass tubing or a rod through a
perforated rubber or cork stopper,
select the correct bore so that the
insertion can be made without excessive
strain.
5 Use electric mantels for heating
distillation apparatus, etc,
6 To remove glass splinters, use a
whisk broom and a dustpan. Very
small pieces can be picked up with a
large piece of wet cotton.
IV GASES AND FLAMMABLE SOLVENTS
A Gas Cylinders
1 Large cylinders must be securely
fastened so that they cannot be dis-
lodged or tipped in any direction.
2 Connections, gauges, regulators or
fittings used with other cylinders must
not be interchanged with oxygen
cylinder fittings because of the possi-
bility of fire or explosion from a
reaction between oxygen and residual
oil in the fitting.
3 Return empty cylinders promptly with
protective caps replaced.
B Flammable Solvents
1 Store in designated areas well
ventilated.
2 Flash point of a liquid is the temperature
at which it gives off vapor sufficient to
form an ignitible mixture with the air
near the surface of the liquid or within
the vessel used.
3 Ignition temperature of a substance is
the minimum temperature required to
initiate or cause self-sustained com-
bustion independently of the heating or
heated element.
4 Explosive or flammable limits. For
most flammable liquids, gases and
solids there is a minimum concentration
of vapor in air or oxygen below which
propagation of flame does not occur on
contact with a source of ignition.
There is also a maximum proportion of
vapor or gas in air above which
1-3
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Laboratory Safety Practices
propagation of flame does not occur.
These limit mixtures of vapor or gas
with air, which if ignited will just
propagate flame, are known as the
"lower and higher explosive or flammable
limits."
5 Explosive Range. The difference
between the lower and higher explosive
or flammable limits, expressed in
terms of percentage of vapor or gas in
air by volume is known as the "explosive
range."
6 Vapor Density is the relative density
of the vapor as compared with air.
7 Underwriter's Laboratories Classification
is a standard classification for grading
the relative hazard of the various
flammable liquids. This classification
is based on the following scale:
Ether Class 100
Gasoline Class. 90 - 100
Alcohol (ethyl) Class. ... 60 - 70
Kerosene Class 30 - 40
Paraffin Oil Class 10 - 20
8 Extinguishing agents
V CHEMICAL HAZARDS
A Acids and Alkalies
1 Some of the most hazardous chemicals
are the "strong" or "mineral" acids
such as hydrochloric, hydrofluoric,
sulfuric and nitric.
2 Organic acids are less hazardous
because of their comparatively low
ionization potentials. However, such
acids as phenol (carbolic acid),
hydrocyanic and oxalic are extremely
hazardous because of their toxic
properties.
3 Classification of acids
B Oxidizing Materials
1 Such oxidizing agents as chlorates,
peroxides, perchlorates and perchloric
acid, in contact with organic matter
can cause explosions and fire.
2 They are exothermic and decompose
rapidly, liberating oxygen which reacts
with organic compounds.
3 Typical hazardous oxidizing agents arc:
Chlorine Dioxide
Sodium Chlorate
Potassium Chromate
Chromium Trioxide
Perchloric Acid
C Explosive Power
1 Many chemicals are explosive or form
compounds that are explosive and
should be treated'accordingly.
2 A few of the more common examples
of this class of hazardous materials are:
Acetylides
Silver Fulminate
Peroxides
Peracetic Acid
Nitroglycerine
Picric Acid
Chlorine and Ethylene
Sodium Metal
Calcium Carbide
D Toxicity
1 Laboratory chemicals improperly
stored or handled can cause injury to
personnel by virtue of their toxicity.
2 Types of exposure. There are four
types of exposure to chemicals:
a Contact with the skin and eyes
b Inhalation
c Swallowing
d Injection
1-4
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Laboratory Safety Practices
VI PRECAUTIONARY MEASURES
A Clothing and Personal Protective Equipment
1 "Chemical laboratories should have
special protective clothing and equipment
readily available for emergency use and
for secondary protection of personnel
working with hazardous materials.
2 Equipment should be provided for adequate:
a Eye protection
b Body protection
c Respiratory protection
d Foot protection
e Hand protection
B Bodily Injury
1 Burns, eye injuries, and poisoning are
the injuries with which laboratory
people must be most concerned.
2 First emphasis in the laboratory
should be on preventing accidents.
This means observing all recognized
safe practices using necessary personal
protective equipment and exercising
proper control over poisonous sub-
stances at the source of exposure.
3 So that a physician can be summoned
promptly, every laboratory should have
posted the names, telephone numbers,
and addresses of doctors to be called
in an emergency requiring medical care.
REFERENCES
Guide for Safety in the Chemical Laboratory,
the General Safety Committee of the
Manufacturing Chemists Association, Inc.,
Van Nostrand, New York (1954).
This outline was prepared by Paul F. I lallbach,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268
Descriptors; Safety, Laboratory, Practices
Safety, Laboratory Design Chemical Storage,
Gas Cylinders, Flammable Solvents
1-5
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SAMPLE HANDLING - FIELD THROUGH LABORATORY
I PLANNING A SAMPLING PROGRAM
A Factors to Consider;
1 Locating sampling sites
2 Sampling equipment
3 Type of sample required
a grab
b composite
4 Amount of sample required
5 Frequency of collection
6 Preservation measures, if any
B Decisive Criteria
1 Nature of the sample source
2 Stability of constituent(s) to be measured
3 Ultimate use of data
II REPRESENTATIVE SAMPLES
If a sample is to provide meaningful and
valid data about the parent population, it
must be representative of the conditions
existing in that parent source at the sam-
pling location.
A The container should be rinsed two or
three times with the water to be collected,
B Compositing Samples
1 For some sources, a composite of
samples is made which will represent
the average situation for stable con-
stituents.
2 The nature of the constituent to be
determined may require a series of
separate samples.
C The equipment used to collect the sample . .
is an important factor to consider. ASTM
has a detailed section on various sampling
devices and techniques.
D Great care must be exercised when collect-
ing samples in sludge or mud areas and
near benthic deposits. No definite proce-
dure can be given, but careful effort should
be made to obtain a representative sample.
Ill SAMPLE IDENTIFICATION
A Each sample must be unmistakably identified,
preferably with a tag or label. The required
information should be planned in advance.
B An information form preprinted on the tags
or labels provides uniformity of sample
records, assists the sampler, and helps
ensure that vital information will not be
omitted.
C Useful Identification Information includes:
1 sample identity code
2 signature of sampler
3 signature of witness
4 description of sampling location de-
tailed enough to accommodate repro-
ducible sampling. (It may be more
convenient to record the details in the
field record book).
5 sampling equipment used
6 date of collection
7 time of collection
8 type of sample (grab or composite)
9 water temperature
10 sampling conditions such as weather,
water level, flow rate of source, etc.
11 any preservative additions or techniques
12 record of any determinations done in
the field
13 type of analyses to be done in laboratory
WP.SUR.sg. 6a. 10.80
2-1
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Sample Handling - Field Through Laboratory
IV SAMPLE CONTAINERS
A Available Materials
1 glass
2 plastic
3 hard rubber
B Considerations
1 Nature of the sample - Organics attack
polyethylene.
2 Nature of constituent(s) to be determined
Cations can adsorb readily on some
plastics and on certain glassware. Metal
or aluminum foil cap liners can inter-
fere with metal analyses.
3 Preservatives to be used - Mineral acids
attack some plastics,
4 Mailing Requirements - Containers
should be large enough to allow extra
volume for effects of temperature
changes during transit. All caps should
be securely in place. Glass containers
must be protected against breakage.
Styrofoam linings are useful for protect-
ing glassware,
C Preliminary Check
Any question of possible interferences re-
lated to the sample container should be
resolved before the study begins. A pre-
liminary check should be made using cor-
responding sample materials, containers,
preservatives and analysis.
D Cleaning
If new containers are to be used, prelim-
inary cleaning is usually not necessary.
If the sample containers have been used
previously, they should be carefully
cleaned before use.
There are several cleaning methods avail-
able. Choosing the best method involves
careful consideration of the nature of the
sample and of the constituent(s) to be
determined.
1 Phosphate detergents should not be
used to clean containers for phosphorus
samples.
2 Traces of dichromate cleaning solution
will interfere with metal analyses.
E Storage
Sample containers should be stored and
transported in a manner to assure their
readiness for use.
V SAMPLE PRESERVATION
Every effort should be made to achieve the
shortest possible interval between sample
collection and analyses. If there must be
a delay and it is long enough to produce
significant changes in the sample, preser-
vation measures are required.
At best, however, preservation efforts
can only retard changes that inevitably
continue after the sample is removed
from the parent population.
A Functions
Methods of preservation are relatively
limited. The primary functions of those
employed are:
1 to retard biological action
2 to retard precipitation or the hydrolysis
of chemical compounds and complexes
3 to reduce volatility of constituents
B General Methods
1 pH control - This affects precipitation
of metals, salt formation and can in-
hibit bacterial action.
2 Chemical Addition - The choice of
chemical depends on the change to be
controlled.
Mercuric chloride is commonly used
as a bacterial inhibitor. Disposal of
the mercury-containing samples is a
problem and efforts to find a substitute
for this toxicant are underway.
2
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Sample Handling - Field Through Laboratory
To dispose of solutions of inorganic
mercury salts, a recommended pro-
cedure is to capture and retain the
mercury salts as the sulfide at a high
pH. Several firms have tentatively
agreed to accept the mercury sulfide
for re-processing after preliminary
conditions are met.
3 Refrigeration and Freezing - This is
¦ > I IB mmnmrnm ill I 11 !¦ !¦¦¦ i Mini i i i i
the best preservation technique avail-
able, but it is not applicable to all
types of samples. It is not always a
practical technique for field operations.
C Specific Methods
The EPA Methods Manual^) includes a
table summarizing the holding times and
preservation techniques for several ana-
lytical procedures. This information also
can be found in the standard references
(1,2, 3) as part 0f the presentation of the
individual procedures.
D Federal Register Methods
When collecting samples to be analyzed
for National Pollutant Discharge Elimi-
nation System or State Certification
report purposes, one must consult the
appropriate Federal Register^ for
information about sample handling pro-
cedures. When collecting samples to be
analyzed for compliance with maximum
contaminant levels in drinking water,
consult the EPA Report^ which includes
this information.
VI METHODS OF ANALYSIS
Standard reference books of analytical
procedures to determine the physical
and chemical characteristics of various
types of water samples are available.
A EPA Methods Manual
The Environmental Monitoring and
Support Laboratory of the Environmental
Protection Agency, has published a
manual of analytical procedures
to provide methodology for monitoring the
quality of our Nation's Waters and to deter-
mine the impact of waste discharges. The
title of this manual is "Methods for Chemical
Analysis of Water and Wastes. "(2)
For some tests, this manual refers the
analyst to Standard Methods and/or to
ASTM for the stepwise procedure.
B Standard Methods
The American Public Health Association,
the American Water Works Association
and the Water Pollution Control Federation
prepare and publish a volume describing
methods of water analysis. These include
physical and chemical procedures. The
title of this book is "Standard Methods
for the Examination of Water and Waste-
water. "(3)
C ASTM Standards
The American Society for Testing and
Materials publishes an annual "book"
of specifications and methods for testing
materials. The "book" currently consists
of 47 parts. The part applicable to water
is a book titled, "Annual Book of ASTM
Standards", Part 31, Water
D Other References
Current literature and other books of
analytical procedures with related in-
formation are available to the analyst.
E Federal Register Methodology
The analyst must consult the appropriate
Federal Register for a listing of approved
methodology if he is gathering data for
National Pollutant Discharge Elimination
System^ or State Certification'5' report
purposes, or to document compliance
with maximum contaminant levels in
drinking water'7 The Federal Register
directs the user to pages in the above
cited reference books where acceptable
procedures can be found. The Federal
Register also provides information con-
cerning the protocol for obtaining approval
to use analytical procedures other than
those listed.
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Sample Handling - Field Through Laboratory
VII ORDER OF ANALYSES
VIII RECORD KEEPING
The ideal situation is to perform all
analyses shortly after sample collection.
In the practical order, this is rarely
possible. The allowable holding time for
preserved samples is the basis for sched-
uling analyses.
A The allowable holding time for samples de-
pends on the nature of the sample, the sta-
bility of the constituent(s) to be determined
and the conditions of storage.
1 For some constituents and physical
values, immediate determination is
required, e.g. dissolved oxygen, pH.
2 Using preservation techniques, the
holding times for other determinations
range from 24 hours (BOD) to 7 days
(COD). Metals may be held up to 6
months.
3 The EPA Methods Manual^) and standard
Methods(5) include a table summarizing
holding times and preservation techniques
for several analytical procedures. Addi-
tional information can be found in the
standard references^* ^ as part of
the presentation of the individual procedures.
4 A table with proposed holding times and
preservation techniques applicable to sam-
ples collected for National Pollutant Dis-
charge Elimination System or State
Certification purposes was published in the
December 18, 1979 Register^), A similar
table for drinking water samples can be
found in a May, 1978 EPA Report^6).
5 If dissolved concentrations are sought,
filtration should be done in the field if
at all possible. Otherwise, the sample
is filtered as soon as it is received in
the laboratory. A 0. 45 micron mem-
brane filter is recommended for re-
producible filtration.
B The time interval between collection and
analysis is important and should be re-
corded in the laboratory record book.
The importance of maintaining a bound,
legible record of pertinent information
on samples cannot be over-emphasized.
A Field Operations
A bound notebook should be used. Informa-
tion that should be recorded includes;
1 Sample identification records (See Part III)
2 Any information requested by the analyst
as significant
3 Details of sample preservation
4 A complete record of data on any deter-
minations done in the field. (See B, next)
5 Shipping details and records
B Laboratory Operations
Samples should be logged in as soon as
received and the analyses performed as
soon as possible.
A bound notebook should be used. Preprinted
data forms provide uniformity of records and
help ensure that required information will be
recorded. Such sheets should be permanent-
ly bound.
Items in the laboratory notebook would include:
1 sample identifying code
2 date and time of collection
3 date and time of analysis
4 the analytical method used
5 any deviations from the analytical method
used and why this was done
6 data obtained during analysis
7 results of quality control checks on the
analysis
8 any information useful to those who
interpret and use the data
9 signature of the analyst
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Sample Handling - Field Through Laboratory
IX SUMMARY
Valid data can be obtained only from a repre-
sentative sample, unmistakably identified,
carefully collected and stored. A skilled
analyst, using approved methods of analyses
and performing the determinations within
the prescribed time limits, can produce data
for the sample. This data will be of value
only if a written record exists to verify sample
history from the field through the laboratory.
REFERENCES
1 ASTM Annual Book of Standards,
Part 31, Water, 1975.
2 Methods for Chemical Analysis of Water
and Wastes, EPA-EMSL
Cincinnati, Ohio 45268, 1979.
3 Standard Methods for the Examination of
Water and Wastewater, 14th edition
APHA-AWWA-WPCF, 1S75.
4 Dean, R., Williams, R. and Wise, R.,
Disposal of Mercury Wastes from
Water Laboratories, Environmental
Science and Technology, October,
1971.
5 Federal Register, "Guidelines Esta-
blishing Test Procedures for the
Analysis of Pollutants, " Vol. 41,
No. 232, December 1, 1976, pp
pp 52780-52786. Also, Vol. 44,
No. 244, December 18, 1979
pp 75028-75052 presents proposed
changes. The latter is scheduled
for finalization after January, 1981.
6 "Manual for the Interim Certification of
Laboratories Involved in Analyzing
Public Drinking Water Supplies -
Criteria and Procedures, " U.S. EPA
Report No. 600/8-78-008, May, 1978.
7 Federal Register, "National Interim
Primary Drinking Water Regulations, "
Vol. 40, No. 248, December 24, 1975,
pp 59566-59574. Also, 'interim Primary
Drinking Water Regulations; Amendments,
Vol. 45, No. 168, August 27, 1980,
pp 57332-57346.
This outline was prepared by Audrey Kroner,
Chemist, National Training and Operational
Technology Center, OWPO, USE PA,
Cincinnati, Ohio 45268
Descriptors: On-Site Data Collections,
On-Site Investigations, Planning, Handling,
Sample, Sampling, Water Sampling, Surface
Waters, Preservation, Wastewater
2-5
-------
METHODOLOGY FOR CHEMICAL ANALYSIS OF WATER AND WASTEWATER
I INTRODUCTION
This outline deals with chemical methods which
are commonly performed in water quality
laboratories. Although a large number of
constituents or properties may be of interest
to the analyst, many of the methods employed
to measure them are based on the same
analytical principles. The purpose of this
outline is to acquaint you with the principles
involved in commonly-used chemical methods
to determine water quality.
II PRE-TREATMENTS
For some parameters, a preliminary treatment
is required before the analysis begins. These
treatments Serve various purposes.
A Distillation - To isolate the constituent by
heating a portion of the sample mixture to
separate the more volatile part(s), and then
cooling and condensing the resulting vapor(s)
to recover the volatilized portion.
B Extraction - To isolate/concentrate the
constituent by shaking a portion of the
sample mixture with an immiscible solvent
in which the constituent is much more
soluble.
C Filtration - To separate undissolved matter
from a sample mixture by passing a portion
of it through a filter of specified size.
Particles that are dissolved in the original
mixture are so small that they stay in the
sample solution and pass through the filter.
D Digestion - To change constituents to a form
amenable to the specified test by heating a
portion of the sample mixture with chemicals.
Ill METERS
For some parameters, meters have been
designed to measure that specific constituent
or property.
CH. 14b. 11. 77
A pH Meters
pH (hydrogen ion concentration) is meas-
ured as a difference in potential across a
glass membrane which is in contact with
the sample and with a reference solution.
The sensor apparatus might be combined
into one probe or else it is divided into an
indicating electrode (for the sample) and a
reference electrode (for the reference
solution). Before using, the meter must
be calibrated with a solution of known pi I
(a buffer) and then checked for proper
operation with a buffer of a different pH
value.
B Dissolved Oxygen Meters
Dissolved oxygen meters measure the
production of a current which is proportional
to the amount of oxygen gas reduced at a
cathode in the apparatus. The oxygen gas
enters the electrode through a membrane,
and an electrolyte solution or gel acts as a
transfer and reaction media. Prior to use
the meter must be calibrated against a known
oxygen gas concentration.
C Conductivity Meters
Specific conductance is measured with a
meter containing a Wheatstone bridge which
measures the resistance of the sample
solution to the transmission of an electric
current. The meter and cell are calibrated
according to the conductance of a standard
solution of potassium chloride measured
at 25°C by a "standard" cell with electrodes
one cm square spaced one cm apart. This
is why results are called "specific" con-
ductance.
D Turbidimeters
A turbidimeter compares the intensity of
light scattered by particles in the sample
under defined conditions with the intensity
of light scattered by a standard reference
suspension.
3-1
-------
Methodology for Chemical Analysis of Water and Wastewater
IV SPECIFIC ION ELECTRODES
Just as the conventional glass electrode
for pi I develops an electrical potential in
response to the activity of hydrogen ion
in solution, the' specific ion electrode
develops an electrical potential in response
to the activity of the ion for which the electrode
is specific. The potential and activity are
related according to the Nernst equation.
Simple analytical techniques can be applied
to convert activity to an expression of con-
centration.
These electrodes are used with a pH meter
with an expanded mV scale or with a specific
ion meter. Two examples are the ammonia
and fluoride electrodes.
A Ammonia
The ammonia electrode uses a hydrophobic
gas-permeable membrane to separate the
sample solution from an ammonium chloride
internal solution. Ammonia in the sample
diffuses through the membrane and alters
the pH of the internal solution, which is
sensed by-a pH electrode. The constant
level of chloride in the internal solution is
sensed by a chloride selective ion electrode
which acts as the reference electrode.
B Fluoride
The fluoride electrode consists of a lanthanum
fluoride crystal across which a potential is
developed by fluoride ions. The cell may be
represented by Ag/Ag CI, CI (0.3), F (0.001)
LaF/test solution/SCE/. It is used in con-
junction with a standard single junction
reference electrode.
V GENERAL ANALYTICAL METHODS
A Volumetric Analysis
Titrations involve using a buret to measure
the volume of a standard solution of a sub-
stance required to completely react with
the constituent of interest in a measured
volume of sample. One can then calculate
the original concentration of the constituent
of interest.
3, There are various ways to detect the end
point when the reaction is complete.
1 Color change indicators
The method may utilize an indicator which
changes color when the reaction is
complete. For example, in the Chemical
Oxygen Demand Test the indicator,
ferroin, gives a blue-green color to the
mixture until the oxidation-reduction
reaction is complete. Then the mixture
is reddish-brown.
Several of these color-change titrations
make use of the iodometric process
whereby the constituent of interest quan-
titatively releases free iodine. Starch
is added to give a blue color until enough
reducing agent (sodium thiosulfate or
phenylarsine oxide) is added to react
with all the iodine. At this end point,
the mixture becomes colorless.
2 Electrical property indicators
Another way to detect end points is a
change in an electrical property of the
solution when the reaction is complete.
In the chlorine titration a cell containing
potassium chloride will produce a small
direct current as long as free chlorine
is present. As a reducing agent (phen-
ylarsine oxide) is added to reduce
the chlorine, the micro ammeter which
measures the existing direct current
registers a lower reading on a scale.
By observing the scale, the end point of
total reduction of chlorine can be
determined because the direct current
ceases.
3 Specified end points
For acidity and alkalinity titrations, the
end points are specified pH values for
the final mixture. The pH values are
those existing when common acidity or
alkalinity components have been neutral-
ized. Thus acidity is determined by
titrating the sample with a standard
alkali to pH 8. 2 when carbonic acid
would be neutralized to (HCOg)~. Alka-
linity (except for highly acidic samples)
is determined by titrating the sample
with a standard acid to pH 4. 5 when the
carbonate present has been converted
to carbonic acid. pH meters are used to
detect the specified end points.
-------
Methodology for Chemical Analysis of Water and Wastewater
B Gravimetric Procedures
Gravimetric methods involve direct
weighing of the constituent in a container.
An empty container is weighed, the
constituent is separated from the sample
mixture and isolated in the container, then
the container with the constituent is weighed.
The difference in the weights of the container
before and after containing the constituent
represents the weight of the constituent.
The type of container depends on the method
used to separate the constituent from the
sample mixture. In the solids determinations,
the container is an evaporating dish (total or
dissolved) or a glass fiber filter disc in a
crucible (suspended). For oil and grease,
the container is a flask containing a residue
after evaporation of a solvent.
C Combustion
Combustion means to add oxygen. In the
Total Organic Carbon Analysis, combustion
is used within an instrument to convert
carbonaceous material to carbon dioxide.
An infrared analyzer measures the carbon
dioxide.
VI PHOTOMETRIC METHODS
These methods involve the measurement of light
that is absorbed or transmitted quantitatively
either by the constituent of interest or else by
a substance containing the constituent of interest
which has resulted from some treatment of
the sample. The quantitative aspect of these
photometric methods is based on applying the
Lambert-Beer Law which established that the
amount of light absorbed is quantitatively
related to the concentration of the absorbing
medium at a given wavelength and a given
thickness of the medium through which the
light passes.
Each method requires preparing a set of
standard solutions containing known amounts
of the constituent of interest. Photometric
values are obtained for the standards. These
are used to draw a calibration (standard) curve
by plotting photometric values against the
concentrations. Then, by locating the photo-
metric value for the sample on this standard
curve, the unknown concentration in the
sample can be determined.
A Atomic Absorption
Atomic Absorption (AA) instruments utilize
absorption of light of a characteristic wave-
length. This form of analysis involves
aspirating solutions of metal ions (cations)
or molecules containing metals into a
flame where they are reduced to individual
atoms in a ground electrical state. In this
condition, the atoms can absorb radiation
of a wavelength characteristic for each
element. A lamp containing the element of
interest as the cathode is used as a source
to emit the characteristic line spectrum, for
the element to be determined.
The amount of energy absorbed is directly
related to the concentration of the element
of interest. Thus the Lambert-Beer Law
applies. Standards can be prepared and
tested and the resulting absorbance values
can be used to construct a calibration
(standard) curve. Then the absorbance
value for the sample is located on this curve
so determine the corresponding concentration.
Once the instrument is adjusted to give
optimum readings for the element of interest,
the testing of each solution can be done in
a matter of seconds. Many laboratories
wire recorders into their instruments to
rapidly transcribe the data, thus conserving
time spent on this aspt.ct of the analysis.
Atomic absorption techniques are generally
used for metals and semi-mctals in solution
or else solubilized through some form of
sample processing. For mercury, the
principle is utilized but the absorption of
light occurs in a flameless situation with
the mercury in the vapor state and contained
in a closed glass cell.
B Flame Emission
Flame emission photometry involves
measuring the amount of light given off by
atoms drawn into a flame. At certain
temperatures, the flame raises the electrons
in atoms to a higher energy level. When
the electrons fall back to a lower energy
level, the atoms lose (emit) radiant energy
which can be detected and measured.
Again standards must be prepared and
tested to prepare a calibration (standard)
curve. Then the transmission value of the
sample can be located on the curve to
determine its concentration.
Many atomic absorption instruments can be
used for flame emission photometry.
Sodium and potassium are very effectively J-3
determined by the emission technique.
-------
Methodology for Chemical Analysis of Water and Wastewater
However, for many elements, absorption
analysis is more sensitive because there are
i great number of unexcited atoms in the
flame which are available to absorb the
radiant energy.
C Colorimetry
Colorimetric analyses involve treating
standards which contain known concentrations
of the constituent of interest and also the
sample with reagents to produce a colored
solution. The greater the concentration of
the constituent, the more intense will be
the resulting color.
The Lambert-Beer Law which relates the
absorption of light to the thickness and
concentration of the absorbing medium
applies. Accordingly, a spectrophotometer
is used to measure the amount of light of
appropriate wavelength which is absorbed
by the same thickness of each solution.
The results from the standards are used to
construct a calibration (standard) curve.
Then the absorbance value for the sample
is located on this curve to determine the
corresponding concentration.
Many of the metals and several other
parameters (phosphorus, ammonia, nitrate,
nitrite, etc. ) are determined in this
manner.
VII GAS-LIQUID CHROMATOGRAPHY
Chromatography techniques involve a separa-
tion of the components in a mixture by using
a difference in the physical properties of the
components. Gas-Liquid Chromatography
(GLC) involves separation based on a differ-
ence in the properties of volatility and solu-
bility. The method is used to determine
algicides, chlorinated organic compounds
and pesticides.
The sample is introduced into an injector
block which is at a high temperature (e. g.
210°C), causing the liquid sample to volatilize.
An inert carrier gas transports the sample
components through a liquid held in place as
a thin film on an inert solid support material
in a column.
3-i
Sample components pass through the column
at a speed partly governed by the relative
solubility of each in the stationary liquid.
Thus the least soluble components are the
first to reach the detector. The type of
detector used depends on the class of compounds
involved. All detectors function to sense and
measure the quantity of each sample component
as it comes off the column. The detector
signals a recorder system which registers
a response.
•\h with other iii.su methods, standards
with known concentrations of the substance of
interest are measured on the instrument. A
calibration (standard) curve can be developed
and the concentration in a sample can be
determined from this graph.
Gas-liquid chromatography methods are very
sensitive (nanogram, picogram quantities) so
only small amounts of samples are required.
On the other hand, this extreme sensitivity
often necessitates extensive clean-up of
samples prior to GLC analysis,
VII i AUTOMATED METHODS
The increasing number of samples and
measurements to be made in water quality
laboratories has stimulated efforts to automate
these analyses. Using smaller amounts of
sample (semi-micro techniques), combining
reagents for fewer measurements per analysis,
and using automatic dispensers are all means
of saving analytical time.
However, the term "automated laboratory
procedures" usually' means automatic intro-
duction of the sample into the instrument,
automatic treatment of the sample to test for
a component of interest, automatic recording
of data and, increasingly, automatic calculating
and print-out of data. Maximum automation
systems involve continuous sampling direct
from the source (e. g. an in-place probe) with
telemetering of results to a central computer.
Automated methods, especially those based on
colorimetric methodology, are recognized for
several water quality parameters including
alkalinity, ammonia, nitrate, nitrite, phosphorus,
and hardness.
-------
Methodology for Chemical Analysis of Water and Wastewater
IX SOURCES OF PROCEDURES
Details of the procedure for an individual
measurement can be found in reference books.
There are three particularly-recognized books
of procedures for water quality measurements.
A Standard Methods^
The American Public Health Association,
the American Water Works Association
and the Water Pollution Control Federation
prepare and publish "Standard Methods for
the Examination of Water and Wastewater. "
As indicated by the list of publishers, this
book contains methods developed for use by
those interested in water or wastewater
treatment.
B ASTM Standards(2)
The American Society for Testing and
Materials publishes an "Annual Book of
ASTM Standards" containing specifications
and methods for testing materials. The
"book" currently consists of 47 parts.
The part applicable to water was formerly
Part 2 3. . It is now Part 31 j Water.
The methods are chosen by approval of the
membership of ASTM and are intended to
aid industry, government agencies and the
general public. Methods are applicable to
industrial waste waters as well as to other
types of water samples.
C EPA Methods Manual3)
The United States Environmental Protection
Agency publishes a manual of "Methods for
Chemical Analysis of Water and Wastes. "
EPA developed this manual to provide
methodology for monitoring the quality of
our Nation's waters and to determine the
impact of waste discharges. The test pro-
cedures were carefully selected to meet
these needs, using Standard Methods and
ASTM as basic references. In many cases,
the EPA manual contains completely
described procedures because they modified
methods from the basic references. Other-
wise, the manual cites page numbers in
the two references where the analytical
procedures can be found.
X ACCURACY AND PRECISION
A Of the Method
One of the criteria for choosing methods
to be used for water quality analysis is that
the method should measure the desired
property or constituent with precision,
accuracy, and specificity sufficient to meet
data needs. Standard references, then,
include a statement of the precision and
accuracy for the method which is obtained
when (usually) several analysts in different
laboratories used the particular method.
B Of the Analyst
Each analyst should check his own precision
and accuracy as a test of his skill in per-
forming a test. According to the U. S. EPA
Handbook for Analytical Quality Control^,
he can do this in the following manner.
To check precision, the analyst should
analyze samples with four different
concentrations of the constituent of interest,
seven times each. The study should cover
at least two hours of normal laboratory
operations to allow changes in conditions
to affect the results. Then he should
calculate the standard deviation of each of
the sets of seven results and compare his
values for the lowest and highest concen-
trations tested with the standard deviation
value published for that method in the reference
book. (It may be stated as % relative
standard deviation. If so, calculate
results in this form.) An individual
should have better values than those
averaged from the work of several
analysts.
To check accuracy, he can use two of the
samples used to check precision by adding
a known amount (spike) of the particular
constituent in quantities to double the lowest
concentration used, and to bring an inter-
mediate concentration to approximately 75%
of the upper limit of application of the
method. He then analyzes each of the spiked
samples seven times, then calculates the
average of each set of seven results. To
calculate accuracy in terms of % recovery,
he will also need to calculate the average of
3-5
-------
Methodology for Chemical Analysis of Water and Wastewater
the results he got when he analyzed the
unspiked samples (background). Then:
% Recovery
observed - background
n X 100
spike I
The actual calculation involves volume -
concentration calculations for each term.
If the published accuracy is stated as
% bias, subtract 100% from % recovery
to compare results. Again, the individual
result should be better than the published
figure derived from the results of several
analysts.
C Of Daily Performance
Even after an analyst has demonstrated his
personal skill in performing the analysis,
a daily check on precision and accuracy
should be done. About one in every ten
samples should be a duplicate to check
precision and about one in every ten samples
should be spiked to check accuracy.
It is also beneficial to participate in inter-
laboratory quality control programs. The
U. S. EPA provides reference samples at
no charge to laboratories. These samples
serve as independent checks on reagents,
instruments or techniques; for training
analysts or for comparative analyses within
the laboratory. There is no certification
or other formal evaluative function resulting
from their use.
XI SELECTION OF ANALYTICAL
PROCEDURES
Standard sources^1, "> 3) will, for most
parameters, contain more than one analytical
procedure. Selection of the procedure to be
used in a specific instance involves consider-
ation of the use to be made of the data. In
some cases, one must use specified procedures.
In others, one may be able to choose among
several methods.
A NPDES Permits and State Certifications
A specified analytical procedure must be
used when a waste constituent is measured:
1 For an application for a National Pollutant
Discharge Elimination System (NPDES)
permit under Section 402 of the Federal
Water Pollution Control Act (FWPCA),
as amended.
2 For reports required to be submitted by
dischargers under NPDES.
3 For certifications issued by States
pursuant to Section 401 of the FWPCA,
as amended.
Analytical procedures to be used in these
situations must conform to those specified
in Title 40, Chapter 1, Part 136, of the
Code of Federal Regulations (CFR). The
listings in the CFR usually cite two different
procedures for a particular measurement.
The CFR also provides a system of
applying to EPA for permission to
use methods not cited in the CFR.
Approval of alternative methods for
nationwide use will be published in
the Federal Register.
B Ambient Water Quality Monitoring
For Ambient Water Quality Monitoring,
analytical procedures have not been
specified by regulations. However, the
selection of procedures to be used should
receive attention. Use of those listed in
the CFR is strongly recommended. If
any of the data obtained is going to be used
in connection with NPDES permits, or may
be used as evidence in a legal proceeding,
use of procedures listed in the CFR is
again strongly recommended.
3-6
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Methodology for Chemical Analysis of Water and Wastewater
C Drinking Water Monitoring
In December, 1975, National Interim
Primary Drinking Water Regulations
to be effective June 24, 1977 were
published in the Federal Register in
Title 40, Chapter 1, Subchapter D,
Part 141. The publication includes
specification of analytical procedures
to be used when determining compliance
with the maximum contaminant levels
of required parameters.
Because of the low concentrations in-
volved in the regulations, there is often
just one analytical method cited for
each parameter.
Individuals or organizations may apply
to EPA for permission to use methods
not cited in the above. Approval of
alternative methods for nationwide use
will be published in the Federal Register.
XII FIELD KITS
Field kits have been devised to perform
analyses outside of the laboratory. The kit
may contain equipment and reagents for only
one test or for a variety of measurements.
It may be purchased or put together by an
agency to serve its particular needs.
Since such kits are devised for performing
tests with minimum time and maximum
simplicity, the types of labware and reagents
employed usually differ significantly from the
equipment and supplies used to perform the
same measurement in a laboratory.
A Shortcomings
Field conditions do not accommodate the
equipment and services required for pre-
treat ments like distillation and digestion.
Nor is it practical to carry and use calibrated
glassware like burets and volumetric pipets.
Other problems are preparation, transport
and storage of high quality reagents, of
extra supplies required to test for and remove
sample interferences before making the
measurement, and of instruments which
are very sensitive in detecting particular
constituents. One just cannot carry and
set up laboratory facilities in the field which
are equivalent to stationary analytical
facilities.
B Uses
Even though the results of field tests are
usually not as accurate and precise as those
performed in the laboratory, such tests do
have a place in water quality programs.
In situations where only an estirra te of the
concentrations of various constituents is
required, field tests serve well. They are
invaluable sources of information for
planning a full-scale sampling/testing
program when decisions must be made
regarding location of sampling sites,
schedule of sample collection, dilution of
samples required for analysis, and treat-
ment of samples required to remove inter-
ferences to analyses.
C NPDES Permits and State Certification
Kit methods are not approved for obtaining
data required for NPDES permits or State
construction certifications. If one judges
that such a method is justifiable for these
purposes, he must apply to EPA for per-
mission to use it.
I) Drinking Water Monitoring
The DPD test kit for residual chlorine is
approved in the December, 1975 Federal
Register for monitoring drinking water.
3-7
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Methodology for Chemical Analysis of Water and Wastewater
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater, 14th Edition.
1976, APHA-AWWA-WPCF, 1015 18th Street,
N.W., Washington, D. C. 20036
2 1975 Annual Book of ASTM Standards,
Part 31, Water. ASTM, 1916 Race Street,
Philadelphia, PA 19103.
3 Methods for Chemical Analysis of Water
and Wastes. 1979, U. S. EPA, EMSL.
Cincinnati, OH 45268.
4 Handbook for Analytical Quality Control
in Water and Wastewater Laboratories,
1972. u. S. EPA. EMSL, Cincinnati,
Ohio 45268.
This outline was prepared by Audrey Kroner,
Chemist, National Training and Operational
Technology Center, OWPO, U. S. EPA,
Cincinnati, Ohio 45268,
Descriptors: Analysis, Chemical Analysis,
Methodology, Wastewater, Water Analysis
-------
I INTRODUCTION
A Statistics may be defined, for our purpose,
as a collection of methods which have been
developed for handling numerical data
pertaining to samples or portions of entire
"populations.
B The statistical methods with which we will
concern ourselves deal with the presentation
and analysis of numerical data from samples.
II FREQUENCY
A Definitions
1 Frequency - indicates how many times
a particular score occurs in a collection
of data
table - a tabular arrange-
ment of data, ranked in ascending or
descending order of magnitude,
together with the corresponding
frequencies
3 Frequency histogram - a set of
rectangles having bases on a horizontal
axis with centers at the given scores
and heights equal to the corresponding
frequencies (See Figure 1)
4 Frequency polygon - a line graph of
frequencies plotted against scores
(can be obtained by connecting mid-
points of tops of rectangles in the
frequency histogram) (See Figure 1)
STATISTICS FOR CHEMISTS
2 Frequency
Figure 1
Frequency Histogram & Frequency Polygon
V Q
c 0
0)
&
£ 2
£
7^
98
100 101 102
Chloride |jg/l
ST. 25b. 11. 77
4-1
-------
Statistics For Chemists
B Application
Consider the application of the above
definitions to the following set of data,
obtained from twelve determinations for
chloride in water.
Results (tig/1)
100 101 99
101 100 100
99 102 100
98 101 102
number of observations the median is
Xn + Xn
+ 1
, the average of the
middle two scores.
4 Mean - arithmetic average of all the
values in the sample distribution, de-
noted by X. The formula for calcula-
ting the sample mean is
• xn
X
+ Xg
n
X =
n
ifix'
n
Table 1
Frequency Table
Chloride (|xg/l) Frequency
98
99
100
101
102
1
2
4
3
2
III MEASURES OF CENTRAL TENDENCY
A Definitions
1 Central tendency - the tendency of
values to cluster about a particular
value in the distribution
2 Mode - that value which occurs most
fre quently
3 Median - midpoint of an array of
scores. If there is an odd number of
observations, n, the median is
X.
the
n +1
~2
n + 1
where
X
n' + 1
represents
value in the frequency
distribution, jf there is an even
— 2X*
X = —i where there are n number
of values.
B Aids in calculation of the mean
Application of the following two statements
can reduce errors and amount of time
spent in calculating the mean of a
distribution.
1 Adding or subtracting a constant to or
from each score in a distribution is
equivalent to adding or subtracting the
same constant to or from the mean of
the distribution. Thus the following
formula:
X = X± c
c
where the X^'s are the
values in the distribution with mean X,
and the X^ ± Cs are the values in the
distribution with mean Xn.
Multiplying or dividing each score in
a distribution by a constant is equivalent
to multiplying or dividing the mean of
the distribution by the same constant.
Thus the following formulas:
(1) X,, = CX
or
(2) Xr
- 5
-c ~ c _
values in the distribution with mean X,
where the X-'s are the
4-2
-------
Statistics for Chemists
X'
and the CX-'s or the 's are the
values in the distribution with mean
X
C Application
Consider the application of the above
definitions to the previously mentioned
set of data, obtained from twelve deter-
minations for chloride in water, shown
in Table 1.
1 Mode = 100
2 Median =
100 + 100
+ Xn
2 2 _ Xfi + X7
2 2
= 100
X
Denote the mean of the distribution in
Table 1 by X . If we add 100 to each
score in the (distribution in Table 2, we
obtain the scores in the distribution in
Table 1; likewise if we add 100 to the
mean, X, of the distribution in Table 2,
we obtain the mean, X , of the distri-
bution in Table 1.
Thus X = X + 100
c
x =— + 100
c n
l(-2) + 2(-1) + 4(0) + 3(1) + 2(2)
12
.25 + 100 = 100.25
+ 100
3 Mean =
EX}
98 + 2 (99) + 4 (100) + 3 (101) + 2 (102)
12
100.25
4 Aid in Calculation
Consulting Table 1 and observing that
the values are in the neighborhood of
100 we might subtract 100 from each
score and obtain the following distribution:
Table 2
Frequency Table
Chloride (txg/1) Frequency
-2 1
-1 2
0 4
1 3
2 2
IV MEASURES OF DISPERSION
A Definitions
1 Dispersion - spread or variability of
observations in a distribution
2 Range - the difference between the
highest value and the lowest value
R
max - mm
3 Average deviation - the sum of the
deviations of the values from their
mean, without regard to sign, divided
by the total number of data values (n)
The formula for calculating the average
deviation is;
D|X. - Xl
d - 1
4-3
-------
Statistics for Chemists
4 Average deviation of the mean (D) -
the average deviation of individual
data items from the mean (d) divided
by the square root of the number of
data items (n)
The definition of the average deviation
of the mean can be expressed by the
formula:
d '
D =
5 Variance - the sum of the squares of
the deviations of the values from their
mean divided by the total number of
data values (n) minus 1
The definition of the variance can be
expressed by the following formula:
E(Xi - X)2
6 Standard deviation - the square root
of the variance
The definition of the standard
deviation of the mean can be
expressed by the formula:
S =
\Tn~ '
8 Relative standard deviation - the
standard deviation (s) expressed as
a fraction of the mean, s
X
The relative standard deviation is
often expressed as a percent. It is
then referred to as the coefficient
of variation (V), or % relative standard
deviation:
V = - x 100
X
The relative standard deviation is .
particularly helpful when comparing
the precision of a number of deter-
minations on a given substance at
different levels of concentration.
B Aids in Calculation
The definition of the standard deviation
can be expressed by the following
formula:
E(Xi - X)
n
However, the formula commonly used
because of its adaptability to the hand
calculator is the following:
SX-1
(2Xi>
n
n
where there are
n number of values.
Standard deviation of the mean (S) - the
standard deviation of individual data
items (s) divided by the square root of
the number of data items (n)
Application of the following statements
can reduce errors and amount of time
spent in calculating the variance or
standard deviation of a distribution.
1 Adding or subtracting a constant to or
from each score in a distribution
doesn't affect the variance or standard
deviation of the distribution.
Thus the following formulas;
(1)
2 2
s a s
c
(2) s = s
c
where the X^'s are the values in
the distribution with variance s2
and standard deviation s, and the
Xj + C's are the values in the
distribution with variance s ^
and standard deviation s„. C
4-4
-------
Statistics for Chemists
Multiplying or dividing each score In
a distribution by a constant is equivalent
to multiplying or dividing the variance
of that distribution by the square of the
same constant.
Thus the following formulas;
(1) s/
(2) sj3 = 4
C2s2
where the Xj's are the values in
o
the distribution with variance s ,
X ¦
and the CX^'s or the * "s are
the values in the distribution with
9
variance s„ .
c
Multiplying or dividing each score in a
distribution by a constant is equivalent
to multiplying or dividing the standard
deviation of that distribution by the
same constant.
Thus the following formulas:
s = Cs
(1)
or
(2)
s
C
where the Xj's are the values in
the distribution with standard
deviation s, and the CX.'s or the
Y,
i 's are the values in the
C
distribution with standard
deviation s .
C Application
Consider the application of the above
definitions to the previously mentioned
set of data, obtained from twelve
determinations for chloride in water,
shown in II B. Table 1.
2 Average deviation - d
ZlXi- XI
n
n Xi
|Xi
- x!
nlXi- X|
1 98
2.
25
iO
CM
CM
2 99
1.
25
2. 50
4 100
.
25
1. 00
3 101
-
75
2. 25
2 102
1.
75
3. 50
X = 100.25
11. 50
,Z|Xj- XI
n
11. 50
12
. 96
3 Average deviation of the mean
d
\T~n
D
Using calculations from number 2,
0,96 "* 0. 96 — q 20
4T 3'4G
4 Variance - s
2 _ S(Xi- X)'
n-1
n
Xi
Xi-X
(Xi- X)2
n(Xi- X)
1
98
-2. 25
5.06
5. 06
2
99
-1. 25
1. 56
3. 12
4
100
- . 25
. 06
. 24
3
101
+ . 75
. 56
1. 68
2
102
+1. 75
3. 06
6. 12
16. 22
S(Xi- X)2 16.22
n
11
1. 47
1 Range = 102 -98 = 4
4-5
-------
Statistics for Chemists
Standard deviation
n
Si
nXj
X2-
nX2i
1
98
98
9604
9604
2
99
198
9801
19602
4
100
400
10000
40000
3
101
303
10201
30603
2
102
204
10404
20808
1203
120617
120617
120617 - 120601
• 11
17
2 12
s
11
16. 25
11
1. 48
EX, - (LX.)
i r_ = /1.48
1. 22
6 Aid in calculation
n - 1
7 Standard deviation of the mean -
S = —
•J n
Using calculations from number 6,
S =-§_= hiI- hll - n ^
b — —— - 0.35
^^n ^^T2
8 Relative standard deviation expressed
as a percent (coefficient of variation)
Recalling that adding or subtracting a
constant to each score in the distri-
bution doesn't affect the variance or
the standard deviation of the distribu-
tion we can simplify the computations
by first subtracting 100 from each
score in the distribution, thus obtain-
ing the frequency distribution shown
in Table 2.
n
3z
C n(XrC)
(xrc)2
n(Xr
1
_2
-2
4
4
2
-1
-2
1
2
4
0
0
0
0
3
1
3
1
3
2
2
4
4
8
3
17
2
2X. -
(ZXi)2
2
c
l
n
D
n
- 1
V = X100
X
Using calculations from number 6 for
s = 1. 22 and from number 2 for
X = 100.25,
V =-
X
1.22
100.25
X 100 = 1.21%
Figure 2
Normal Distribution Curve
Quantity Measured
4-6
-------
Statistics for Chemists
V INTRODUCTION TO NORMAL
DISTRIBUTION CURVE
A Statistics deals with theoretical curves
which are smoother than frequency
polygons, obtained from experiments in
real life. However, frequency distribu-
tions or frequency polygons of experimental
data often approximate a mathematical
function called the normal distribution
curve. (See Figure 2)
As shown in Figure 3, the frequency polygon
for the 12 determinations for chloride in
water is a fairly good approximation of the
normal curve. If, however, in the chloride
determinations we had obtained 103 instead
of-98 and 104 instead of 99 this distribution
would not have- been a good approximation of
the normal curve, as is shown in Figure 4.
Figure 3
Comparison of Normal Curve and Frequency Polygon
4
3
2
1
102
101
100
98
99
97
Chloride -jg/1
Figure 4
Comparison of Normal Curve and Frequency Polygon
4
3
2
1
102
103
104
100
101
99
Chloride (ig/1
4-7
-------
Statistics for Chemists
If a frequency distribution is a good
approximation of the normal curve, we
can use some facts about the normal
curve to give us information about the
frequency distribution.
Figure 5 shows the normal distribution
in terms of the population mean u, and
the standard deviation of the population
o , and gives the percent of area under
the curve between certain points.
Figure 5
Normal Distribution Curve
U +lcr
Figure 6
Frequency Distribution Polygon
o
d
6)
&
-------
Statistics for Chemists
We may check the distribution, of sample
data to see if it is a "normal" distribution
in the following manner. Substitute the
value of the sample mean (X) for the value
of the midline and substitute the value of
the sample standard deviation (s) for the
limits of the value spans where we might
expect certain percentages of the data
items to occur. Then we can check the
number of data items which actually do
occur within these value spans.
Figure 6 demonstrates this application
using the chloride data values from Table 1.
The data values are marked on the hori-
zontal line and the frequency of the
occurrence of each value is marked on the
vertical. The midline of the distribution
is marked at the value of the sample mean
(3T = 100, See III C 3), The value of the
sample standard deviation (s = 1.21, See
IV C 5) is used to mark value areas under
the curve where different percentages of
data values will probably occur. Thus,
for the area X + Is, X - Is = 98. 79 and
X + 1 s = 101.21. Therefore, according
to the normal distribution curve shown in
Figure 5, we might expect about 68% of the
data items to have values between 99 and
101. (The values are rounded to whole
numbers since the data values are thus
recorded).
It would be good to become as familiar as
possible with the normal distribution since
an underlying normal distribution is
assumed for many statistical tests of
hypothesis.
REFERENCES
1 Bennett, C. A. and Franklin, N. L.
Statistical Analysis in Chemistry and
the Chemical Industry. John Wiley
& Sons, Inc., New York. 1954.
2 Crow, E. L., Davis, F. A., and Maxfield,
M.W. Statistics Manual. Dover
Publications, Inc., New York. 1960.
3 Dixon, W. J. and Massey, F. J.
Introduction to Statistical Analysis.
McGraw-Hill Book Co., Inc., New
York. 1957.
4 Ostle, B. Statistics in Research. The
Iowa State University Press. Iowa,
1963.
Youdon, W. J.
Chemists.
New York.
Statistical Methods for
John Wiley & Sons, Inc.,
1951.
Consulting Table 1, we find that 75% or 9
of the 12 data items have values in this
range. This percentage is shown in
Figure 6 by the frequency polygon for the
data shown earlier in Figure 3.
Likewise assuming a normal distribution,
we would expect 95% of the observations
to lie within + 2 a ' s from the population
mean. In fact, 100% of the observations
were within + 2 s's from the sample mean.
In both cases the observed percentages are
reasonably close to the expected percentages.
Other tests exist for determining whether
or not a frequency distribution might
reasonably be assumed to approximate
the normal distribution.
This outline was prepared by L. A. Lederer,
Statistician, formerly with Analytical
Reference Service, Training Program,
NCUIH, SEC. Revised by Audrey Kroner,
Chemist, National Training and Operational
Technology Center, OWPO, USEPA,
Cincinnati, Ohio 45268.
Descriptors: Graphic Methods, Quality
Control, Statistical Methods, Statistics
4-9
-------
ACCURACY-PRECISION-ERROR
I INTRODUCTION
An analytical method is subject to errors.
These errors may affect the accuracy of the
method because they introduce bias into the
results. There are other types of errors
which affect the precision of the method
because they produce random fluctuations in
the data. The most desirable situation for
the analyst is shown in the diagram in
Figure 1 where the results are both precise
and accurate.
IMPRECISE AND INACCURATE
PRECISE BUT INACCURATE
ACCURATE BUT IMPRECISE
PRECISE AND ACCURA
Figure 1. PRECISION AND ACCURACY
A Accuracy
For results to be accurate, the analysis
used must give values close to the true
value.(See Figure 1)
B Precision
Precision is the degree of agreement
among results obtained by repeated
measurements on a single sample under
a given set of conditions. It is a measure,
of the degree to which results "check. "
(See Figure 1)
C Note
It is possible to have precision without
accuracy. (See Figure 1)
II DETERMINATE ERROR AND ACCURACY
A determinate error is one which con-
tributes a constant error or bias to results,
causing them to be inaccurate. This
constant error makes it possible for results
to agree with each other (be precise) and
still be inaccurate.( See Figure 1)
Determinate errors have "assignable"
causes which can usually be identified and
either eliminated or controlled. (The terms
"determinate" error, "assignable" error,
and "systematic" error are synonymous).
A Sources of Determinate Error
1 Method error
Method errors are those that are
inherent in the procedure. These are
very serious and the hardest to detect
and correct. The most common
method error is the presence of inter-
ferences in the sample. Other
examples would be precipitation of
substances other than the desired
, material, partial solubility of pre-
cipitates, and entrainment as in a
solvent extraction procedure.
CH. MET. con. Id. 11. 77
5-1
-------
A ccuracy-Precision-Error
2 Personal errors
Personal errors are attributable to
individual mistakes which are con-
sistently made by an analyst. These
errors are the result of consistent
carelessness, lack of knowledge or
personal bias. Examples are errors
in calculations, use of contaminated
reagents, non-representative sampling,
or poor calibration of standards and
instruments.
3 Instrumental errors
Instrumental errors are those which
are caused by an analytical instrument
or by the effects of the environment
acting on the instrument. Moisture in a
G. C. column, improper wavelength
markings on a spectrophotometer or
incorrect scoring on a buret would be
examples.
B Effects of Determinate Error
1 Additive
An additive determinate error is one
which has a constant value regardless of
the amount of analytically sought con-
stituent present in the sample.
(See Figure 2)
2 Proportional
A proportional determinate error
changes value according to the amount
of analytically sought constituent in the
sample. (See Figure 3)
III DETECTION OF DETERMINATE ERROR
A Spiked Samples
1 Samples which can be determined before
and after the addition of a known con-
stituent ( in the concentration range of
interest) provide a way to detect
determinate errors. Spiked samples
should be representative and resemble
actual conditions as closely as possible,
¦ The quantitation of bias can then be
obtained with the following measures.
a Mean error - the difference between
the mean of the data and the true
result.
b Relative error - the mean error of
a set of data expressed as a per-
centage of the true result.
EXAMPLE; An analyst determines
the nitrate content of the effluent
from his sewage treatment plant to be
0. 50 mg/1. He then adds 1 mg/ 1 of
standard nitrate solution to the
sample. Table 1 shows the replicate
results obtained on the spiked
sample, and calculation of both
mean and relative errors.
Table 1
Sample: Effluent
Determination: Nitrate (Modified Brucine)
Xi
1.35 1.56
1.47 1,59
1.49 1.60
1.55
1 Calculation of mean error
X « 10^61 = 1. 51 mg/l
Mean Error = 1,51-1.50
= + 0. 01 mg/ 1
2 Calculation of relative error
% Relative Error =+ °' ^ fn1Q° =+0- 7 >'<
1.50
2 Control charts
Trends and shifts on control charts
may also indicate determinant error.
Using spiked samples, the standard
deviation is calculated and control
limits (usually + 3 standard deviations)
for the analysis are set (see Figure 4.
For further discussion of control limits,
see reference 3, p. 62.
5-2
-------
Accuracy-Precision-Error
LU
3
<
>
<
U
l—
>-
_i
<
Z
<
LU
3
<
>
<
VJ
H-
>-
I
<
z
<
2
THEORETICAL VALUE THEORETICAL VALUE
Figure 2. ADDITIVE ERROR Figure 3. PROPORTIONAL ERROR
UPPER
CONTROL LIMIT
LOWER
CONTROL LIMIT
¦g, OUT OF
CONTROL
12 3456789 tO
DAY NUMBER
Figure 4. CONTROL CHART
£-3
-------
A c cura cy- Pre cision- Error
3 In some cases, it is impossible to
spike a sample so that it resembles
actual conditions (e.g., BOD and
pesticide samples). Youden
provides excellent techniques for
detecting bias in this situation.
B Unknown Samples
1 Independent method
Analysis of a sample for a desired
constituent by two or more methods
that are entirely different in principle
(gravimetric and volumetric) may aid
in the estimation of determinate error.
However, another reliable method may
not be available or may be laborious to
perform.
2 Control charts
It is possible to plot a control chart
(Figure 4) even when it is not possible
to spike a sample. One can use as a
reference value an average of a series
of replicate determinations performed
on a composite check sample. Such a
sample must be preserved or stabilized
in such a way that the concentration
of the constituent being measured will
not change from day to day (see
reference 1).
3 Aliquoting
If the determinate error is additive,
the magnitude may be estimated by
plotting the measured quantity versus
a range of sample volumes or sample
weights. If the error has a constant
value regardless of the amount of
analytically sought constituent, then
a straight line fitted to the points will
not go through the origin. (See Figure 5)
C You den's Graphical T echnique (10, 11, 12)
Dr. W. J. Youden has devised an
approach to test for determinant errors
with a minimum of effort on the part
of the analyst and his laboratory.
Samples used may be of known (spiked)
or unknown composition.
1 Technique
Two different test samples (X and Y)
are prepared and distributed for
analysis to as many individuals or
laboratories as possible. Each
participant is asked to perform
only one determination on each
sample (NOTE; It is important
that the samples be relatively
similar in concentration of the
constituent being measured).
Table 2 shows the results on two
such samples analyzed for percent
potassium by 14 different laboratories.
The mean for each sample lias been
calculated.
2 Interpretation - Figure 6
The vertical line drawn on the
graph represents the mean (X)
of all the results obtained on
Sample X; the horizontal line was
drawn through the mean (Y) of all
the results obtained on sample Y.
Each pair of laboratory results
can then be plotted as a point on
the graph (marked X. 1 etc.)
If the ratio of the bias (error) to
standard deviation is close to zero
for the determinations submitted
by the participants, then one would
expect the distribution of the paired
values (or points) to be close to
equal among the four quadrants.
The fact that the majority of the
points fall in the {+, +) and (-, -)
quadrants indicates that the results
have been influenced by some source
of bias or determinate error.
Furthermore, one can even learn
something about a participant's
precision from the graph. If all
participants had perfect precision
(no indeterminate error), then all
the paired points would fall on a
450 line passing through the origin.
Consequently the distance from such
a 45o line of each participant's
point provides an indication of that
participant's precision.
5-4
-------
Accuracy-Precision-Error
Table 2
Laboratory
Sample X
Sample Y
1
9. 74%
8
50%
2
9.92
8
28
3
9. 98
8
84
4
9. 99
8
24
5
10.00
8
73
6
10.11
8
54
7
10. 12
8
64
8
10.14
8
82
9
10.19
9
04
10
10,23
8
93
11
10.25
8
97
12
10.29
8
80
13
10.55
9
21
14
10.62
8
95
X = 10,15
8. 75
3, Examples of You den two-sample
charts are in the Appendix (p4-13).
4, A quantitative treatment of this
subject can be found in references
11 and 12.
12 3 4
SAMPLE VOLUME (ml)
Figure 5
-
X
n
A
13
>-
Y
X
X
y
X
10
X
4
uj
a.
s
<
3
8
X
<
X
I L"
X
1
7
X
6
X
2 X
1
<
# ¦ •
SAMPLE X (
1
7„K)
9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00
Figure 6. YOUDEN'S GRAPHICAL TECHNIQUE
5-5
-------
A c curacy-Precision-Error
IV ELIMINATION OF DETERMINATE
ERROR
Various approaches can be used to eliminate
the source of determinate error. The
approach used depends upon whether the
source is personal, method, or instrumental.
A Personal
Great care must be used to avoid producing
an unconfident attitude in a technician. It
is undesirable for the analyst to feel he is
being "policed."
B Method and Instrumental
1 Blanks
Blanks can be used to correct for
interferences from reagents, sample
color, etc.
2 Correction factors
Figure 7. METHOD OF STANDARD
ADDITION GRAPHIC AT, METHOD OF
COMPUTING Sr CONCENTRATION
t 90
in
r 70
o 50
l/l
* 3 0*
~
~
w
/
j
>
/
/
y
M—j
Sr + +CONC (mg/l)
NOTE: The X value appearing along the
abscissa fi, e., X + 20, X + 40, etc.) re-
fers to the unknown added to the strontium
standard
Examples of correction factors used
in environmental analyses to eliminate
determinate errors are the following:
a Recovery factors in organic
extractions
b Chemical yield values in gravimetric
analyses
c Counting efficiencies for radiation
counters
3 Standard addition
V
4 Standard compensation
Another approach is to prepare the
standard so that its composition
resembles that of the sample as
closely as possible. The objective
of the approach is exactly the same
as that of standard addition - to
compensate for the presence of
interfering substances in the unknown.
INDETERMINATE ERROR AND
PRECISION
Sample interferences producing deter-
minate errors can be overcome by
adding equal amounts of unknown
sample to a series of standards. The
concentration of the unknown can then
be determined graphically from a plot
of the measured quantity (absorption,
emission, etc.) versus the standard
concentration. (See Figure 7)
Even when all determinate errors are
eliminated, every replicate analysis will
not give the same value. Such variation
in results is due to indeterminate error,
also known as random, chance or
uncontrollable error. Indeterminate
error affects the precision or agreement
among results.
5-6
-------
Accuracy-Precision-Error
A Sources of Indeterminate Error
Indeterminate errors are due to
unassignable or "chance causes."
Examples are inadvertent contamination
of sample or glassware, variation in
reagent additions, or variations in
instrument response (see Figure 8).
B Effects of Indeterminate Error
Since the causes of these errors are
random, the effects are also random.
Fortunately, these random variations
conform to the "Laws of Chance" so
statistical measures of precision can be
used to quantitate indeterminate errors.
VI DETECTION OF INDETERMINATE
ERROR
A measure of the degree of agreement among
results can be obtained by analyzing a single
sample repeatedly under a given set of
conditions.
line (those values encircled) should be
rejected in the calculation of standard
deviation. Other statistical tests (8)
can be used to objectively evaluate the
rejection of outliers. The value obtained
for standard deviation for a particular
method may vary with the analyst, the
concentration range of the constituent,
and the composition of the sample
analyzed. The confidence of the
estimate is increased as the number of
results (n) used to compute the standard
deviation is increased.
1 Replicate results on the same sample:
(1)
' E(X. - X)
n-1
Xi =
X =
value of single result
average (mean) of results on
same sample
n =
= number of results
A Range
The range of the replicate results
(difference between the lowest and the
highest value) provides a measure of
indeterminate variations.
B Standard Deviation
An estimation of indeterminate error can
be obtained through a calculation of the
standard deviation. The following formulas
should be applied to random data which
follows a normal distribution. Normality
can be checked by ranking and plotting the
data on normal probability paper; it should
fall on a straight line (see Figure 9). Any
values which do not fall close to the straight
Example: Table 3 contains a set of 5-day
BOD results obtained on a synthetic
sample containing 15 0 mg/1 of glucose
and 150 mg/1 glutamic acid. (Note: A 1%
dilution was used in the actual test). The
results are those submitted by laboratories
participating in a culaborative study.
Calculate the standard deviation of these
results.
X = 192 mg/1
n = 36.0
E(Xi-X)2 » 58,200
I 58, 200
S ~J 35
s = 41 mg/1
5-7
-------
Accuraey-Precision-Error
>-
U
3
o
BECKMAN B (650 m/<)
METHYLENE BLUE SOLUTION
1cm CELL
25 REPLICATE READINGS
.528 .530 .532 .534 .536
ABSORBANCE
Figure 8. VARIATIONS IN SPECTROPHOTOMETER READINGS
FIGURES NORMAL PROBABILITY CURVE
4.40
4 20
400
2.80
360
3.40
320
3.00
| 2.80
- 2.60
r 2.40
2.20
2.00
1.80
1.60
1,40
1.20
1,00
0.80
99,99 M9 9U 94
99 90 BO 70 CO SO 40 30 HJ JO 5 2 ( 0.5 02 0! OH OOl
It 1
1
%
4
• •
¦**'
i *
*
•
3
OD\ COS 0.! 02 0.5
plotting inl«rvgl "
12 3 10 20 90 40 90 60 70 BO 90 » 99
^ = 2.083
MS 995 99-99
5-8
-------
Accuracy- Pre cision.- E r ror
Table 3
SAMPLE; 150 mg/1 glucose + 150mg /I glutamic acid (1% dilution)
DETERMINATION: 5-day Biochemical Oxygen Demand
X. X - X (X, -X)2 X. X. - X (X. - X)2
1 1 1 1 1 1
100 mg/1 -92 mg/1 8464 (mg/1)2 198mg/I 6 mg/1 36 (mg/1)^
117 -75 5625 199 7 49
125 -67 4480 200 8 64
132 -60 3600 200 8 64
142 -50 2500 204 12 144
147 -45 2025 210 18 326
153 -33 1521 211 19 361
160 -32 1024 212 20 400
165 -27 729 215 23 529
165 -27 729 223 31 961
167 -25 625 224 32 1024
173 -19 361 227 35 1225
178 -14 196 229 37 1369
189 - 3 9 238 46 2116
190 - 2 4 247 55 3025
106 4 16 250 58 3344
ISfi 4 16 259 67 4489
197 5 25 274 G2 6724
-Data taken from Water, Oxygen Demand Report (July, i960), Analytical Reference
Service, Training Program, R. A. Taft Sanitary Engineering Center, Cincinnati, Ohio.
Table 4 * concentration
SAMPLE: Aqueous RANGE: 30 - 70 mg/l
DETERMINATION: Phosphate (Lucerna, Ccr.de, and Prat Method)
Sample
Mg/1 P
d
d2
Sample
Mg/I P
d
d2
A
51
2
4
L
54
2
4
53
56
B
39
0
0
M
38
1
1
39
37
C
53
1
1
N
52
0
0
54
52
D
47
0
0
O
58
0
0
47
58
E
50
1
1
P
54
0
0
51
54
F
48
1
1
Q
54
0
0
47
54
G
50
0
0
R
48
0
0
50
48
H
47
0
0
S
52
1
1
47
51
I
42
0
0
T
53
0
0
42
53
J
50
1
1
U
4S
1
1
51
47
K
59
1
1
V
42
0
0
60
42
*Data obtained from Frank Schickner, Proctor and Gamble Company,
-------
Accuracy-Precision-Error
2 Duplicate results on different samples
In a laboratory where duplicates are
routinely run, it would be simple to
use the following formula for evaluation
of standard deviation.
d
k
E(d ) (See reference 5,
2k page 654) (2)
difference between duplicates
number of samples
Example: Table 4 contains a set of
phosphate results obtained on aqueous
samples in the range 100-200 rag/1,
Calculate the standard deviation of these
results.
m2)
16
22
s = . 61 mg/1
3 Duplicate and triplicate results on
different samples
£(X. - X)
(See reference 7,
page 73) (3)
x = average of results on the
same sample
n = total number of results
k = number of different samples
Example: Table 5 contains a set of %
nitrogen results obtained on unknown
organic compounds.
Calculate the standard deviation.
E(X. - X)
i
n - k
s
s
. 2401
22
* V
. 2401
22
104%
Use of range to estimate standard
deviation
For a small number of replicates
(n < 10), the range can be used to
estimate the standard deviation (See
Table 6 ) .
_R
d
(4)
N
2(X,
Example: Table 7 contains a set of
replicate nitrate results. Calculate
the standard deviation of these results.
a Use of formula (1)
X = 0. 72 mg/1
n ~ 5
-x)2 = .0134
. 0134
s ^
s = . 058 mg/l
b Use of formula (4)
R = . 14 mg/1
2. 33
N
. 14 mg/1
2. 33
s = . OSO mg/1
C Coefficient of Variation
An estimation of indeterminate error
can also be made by calculating the
coefficient of variation (V), also
known as % relative standard
deviation:
V = ® x 100
X
By comparing the standard deviation
(dispersion) to the average or mean
value (central tendency) in a set of data
and expressing this relative standard
deviation as a percentage, the analyst
has a meaningful interpretation of the
degree of dispersion present. Indeter-
5-10
-------
Accuracy-Precision-Error
Table 5*
SAMPLE: Unknown Organic Compounds CONCENTRATION
DETERMINATION: % Nitrogen (Kjeldahl) RANGE: 10% - 2a<&
Sample
%N
X
X. - X
l
(X, - X)2
l
Sample
X
X - X
1
(X. - X)2
l
A
16.43
16.47
.01
.0001
J
13. 33
13, 75
. 18
.0324
16, 45
.01
.0001
13, 56
, IS
.0361
B
16, 50
16. 50
0
0
K
10.34
10.27
.07
.0049
16, 48
.01
,0001
10, 19
.08
,0064
C
16.72
16,65
.07
.0043
L
17. 16
17.15
.01
.0001
16.57
.08
. 0064
17. 13
.02
.0004
D
17.52
17.58
,06
.0035
M
15. 01
15.03
.02
.0004
17, 50
.02
.0004
15. 05
. 02
. 0004
17. 63
.05
.0025
E
16. 31
16.33
0
0
N .
12.44
12. 62
. 18
. 0324
ISs 30
,01
.0001
12.70
,08
, 0064
12. 73
. 11
.0121
F
16.4Q
16. 35
. 05
.0025
O
14. 37
14.37
0
0
16, 31
.04
.0016
14. 35
.01
, QQ01
16. 35
0
0
P
11. 85
11.85
0
0
G
17. 56
17. 55
, CI
. 0001
11, 83
0
0
17, 54
. 01
.0001
Q
14.79
14.73
.06
.0036
H
14.95
14, 81
. 15
,0225
14. 70
.03
. ooos
14, 65
. 15
,0225
14, 70
.03
.0009
I
R
17. 19
17. 17
.02
.0004
19. 15
19,02
. 13
.0169
17. 14
,03
. oooa
18.89
. 13
.0169
*Data obtained from Fraiilt Schickne r, Proctor and Gamble Company,
Table S*
FACTORS USED TO ESTIMATE THE STANDARD
DEVIATION FROM RANGE
Si a 2
ample (n)
dN
1
dN
2
1. 13
.837
3
1.60
,591
4
2,06
.486
5
2, 33
.430
6
2, 53
.395
7
2.7D
.370
8
2. 85
. 351
8
2. 97
. 337
10
3. 08
,325
<8>
^Natrella, Experimental Statistics, pp. 2-5,
Table 7
SAMPLE: Ohio River "Water
DETERMINATION; Nitrate (Modified Brnciiie)
X.
l
X.-X
i
(X{-X)'
0. 65 rng/lN
-0.C7
.0049
0.68
-0.04
. DDIS
D. 70
-Q.U2
, 0004
0.76
+0. 04
, 0016
0.73
+0. 07
, 0049
-------
Accuracy-Precision-Error
ruinate error would be the cause of undue
dispersion.
Example: Table 3
s = 41 mg/1
X = 192 mg/1
V = 21%
D An indication of an individual's analytical
precision can be obtained by his partic-
ipation in an interlaboratory study. In
this case, the analyst performs only
one determination on each of two samples,
' Using Youden1 s Graphical Technique as
an indication of an individual's precision
was discussed previously in this outline
in III C.
REFERENCES
1 Accuracy in Clinical Chemistry. Dade
Reagents, Inc., Miami, Florida.
2 Allan, Douglas H, Statistical Quality
Control. Reinhold Publishing Corp.,
New York. 1959.
3 American Society for Testing Materials
ASTM Manual on Quality Control of
Materials. Special Technical
Publication 15-C. 1951.
7 Mickley, Harold S., Sherwood, Thomas K.
and Reed, Charles E. Applied
Mathematics in Chemical Engineering.
McGraw-Hill Book Company, New York.
1957,
8 Natrella, M.G. Experimental Statistics,
National Bureau of Standards Handbook
91. U.S. Dept. of Commerce. 1963.
9 Schickner, Frank A. Personal Com-
munication. The Procter & Gamble
Company, Miami Valley Laboratories,
Research & Development Department,
P.O. Box 39175, Cincinnati, OH 45239.
10 Youden, W.J. The Collaborative Test.
JAOAC 46:55-62. January 1963.
11 Youden, W.J. The Sample, The
Procedure, and the Laboratory.
Anal. Chem. 32;23~37A. Dec. 1960.
12 Youden, W.V. Statistical Techniques
for Collaborative Tests. Association
of Official Analytical Chemists, Box 540,
Benjamin Franklin Station, Washington,
DC . 1967.
This outline was prepared by Betty A.
Punghorst, former Chemist, National
Training Center, and revised by Audrey D,
Kroner, Chemist, National Training and
Operational Technology Center, MOTD,
OWPO, USE PA, Cincinnati, Ohio 45268.
4 Bauer, E. L. A Statistical Manual for
Chemists. Academic Press, New York.
1960.
5 Bennett, Carl A. and Franklin, Norman L.
Statistical Analysis in Chemistry and
the Chemical Industry. John Wiley &
Sons, Inc., New York. 1954.
6 Chase and Rabinocvitz. Principles of
Radioisotope Methodology. Minneapolis
Burgess Publishing Company. 1964.
Descriptors: Accuracy, Data Collections,
Data Processing, Error Analysis, Errors,
Graphic Methods, Measurement, Monitoring,
Precision, Quality Control
5-12
-------
Accuracy-Precis ion-Error
APPENDIX
I" '
~ pN DATA. GOOD PRE
FROM: METHOD STll
Al
J
'
CISION AND GOOD ACCURACY. ~
)Y 1, MINERAL AND PHYSICAL
AIYSES
tf
*
UJ
a.
X
S SAMPLE 3
i
'1
7,or
6.0
t r
! , r
KJELDANL NITROGEN, «( H/LITEH.
«non PHECISIDW WI1N systematic
ERROR. FROM: METHOD STUB* 2, . •
NITSIENT ANALYSES. MANUAL .
METHODS
SAMPLE 8
2.0 3.0 4.0 5.0 6.0 7.0
.71
.58
.42-
AMMONIA NITROGEN, mg N/LITEB. LIMITED ACCURACY
AS NEGATIVE BIAS. H0M METHOD STUDY 2, NUTRIENT
ANALYSES. MANUAL METHODS
.Oflf
.76
.52
~r
KIELDAHL NITROGEN, mg N/LITtR. POOR PRECISION,
FRDM: METHOD STUDY 2, NUTRIENT ANALYSES.
MANUAL METHODS
-------
ELEMENTS OF A QUALITY ASSURANCE PROGRAM
I WATER QUALITY DATA D
A Importance
1 Criteria for decisions
a Planning
b Permit issuance
c Compliance
d Enforcement
e Evaluation of treatment processes
f Research decisions E
2 Effects of decisions
a Social
b Legal
c Economic
B Requirements for Reliability
1 Specificity
2 Accuracy II
3 Precision A
C Elements of Quality Assurance' *-
1 Valid sample
2 Recognized methodology
3 Control of services, instruments,
equipment and supplies
4 Quality analytical performance
5 Efficient data handling and reporting
Documentation System
1 Complete and permanent records must
be kept by all field and laboratory
personnel.
2 Any procedures undertaken as quality
checks should also be recorded, dated
and signed.
3 The results of any quality checks should
be recorded, dated and signed.
4 Any checks by outside service personnel
should be recorded, dated, and signed.
Quality Assurance Control Coordinator^)
1 Overall responsibility for program;
development, implementation, administration
2 Oantinuing assessment of level of operations
3 Identification of training needs and provi-
sion to accomplish
4 Coordinator for inter-laboratory quality
control programs
SAMPLE
Validity^' "*» 6)
1 Representative
2 Properly collected
3 Clean, appropriate containers
4 Approved preservation measures
5 Analytical checks on containers and
preservatives
6 Holding times observed
CH. MET. con. 9. 11. 77
6-1
-------
Elements of a Quality Assurance Program
B Integrity^2, ^
1 Written procedures for all aspects
of sample handling
2 Field labels, records, seal
3 Appropriate transport to laboratory
4 Logging in system
5 Appropriate storage conditions and
holding time
6 System for distribution for analysis
7 System for storage or discard
8 System for chain-of-custody documen-
tation
III RECOGNIZED METHODOLOGY
A Need for Standardization
1 Within one laboratory
2 Between cooperating laboratories
3 Users of commorj data bank
4 Nation-wide requirements
B Criteria for Selection'®^
1 Specificity with accuracy and
precision
2 Validity established by sufficient
use and evaluation
3 Equipment and skill requirements
normally available
4 Time requirement reasonable
C Sources
1 Annual Book of ASTM Standards^
2 Standard Methods for the Examination
of Water and Wastewater^
3 Methods for Chemical Analysis of Water
and Wastes (*>)
4 U.S. Geological Survey Techniques of
Water Resources Inventory^
5 Others
D Commonly-Used Types^
1 Various sample treatments (filtration,
digestion, etc.)
2 Electrode-meters
3 General analytical methods
a Volumetric analysis
b Gravimetric procedures
c Combustion
4 Photometric methods
a Atomic absorption
b Flame emission
c Colorimetry
5 Gas chromatography
E Selection on Basis of Use of Data
1 Compliance monitoring
a National Pollutant Discharge
Elimination System and State
Certifications (10)
1) Use of alternate procedures
2) Procedures for non-listed
parameters
b National Interim Primary Drinking Water
Regulations^1^
1) Use of alternate procedures
2) Procedures for non-listed
parameters
6-2
-------
Elements of a Quality Assurance Program
2 State monitoring programs'' ^
a Fixed station ambient monitoring
b Intensive survey programs
Local regulations
4 I're-survey field investigations
5 Control of treatment processes
F Using Heeognized Procedures
1 Written step-bv-stop laboratory
manuals
2 Strict adherence to reference source
3 Record of modifications and why
G Field Kits
1 Shortcomings
2 Uses
IV CONTROL OF SERVICES, INSTRUMENTS,
EQUIPMENT AND SUPPLIES*
A Services
1 Distilled water
a Ammonia-free
b Carbon dioxide-free
c Ion-free
d Low organic background
2 Compressed air
a Dry
b Oil-free
c No contaminants
3 Elect rical service
a Adequate voltage
b Constant voltage
c. Appropriate grounding
(1 l']lTL<-ient lighting
H Instruments
Applicable to laboratory and field instruments;
and, as possible, fixed continuous monitoring
do vines.
1 Written requirements for daily warm up,
standardization, calibration, and/or
optimization procedures.
2 Standards available to perform daily check
procedures. Some examples;
a Standardized weights
b Certified thermometer
c Filter (or solution) for wavelength
alignment check
d Standard reference materials with
standard absorption curves
e Standard resistor
f Calibration solutions (buffers, con-
ductivity or turbidity standards)
g Parameter standards to establish or
to check calibration curves
h Radioactive standards with date
and count
3 Written trouble-shooting procedures
4 Schedule for required replacement or
cleaning procedures
5 Schedule for check and/or adjustments
by service personnel
6-3
-------
Elements of a Quality Assurance Program
C Laboratory Equipment
1 Great variety
a Of materials (glass, plastic,
porcelain, etc.)
b Of grades
c Of accuracy in calibration
d Of specific properties
e Of unique construction
2 Selection depends on function
a Measurement and delivery of
volumes require varying
degrees of accuracy.
b Storage of reagents and solutions
necessitates composition consider-
ations such as;
1) Polyethylene bottles for solu-
tions of boron, silica and
alkali
2) Glass containers for organics
3)^ Brown glass for light-sensitive
solutions
c Confinement of reactions may
present special requirements
such as:
1) Ground glass joints
2) Teflon plugs
3) Special resistance to thermal
shock
4) Impervious to digestion
conditions
d Volumetric analyses involve:
1) Very accurately calibrated
glassware
2) Consideration of the temperature
at which the apparatus was -
calibrated
e Other laboratory operations like
filtration, ion exchange, absorption
and extractions may require specialized
construction like fritted ware which has
pressure and thermal shock limits.
3 Cleaning procedures
a Basis of selection
1) Appropriate for the composition
material
2) Appropriate for materials to be
removed
3) Appropriate for subsequent use -
(Avoid introducing contaminants)
b Definite program
1) Standardized, consistent,
mandatory
2) Analytical checks on effectiveness
D Laboratory Supplies - Reagents, Solvents
and Gases* ^
1 Required purity depends on:
a What is measured
b Sensitivity of method
c Specificity of detection system
2 General guides
If purity is not specified in the method,
some general guides are:
a General inorganic analyses
1) Analytical reagent (AR) grade
chemicals, except use primary
standard grade for standardizing
solutions.
2) Distilled water and solvents free
of constituent
3) Commercial grade gases
6-4
-------
Elements of a Quality Assurance Program
b Metals analyses by flame
1) Spectroquality chemicals for
standards
2) Spectroquality recommended
for other reagents and solvents,
though analytical reagent grade
may be satisfactory.
3) Acids should be distilled in
glass
4) Deionized distilled water
5) Commercial grade or laboratory-
supplied gases
c Radiological analyses
1) Scintillation grade reagents and
solvents
2) High purity, extra dry gases
with low radioactive background
d Organic analyses
1) Reference grade when available,
AR at minimum
2) For gas chromatography (GC),
various detectors require absence
of certain classes of compounds,
and may necessitate treatment
of chemicals,
3) Pesticide quality solvents
For GC, check assay.
4) Type of detector affects gas
quality required. Molecular-
sieve carrier-gas filters and
drying tubes are required on
combustion gases.
3 Program for assuring quality
a Written purity requirements
according to methods utilized
b Date all on receipt.
c Observe shelf life recommendations.
Dicard date on container.
d Observe appropriate storage
requirements,
e Check assay for possible
interferences,
f Run reagent and solvent blanks,
g As applicable, check background
of reagents and solvents.
h Run method blanks (all reagents
and solvents) with every series
of samples or one for every
nine samples.
i Definite procedures for limits
of error, clean-up procedures
or application of correction
factors
j Replace gas cylinders at
100-200 psi,
4 Procedures for removing impurities
a Recrystallization
b Precipitation
c Distillation
d Washing with solvent(s) used in
' analysis
e Aging (gases)
f Others
5 Reagent and standard solutions
a Preparation
1) Use of primary standard grade
chemicals as required
2) Careful weighing'
3) Class A volumetric glassware
4) Appropriate quality distilled
water or solvent
5) Label listing compound(s),
concentration, date of pre-
paration or discard, preparer
6) Very dilute standards prepared
at time of use R =
-------
Elements of a Quality Assurance Program
b Standardization as appropriate
1) Use reliable primary standards,
2) Restandardize as required by
stability.
c Purchased solutions
1) Should contain chemicals
specified by method
2) Should be checked for
accuracy
d Storage
1) Clean containers of material
suitable for solution to be
stored
2) Tight-fitting stoppers or
caps
3) Safeguards against evaporation
of solvent, adsorption of gases
and water vapor, effects of
light or temperature, etc.
e Signs of deterioration
1) Discoloration
2) Formation of precipitates
3) Significant change in
concentration
V QUALITY ANALYTICAL PERFORMANCE^
A Skilled Analyst
1 Appropriate and continuing training
2 Willingness to follow specified
procedures
3 Skilled in manipulation of laboratory
equipment and techniques required
in analyses
4 Understanding of basic principles
utilized and design of any instruments
s/he uses.
5 Knowledgeable and skilled in performing
the analyses for which responsible
6 Precision and accuracy performance
acceptable
B Establishing Analyst Precision
Applicable except for gas chromato-
graphy and radiological instrumentation.
1 Seven replicates of four samples
covering the concentration range
of applicability for analysis
2 Test among routine samples over
two hours or more in normal operating
conditions.
3 Calculate the standard deviation for
each set.
4 Compare result to precision statement
for method in the source of the procedure.
(It may be stated as % relative standard
deviation. If so, calculate analyst results
in this form).
5 Individual's precision should be better
than round-robin precision results.
C Establishing Analyst Accuracy
Exceptions: gas chromatography and
radiological instrumentation
1 Spike set of 7 precision replicates of
concentration low in applicability range
to bring final to twice original.
2 Spike set of 7 precision replicates of
mid-range concentration to bring final
to about 75% of upper limit of applica-
bility.
3 Test among routine samples over two
hours or more in normal operating
conditions.
4 Calculate % recovery for each set using
average of results from the precision
check and the recorded spike amounts.
5 Compare result to accuracy statement
for method in the source of the procedure.
(It may be stated as % bias, i. e.,
% recovery-100%).
6-6
-------
Elements of a Quality Assurance Program
6 Individual's accuracy should be
better than round-robin accuracy
results.
D Daily Performance Evaluation
1 At least two standards (high and low)
analyzed with a blank to verify an
established standard curve (comparable
operating conditions).
2 Some methods require daily preparation
of a standard curve,
3 One of about every 10 samples should
be a duplicate to check precision
according to acceptable standard
deviation (or % relative std. deviation),
4 One of about every 10 samples should
be a spiked sample to check accuracy
according to acceptable % recovery
(or % bias).
E Documentation of Daily Performance
1 After 20 sets of duplicate data results
or of spiked sample results have been
collected, control charts for precision
and accuracy, respectively, can be
constructed.
2 A variety of construction methods is
available.
3 Plot succeeding results on the
appropriate chart.
4 Charts document reliability of data.
5 Charts give signal of out-of-control
numbers, trends toward out-of-control
conditions, improved performance,
etc.
(2)
F Interlaboratory Checks on Performance
1 Quality Control samples for many
constituents are available from EPA
at no charge through EPA Regional
Quality Assurance Coordinators.
The concentration is provided with
the sample. These might be run
every three to six months.
2 Run split samples and compare
results with the other laboratory.
3 Run performance samples (unknowns)
available from EPA at no charge.
4 Participate in round-robin method and
performance evaluation studies.
5 Participate in laboratory evaluation
programs.
VI DATA HANDLING AND REPORTING*
A laboratory must have a program for
systematic and uniform recording of
data, and for processing and reporting
it in proper form for interpretation and
use.
A The Analytical Value
1 Correct calculation formulas reduced
to simplest factors for quick, correct
calculations.
2 Provisions for cross-checking calculations
3 Rounding-off rules uniformly applied
4 Significant figures established for
each analysis
B Processing
1 Determine control chart approach and
statistical calculations required for
quality assurance and report purposes.
2 Develop report forms to provide
complete data documentation and
permanent records, and also to
facilitate data processing.
a To avoid copying errors, the
number of forms should be minimal.
C Reporting
The program for data handling should provide
data in the form/units required for reporting.
6-7
-------
Elements of a Quality Assurance Program
D Storage
1 For some types of data, laboratory
records must be kept readily available
to regulatory agencies for a period of
time.
2 A bound notebook or preprinted data
forms permanently bound provide good
documentation.
3 STORET is a system for storage and
retrieval of water quality data. It is
a State/Federal cooperative activity
which provides States with direct access
into the central computer system.
4 Many agencies have access to local
systems for storage and retrieval
of data.
Vir SAFETY CONSIDERATIONS*13)
A Laboratory Facilities
B Emergency Equipment
C Program for Health Checks as Required
D Program for Inventory and Control of
Toxic and Hazardous Materials and
Test Wastes
E Safety Officer-Responsibilities
1
Information
2
Planning
3
Inspection
4
Implementation
5
Evaluation
6
Reports
VIII EPA Regional QA Coordinators
A Each of the ten EPA Regions has a
Quality Assurance Coordinator.
1 Implements program in regional
laboratory
2 Maintains relations and serves as
source of information for state and
interstate agencies within the region
3 Serves as liason for EPA's Environmental
Monitoring and Support Laboratory (EMSL).
B The name, address and telephone number
of the regional QA Coordinator can be '
obtained from the EPA Regional Adminis-
trator's Office or from EPA-EMSL,
Cincinnati, Ohio 45268.
IX SUMMARY
Quality Assurance regarding water quality
(or any type of) laboratory data requires
planning, control and checking for every
phase of the operation from sample collec-
tion through storage of the data. This out-
line contains a basic checklist of information
and items to be considered when developing
a program to facilitate quality analytical
performance by laboratory personnel.
To make the program effective, procedures
must be written, responsibilities must be
clearly defined and assigned, and individuals
must be accountable. Development and daily
performance of such a program which meets
the needs of an individual laboratory (or
agency) will take time. Considering the
importance of the data produced, the invest-
ment in assuring its reliability is a sound
one..
REFERENCES
1 Handbook for Analytical Quality Control
in Water and Wastewater Laboratories,
1979, U.S. EPA, EMSL, Cincinnati,
Ohio 45268.
2 Minimal Requirements for a Watex Quality
Assurance Program, EPA-440/9-75-010,
U. S. EPA Office of Water Planning and
Standards, Washington, D.C. 20460.
6-8
-------
Elements of a Quality Assurance Program
3 Annual Book of ASTM Standards (Part 31),
Water, 1980, American Society for Testing This outline was prepared by Audrey Kroner,
and Materials, Philadelphia, PA., 19103. Chemist, National Training and Operational
Technology Center, OWPO, USEPA,
4 Handbook for Monitoring Industrial Waste- Cincinnati, Ohio 45268.
water, 1973, U.S. EPA-Technologv
Transfer, Cincinnati, Ohio 45268.
5 Standard Methods for the Examination of
Water and Wastewater, 14th edition, 1976,
APHA-AWWA-WPCF, Washington, D. C. ,
20036.
6 Methods for Chemical Analysis of Water
and Wastes, 1979, U.S. EI-'A-EMSL, ,
Cincinnati, Ohio 45268.
7 Model State Water Monitoring Program.
EPA-440/9-74-00 2, U.S. EPA Office of
Water and Hazardous Materials, Washington,
D. C.
8 IJ. S. Geological Survey Techniques of
Water Resources Inventory; Book 1, 1975;
BookS, Ch. Al, 1970; Book 5, Ch. A3, 1972;
et. al.; U.S. Government Printing Office,
Washington, D. C. 20402.
9 Kroner, "Methodology for Chemical Analysis
of Water and Wastewater" U. S. EPA-NTOTC,
Cincinnati, Ohio 45268.
10 Federal Register, "Guidelines Establishing
Test Procedures for the Analysis of
Pollutants", Vol. 41, No. 232, December 1,
1976, pp 52780-52786. Also, Vol. 44,
No. 244, December 18, 197 9, pp 75028-
75052 presents proposed changes. The
latter is scheduled for finalization after
January, 1981.
11 Federal Register, "National Interim Primary
Drinking Water Regulations, " Vol. 40,
No. 248, December 24, 1975, pp 59566-
59574. Also, Vol. 45, No 168 August
27, 1980, pp 57332-57346, "Interim
primary Drinking Water Regulations,
Amendments. '
DESCRIPTORS: Analytical Techniques,
Chemical Analyses, Quality Assurance,
Quality Control, Reliability, Water Analysis
12 Federal Register, "State and Local
Assistance, " Vol. 41, No. 82,
April 27, 1976 pp 17694-17700.
13 Safety Management Manual, 1972,
U. S. EPA, Washington, DC 20460.
6-9
-------
USE OF A SPECTROPHOTOMETER
I SCOPE AND APPLICATION
A Colorimetry
Many water quality tests depend on a
treating a series of calibration standard
solutions which contain known concentra-
tions of a constituent of interest, and also
the sample(s) with reagents to produce a
colored solution. The greater the concen-
tration of the constituent, the more intense
will be the resulting color. A spectro-
photometer is used to measure the amount
of light of appropriate wavelength which is
absorbed by equal "thicknesses" of the
solutions. Results from the standards
are used to construct a calibration (standard)
curve. Then the absorbance value for the
sample is located on the curve to determine
the corresponding concentration.
B Lambert Beer Law
States the applicable relationships:
A = e b c
1 A = absorbance
2 e = molar absorptivity
3 b = - light path in cm
4 c = concentration in moles/liter
II APPARATUS
A Requirements
Are given as part of the method write-up
1 The applicable wavelength is
specified. The unit used is
nanometers (nm).
2 The light path (cell dimension)
is often open-ended, e. g., "one
cm or longer. " One must know
the light path length in the
available spectrophotometer,
because it is inversely related
to the usable concentrations in
the test. (Longer path lengths
detect lower concentrations).
3 NOTE: For National Pollutant
Discharge Elimination System
(permit), or for Drinking Water
Regulations test requirements,
check with the issuing/report
agency before using light paths
(cells) that differ from the length
specified in the approved method.
If you have permission to use an
alternate path length, concentra-
tions for the test can be adjusted
accordingly. These adjustments
are discussed in IV and in VII (below).
Ill PREPARATION OF THE SPECTRO-
PHOTOMETER
A Phototube/Filter
1 May have to choose a phototube for
use above or below a particular
wavelength.
2 A filter may be required.
3 If the available instrument involves
a choice, check that the phototube
(and filter, if applicable, ) required
for the wavelength to be used is in
the instrument.
4 Always handle and wipe off the
phototube and/or filter with tissue
to avoid leaving fingerprints.
B Cell compartment
1 This area must be kept clean and
dry at all times.
CH. IN.sp. la. 8. 80
7-1
-------
USE OF A SPECTROPHOTOMETER
C Cells
E Wavelength Alignment
A set must "match" each other
in optical properties. To check
this, use the same solution at
the same wavelength, ami verify
that the absorbance value is the
same for each cell.
Alternatively, a single cell can
be used if it is thoroughly rinsed
after each reading.
Instrument cells should be free
of scratches and scrupulously
clean.
a Use detergents, organic
solvents or 1:1 nitric acid-
water.
b Caustic cleaning compounds
might etch the cells.
c Bichromate solutions are
not recommended because
of adsorption possibilities.
d Rinse with tap, then distilled
water.
e A final rinsing and drying with
alcohol or acetone before
storage is a preferred practice.
An excellent point is the known, maximum
absorption for a dilute solution of potassium
permanganate which has a dual peak at
526 nm and 546 nm. Use t matched cells for the
following steps?
1 Prepare a dilute solution of
potassium permanganate (about
10 mg/1).
2 Follow the steps in VI A, Zeroing
Operation, using a wavelength of
510 nm, and distilled water as a
"reagent blank. " Keep the water in the cell
during this entire procedure.
3 Rinse the matched cell two times
with tap water, then two times with
the permanganate solution.
4 Fill the cell three-fourths full
with the permanganate solution. Keep the
permanganate solution in this cell during this
• entire procedure.
5 Thoroughly wipe the cell with
a tissue. Hold the cell by the
top edges.
6 Open the cover and gently insert
the cell, aligning it to the ridge
as before.
7 Close the cover.
D Warm-Up
1 Plug in the power cord.
2 Turn the power switch on and give
it an additional half-turn to keep
the needle from "pegging. "
3 Wait to use until the recommended
warm-up time has passed. Any-
where from 10 to 30 minutes may
be required.
4 If the instrument drifts during
zeroing, allow a longer time.
7-2
8 Record the wavelength and die
absorbance reading on a sheet of
paper.
9 Remove the cell of permanganate
solution and close the cover.
10 Set the wavelength control at die
next graduation (4- 5nm).
11 If the needle is not at infinite
(symbol co) absorbance, use the
left knob to re-set it.
12 Insert the cell containing distilled
water using the techniques noted in
5, 6 and 7 above.
If necessary, use the right knob
to re-set the needle at zero absorbance.
14 Remove the cell and insert the cell
of permanganate solution using the
techniques noted in 5, 6 and 7 above.
-------
USE OF A SPECTROPHOTOMETER
It) Record toe wavelength and the
absorbance reading.
16 Repeat steps 9 through 15 above
until absorbance readings are
recorded at 5 nm increments
from 510 nm through 5BO nm.
17 Make a graph plotting absorbance
readings against wavelengths.
With very good resolution, there
will be two peaks - one at 525 nm
and one at 545 nm. A single flat
topped "peak" between these two
wavelengths is acceptable.
18 If the maximum abs or ban c e s [pe ak (s jl
occur below or above 526 nm or
5 46 nm, and at a number of scale
units different from the stated
instrument accuracy, the wavelength
control is misaligned. To compensate
for this until the instrument can be
serviced, add or subtract the error
scale units when setting wavelengths
for subsequent tests.
CALIBRATION STANDARDS
Requirements
A set of calibration standards is required,
with concentrations usable in the available
spectrophotometer cell (light path length).
1 The method reference may provide
a table of light path lengths and
the corresponding applicable con-
centration range for calibration
standards, so one can choose the
range required for his instrument
cell or sample concentration,
2 The method reference may give
directions for preparing one range
of concentrations for a given light
path length. If your cell provides
a different length, your concentration
requirements can be easily calculated
by recalling that the light path length
is inversely related to concentration.
Thus, if your cell is twice the given
path length, you need the given con-
centrations divided by two,
3 The method reference may give
directions for preparing only one
range of concentrations for the
calibration standards, and then
not be specific about the associated
path length. You will have to test
if the range is applicable to your
instrument by preparing the giver,
concentrations, obtaining absorbance
values for them and checking the
results according to section VII
(below),
B Preparation
The caliuraiiuti standards required for
spectrophotometric measurements are so
dilute, that they are commonly prepared by
diluting stronger solutions. These are des-
cribed in general terms below. Weights and
volumes involved in preparing these solutions
for a specific test can be found in the method
write-up.
1 Stock Solutions
a Prepare by weighing or measuring
a small amount of a chemical
containing the constituent of
interest and dissolving it to a
one liter volume.
b Common stock solutions have
concentrations in the range of
0. 3 to 1.0 mg/ml.
c Most are refrigerated for storage
and some are further treated by
adding a preservative. Many are
stable up to six months,
2 Standard Solutions
a Prepare by diluting a stock
solution (at room temperature).
Common volumes arc 10. 0 or 20. 0 ml -
of stock diluted to one liter.
b Resulting standard solutions have
concentrations in the range of
1.0 to 10, 0 ugf ml.
c Although some standard solutions
may be stable for a period of time,
it is a recommended practice to
prepare them on the day of use.
A Calibration (Working) Standard Solutions
a Prepare by diluting a standard
solution. Usually a set of cali-
bration standards is required
so that resulting concentrations
give five to seven results within
the sensitivity limits of the instru-
ment. Common volumes are 1 to 10 ml
of standard solution diluted to 100 ml.
-------
USE OF A SPECTROPHOTOMETER
b Resulting solutions might
have concentrations in the
range of 0. 01 and 1. 0 ug/ml.
c A reagent blank (distilled
water) should be included
in the set of standards.
4 Adjusting Concentrations
a You may find it necessary
to adjust preparation quantities
given in the method write-up,
because your cell (light path
length) differs from the
example.
b These adjustments are
discussed in A Requirements
(above), and are usually
applied to the Standard
(intermediate) Solution.
C Chemical Treatment
1 Most colorimetric methods
require that the calibration
standards (including the
reagent blank) are to be
treated as the sample. Thus,
they are to be processed
through pretreatments and
through the test as if they
were samples. Then any
test effects on sample results
will be compensated by the
same effects on results ob-
tained for the treated standards.
2 One should be aware that pH is
a critical condition for most
colorimetric reactions.
Ordinarily, a pH adjustment
is included in the test method
and reagents include chemicals
to control pH. Thus.the pro-
cessed standards correspond to
the samples regarding pH, and
thus they correspond in degree
of color development. If stand-
ards are processed in some
other manner, they must be
pH adjusted to correspond to
the samples at the time of
color development.
V SAMPLE DILUTIONS
A Concentration Limits
The concentration of the sample must
result in an absorbance within the range
of the calibration standards, i. e., accu-
rately detectable in the light path provided
by the instrument. A dilution before analysis
may be required to accomplish this. It is
not accurate to dilute a sample after pro-
cessing in order to obtain a usable absorb-
ance reading.
1 Record dilution volumes so a dilution
factor can be calculated and applied
to results.
2 An analyst often has a good estimate of
the expected concentration of a sample
if s/he routinely tests samples from
the same source. In this case, a single
dilution, if any, is usually sufficient,
3 If a sample is from an unknown source,
the analyst has several choices.
a Process the sample. If the reading
shows it is too concentrated, dilute
it until you get a value in the usable
range. This result is not accurate
enough to report, but you now know
how to dilute the sample to process
it through the test to get usable re-
sults.
b Prepare at least a 50% dilution and
analyze it plus an undiluted aliquot.
c Prepare a variety of dilutions,
d Use some other analytical method
to get a rough estimate of the ex-
pected concentration.
B Final Volumes
1 Dilute to a final volume sufficient to rinse
the measuring glassware and provide the
test volume cited in the referenced method.
2 Save any undiluted sample.
7-4
-------
USE OF A SPECTOPHOTOMETER
VI PROCEDURE FOR USING A SPECTRO-
PHOTOMETER
A Zeroing Operation
The following steps have been written
for spectrophotometers used in this
course. Check the manual for the
available instrument for the steps
applicable for your work.
1 Set the wavelength control to
the setting specified for the
standards you are testing.
Approach the setting by be-
ginning below the number
and dialing up to it.
2 If a cell is in the holder, re-
move it.
3 Close the cell holder cover.
4 Turn the power switch/zero
control (left) knob until the
needle reads infinite (symbol co )
absorbance (on the lower scale).
5 Rinse a cell two times with
tap water, two times with
distilled water, then two
times with the reagent blank
solution.
6 Fill the cell about three -
fourths full with reagent
blank solution.
7 Thoroughly wipe the outside
of the cell with a tissue to
remove fingerprints and any
spilled solution. Hold the
cell by the top edges.
8 Open the cell holder cover and
gently slide the cell down into
the sample holder.
9 Slowly rotate the cell until
the white vertical line on
the cell is in line with the
ridge on the edge of the
sample holder.
10 Close the cover and turn the light
control (right) knob until the needle
reads zero absorbance (on the
lower scale).
11 Record an absorbance of zero for
this zero concentration solution on
a data sheet. (See next page).
12 Slowly remove the cell and close the
cover. (No solution should spill inside
the instrument). Keep the solution in
the cell.
13 The needle should return to the infinite
absorbance setting. It it does not:
a Reset the needle to the co absorbance
mark using the power switch/zero
(left) control knob.
b Re-test the reagent blank solution
using steps 7 through 12 above.
c If the needle does not return to the
oo absorbance mark, another setting
as noted in a. and b. is required.
Additional warm-up time may be
necessary before these settings can
be made.
B Reading Absorbances
Using a single cell in the spectrophotometers
used in this course
1 Discard any solution in the cell.
2 Rinse the cell two times with tap water,
and two times with distilled water. Then
rinse it two times with the lowest concen-
tration standard remaining to be tested,
or with processed sample.
3 Fill the cell about three-fourths full with
the same standard or sample.
4 Use a tissue to remove any fingerprints
from the cell and any droplets on the
outside. Hold the cell by the top edges.
5 Open the cell holder cover and gently
slide the cell down into the sample holder.
7-5
-------
USE OF A SPECTROPHOTOMETER
6 Slowly rotate the cell until the
white vertical line on the cell
is in line with the ridge on the
edge of the sample holder.
7 Close the cover.
8 Record the concentration of
the standard and its absorbance
on a data sheet. (For a sample,
record its identification code and
its absorbance on the data sheet).
DATA SHEET
Concentration
mg/liter
Absorbance
0.00
SAMPLE
SAMPLE
9 Repeat steps 1 through 8 (above) for
each standard and sample to be
tested. If a large number of meas-
urements are to be made, check the
instrument calibration every fifth
reading.
a Use another aliquot of a
solution already tested to
see if the same reading is
obtained. If not, repeat the
zeroing operation in A (above).
b Alternatively, you can use the
blank, if supply permits, and
repeat the zeroing operation
in A. (above).
10 When all the readings have been
obtained, discard any solution
remaining in the cell and rinse
the cell with tap water. Clean
the cell more thoroughly, (III C. 3),
as soon as possible.
11 If no other tests are to be done,
turn off the instrument, pull out
the plug and replace any protective
covering.
VII CHECKING RESULTS
A Readings Greater Than 0. 70
On our instrument, these are considered
to be inaccurate. Check the manual for
your instrument or check the scale divi-
sions to determine the limit for other
models.
1 Do not use readings greater than
0. 70 to develop a calibration curve,
2 From five to eight points (counting
zero) are recommended for constructing
a calibration curve. If you have fewer
than five usable values, you should not
draw a curve.
3 To prevent excessively high values
in future tests, decrease the cell
path length, if possible, by using
an adapter and smaller cell.
4 If you cannot decrease the cell path
length, you can at least obtain
enough values to construct a curve.
Prepare standards with five to eight
concentrations ranging from zero to
the concentration of the standard
having an absorbance nearest to 0. 70.
This gives you more values for a curve,
but it reduces the applicable range of
the test. Usually the sample can be
diluted before testing so the result
will fit on the standard curve.
B Highest Reading is Less Than 0. 6
1 Increase the cell path length by using
a larger cell. A higher reading results.
2 Prepare a different set of standards
with greater concentrations.
7-6
-------
USE OF A SPECTROPHOTOMETER
VIII CONSTRUCTING A CALIBRATION
CURVE
If you have from five to eight usable
absorbance values, you can construct
a concentration curve.
A Graph Paper
Should be divided into squares of
equal size in both directions
B Concentration Axis
1 Labeling
C Absorbance Axis
1 Labeling
The shorter side should be
labeled at equal intervals
with absorbance numbers
marked from 0. 00 to at least
0. 70 absorbance units.
D Plotting the Curve
1 Use the absorbances recorded
for each standard concentration
to plot points for the curve.
The longer side should be
labeled at equal intervals
with the concentrations of
the calibration standards
marked from 0.0 to at least
the highest concentration
recorded for the standards
on the data sheet.
2 Units
a It is most convenient
to express these con-
centrations in the units
to be reported. Otherwise,
a unit-conversion factor
would have to be applied
to obtain final, reportable
values every time you use
the curve.
2 The points should fall in a
reasonably straight line.
3 Use a straight-edge to draw
a line of best fit through the
points. If the points do not
all fall on the line, an acceptable
result is an equal number of
points falling closely above,
as well as below the line.
Experience provides a basis
for judging acceptability.
4 It is not permissable to extra-
polate the curve.
IX USING THE CALIBRATION CURVE
A Finding concentration of the sample
Example: If you dilute a
standard solution to make
100. 0 ml volumes of cali-
bration standards, you
have a choice in expressing
the resulting concentrations.
You can use weight/ 100 ml,
or you can calculate weight/
1 liter. If you are to report
results as weight/liter, but
you construct your curve
using weight/ 100 ml, you will
have to multiply every sample
result from the curve by
iooo ^ • xi.
— or 10 to obtain the re-
portable value. It is much
easier to convert the original
calibration standard concentra-
tions to the desired units and to
use these as labels on the graph.
B
1 Use the absorbance value(s)
recorded for the sample(s), and
the calibration curve to find the
concentrations). If the concentra-
tion units differ from those required
for reporting results, apply a unit
conversion factor (VII IB. 2).
2 If more than one dilution of a sample
was tested, use the result that falls
nearest the middle of the curve.
If a sample was diluted, calculate the
dilution factor and apply it to the con-
centration you find for the sample from
the calibration curve.
?~7
-------
USE OF A SPECTROPHOTOMETER
1 Dilution Factor=
final dilution volume
ml sample used in dilution
! Example: You diluted 10 ml
sample to 50 ml. The con-
centration found by using a
calibration curve was 0. 5
mg/liter.
Then-
This outline was prepared by Audrey D.
Kroner, Chemist, National Training and
Operational Technology Center, MOTD,
OWPO, US EPA, Cincinnati, Ohio 45268
Descriptors; Analytical Techniques,
Chemical Analysis, Colorimetry,
Laboratory Tests, Spectrophotometry
constituent, mg/1
50ml x 0. 5
mg
10 ml liter
2. 5 mg/liter
14
-------
Use
of Spectrophotometer
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7-9
-------
DISSOLVED OXYGEN
Factors Affecting DO Concentration in Water
I The Dissolved Oxygen determination is
a very important water quality criteria for
many reasons:
A Oxygen is an essential nutrient for all
living organisms. Dissolved oxygen is
essential for survival of aerobic
organisms and permits facultative
organisms to metabolize more effectively.
Many desirable varieties of macro or
micro organisms cannot survive at
dissolved oxygen concentrations below
certain minimum values. These values
vary with the type of organisms, stage
in their life history, activity, and other
factors.
13 Dissolved oxygen levels may be used as
an indicator of pollution by oxygen
demanding wastes. Low DO concen-
trations are likely to be associated with
low quality waters.
C The presence of dissolved oxygen
prevents or minimizes the onset of
putrefactive decomposition and the
production of objectionable amounts of
malodorous sulfides, mercaptans,
amines, etc.
D Dissolved oxygen is essential for
terminal stabilization wastewaters.
High DO concentrations are normally
associated with good quality water,
E Dissolved oxygen changes with respect
to time, depth or section of a water
mass are useful to indicate the degree
of stability or mixing characteristics
of that situation,
F The BOD or other respirometric test
methods for water quality are commonly
based upon the difference between an
initial and final DO determination for a
given sample time interval and con-
dition, These measurements are
useful to indicate:
1 The rate of biochemical activity in
terms of oxygen demand for a
given sample and conditions.
2 The degree of acceptability
(a bioassay technique) for bio-
chemical stabilization of a given
mi c rob iota in response to food,
inhibitory agents or test conditions.
3 The degree of instability of a
water mass on the basis of test
sample DO changes over an
extended interval of time.
4 Permissible load variations in
surface water or treatment units
in terms of DO depletion versus
time, concentration, or ratio of
food to organism mass, solids, or
volume ratios,
5 Oxygenation requirements
necessary to meet the oxygen
demand in treatment units or
surface water situations.
G Minimum allowable DO concentrations are
specified in all Water Quality standards.
II FACTORS AFFECTING THE DO
CONCENTRATION IN WATER
A Physical Factors:
1 DO solubility in water for an
air/water system is limited to
about 9 mg DO/liter of water at
2 0°C. This amounts to about
0. 0009% as compared to 21% by
weight of oxygen in air.
2 Transfer of oxygen from air to
water is limited by the interface
area, the oxygen deficit, partial
pressure, the conditions at the
8-1
WP. NAP. 25a. 11. 77
-------
Dissolved Oxygen
interface area, mixing phenomena
and other items.
Certain factors tend to confuse
reoxygenation mechanisms of
water aeration:
a The transfer of oxygen in air
to dissolved molecular oxygen
in water has two principal
variables:
1) Area of the air-water
interface.
2) Dispersion of the oxygen-
saturated water at the
interface into the body liquid.
The first depends upon the surface
area of the air bubbles in the water
or water drops in the air; the
second depends upon the mixing
energy in the liquid. If diffusors
are placed in a line along the wall,
dead spots may develop in the core.
Different diffusor placement or
mixing energy may improve oxygen
transfer to the liquid two or threefold,
b Other variables in oxygen transfer
include:
3) Oxygen deficit in the liquid.
4) Oxygen content of the gas phase.
5) Time.
If the first four variables are
favorable, the process of water
oxygenation is rapid until the liquid
approaches saturation, Much more
energy and time are required to
increase oxygen saturation from
about 95 to 100% than to increase
oxygen saturation from 0 to about
95%. For example: An oxygen-
depleted sample often will pick up
significant DO during DO testing;
changes are unlikely with a sample
containing equilibrium amounts
of DO.
c The limited solubility of oxygen
in water compared to the oxygen
content of air does not require
the interchange of a large mass of
oxygen per unit volume of water
to change DO saturation. DO
increases from zero to 50%
saturation are common in passage
over a weir.
d Aeration of dirty water is practiced
for cleanup. Aeration of clean
water results in washing the air and
transferring fine particulates and
gaseous contaminants to the liquid,
e One liter of air at room temperature
contains about 230 mg of oxygen,
A 5 gal carboy of water with 2 liters
of gas space above the liquid has
ample oxygen supply for equilibration
of DO after storage for 2 or 3 days
or shaking for 30 sec.
f Aeration tends toward evaporative
cooling. Oxygen content becomes
higher than saturation values at
the test temperature, thus
contributing to high blanks.
3 Oxygen solubility varies with the
temperature of the water.
Solubility at 10o c is about two
times that at 30° c. Temperature
often contributes to DO variations
much greater than anticipated by
8-2
-------
Dissolved Oxygen
solubility, A cold water often has
much more DO than the solubility
limits at laboratory temperature.
Standing during warm up commonly
results in a loss of DO due to
oxygen diffusion from the super-
saturated sample. Samples
warmer than laboratory tempera-
ture may decrease in volume due
to the contraction of liquid as
temperature is lowered. The full
bottle at higher temperature will
be partially full after shrinkage
with air entrance around the stopper
to replace the void. Oxygen in the
air may be transferred to raise the
sample DO. For example, a
volumetric flask filled to the 1000 ml
mark at 3C)o C will show a water
level about 1/2 inch below the mark
when the water temperature is
reduced to 20° C. BOD dilutions
should be adjusted to 20° C + or -
1 1/20 before filling and testing.
4 Water density varies with tem-
perature with maximum water
density at 4° C. Colder or warmer
waters tend to promote stratification
of water that interferes with
distribution of DO because the
higher density waters tend to seek
the lower levels.
5 Oxygen diffusion in a water mass is
relatively slow, hence vertical and
lateral mixing are essential to
maintain relatively uniform oxygen
concentrations in a water mass.
6 Increasing salt concentration
decreases oxygen solubility
slightly but has a larger effect
upon density stratification in a
water mass.
7 The partial pressure of the oxygen
in the gas above the water interface
controls the oxygen solubility
limits in the water. For example,
the equilibrium concentration of
oxygen in water is about 9 rag DO/1
under one atmospheric pressure of
air, about 42 mg DO/liter in
contact with pure oxygen and 0 mg
DO/liter in contact with pure
nitrogen (@ 20o C).
B Biological or Bio-Chemical Factors
1 Aquatic life requires oxygen for
respiration to meet energ\
requirements for growth, repro-
duction, and motion. The net
effect is to deplete oxygen resources
in the water at a rate controlled
by the type, activity, and mass of
living materials present, the
availability of food and favor-
ability of conditions.
2 Algae, autotrophic bacteria, plants
or other organisms capable of
photosynthesis may use light
energy to synthesize cell materials
from mineralized nutrients with
oxygen released in process.
a Photosynthesis occurs only
under the influence of adequate
light intensity.
b Respiration of alga is
continuous.
c. The dominant effect in terms
of oxygen assets or
liabilities of alga depends upon
algal activity, numbers and
light intensity. Gross algal
productivity contributes to
significant diurnal DO
variations.
3 High rate deoxygenation commonly
accompanies assimilation of
readily available nutrients and
conversion into cell mass or
storage products. Deoxygenation
due to cell mass respiration
commonly occurs at some lower
rate dependent upon the nature of
the organisms present, the stage
of decomposition and the degree
of predation, lysis, mixing and
regrowth. Relatively high
8-3
-------
Dissolved Oxygen
deoxygenation rates commonly are
associated with significant growth
or regrowth of organisms.
Micro-organisms tend to flocculate
or agglomerate to form settleable
masses particularly at limiting
nutrient levels (after available
nutrients have been assimilated or
the number of organisms are large
in proportion to available food).
a Resulting benthic deposits
continue to respire as bed
loads.
b Oxygen availability is limited
because the deposit is physically
removed from the source of
surface oxygenation and algal
activity usually is more
favorable near the surface.
Stratification is likely to limit
oxygen transfer to the bed load
vicinity.
c The bed load commonly is
oxygen deficient and decomposes
by anaerobic action,
d , Anaerobic action commonly is
characterized by a dominant
hydrolytic or solubilizing action
with relatively low rate growth
of organisms.
e The net effect is to produce low
molecular weight products
from cell mass with a corre-
spondingly large fraction of
feedback of nutrients to the
overlaying waters. These
lysis products have the effect
of a high rate or immediate
oxygen demand upon mixture
with oxygen containing waters.
f Turbulence favoring mixing of
surface waters and benthic
sediments commonly are
associated with extremely
rapid depletion of DO.
Recurrent resuspension of
thin benthic deposits may
contribute to highly erratic
DO patterns.
g Long term deposition areas
commonly act like point
sources of new pollution as
a result of the feedback of
nutrients from the deposit.
Rate of reaction may be low
for old materials but a low
percentage of a large m^ss of
unstable material may produce
excessive oxygen demands.
C Tremendous DO variations are likely
in a polluted water in reference to
depth, cross section or time of day.
More stabilized waters tend to show
decreased DO variations although it is
likely that natural deposits such as leaf
mold will produce differences related
to depth in stratified deep waters.
ACKNOWLEDGMENTS
This outline contains significant materials
from previous outlines by J. W. Mandia.
REFERENCE
1 Methods for Chemical Analysis of
Water & Wastes, U. S. Environmental
Protection Agency, Environmental
Monitoring & Support Laboratory,
Cincinnati, Ohio, 45268, 1974.
This outline was prepared by F. J. Ludzack,
former Chemist, National Training Center,
and revised by Charles R. Feldmann,
Chemist, National Training & Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268
Descriptors ; Aeration, Aerobic Conditions,
Air-Water Interfaces, Anaerobic Conditions,
Benthos, Biolcg ical Oxygen Demand, Dissolved
Oxygen, Water Pollution, Water Quality
8-4
-------
DISSOLVED OXYGEN
Determination by the Winkler Iodometric Titration - Azide Modification
This method is applicable for use with most
wastewaters and streams that contain nitrate
nitrogen and not more than 1 mg/1 of ferrous
iron. Other reducing or oxidizing materials
should be absent. If 1 ml of fluoride solution
is added before acidifying the sample and
there is no delay in titration, the method is
also applicable in the presence of 100-200
mg/ 1 ferric iron.
converting dissolved oxygen in the
water into a form in which it can
be measured.
0„ —»MnO(OH),
Mn(SO )
^ £
Ig —v Thiosulfate (thio) or
phenylarsine oxide (PAO)
titration.
The azide modification is not applicable
under the following conditions: (a) samples
containing sulfite, thiosulfate, polythionate,
appreciable quantities of free chlorine or
hypochlorite; (b) samples high in suspended
solids; (c) samples containing organic
substances which are readily oxidized in a
highly alkaline solution, or which are oxidized
by free iodine in an acid solution; (d) untreated
domestic sewage; (e) biological floes; and
(f) where sample color interferes with
endpoint detection. In instances where the
azide modification is not applicable, the
DO probe should be used.
A Reactions
1 The determination of DO involves
a complex series of interactions
that must be quantitative to provide
a va lid DO result. The number of
sequential reactions also compli-
cates interference control. The
reactions will be presented first
followed by discussion of the
functional aspects.
MnS04 + 2 KOH — Mn(OH)2 + K2S04 (a)
2 Mn(OH)2 + Og —*2 MnO(OH)2
(b)
MnO(OH)2 + 2 H2S04 — Mn(S04)2 + 3^0 (c>
Mn(SO ) + 2 KI-* MnSO + K SO + I (d)
I + 2 Na S„On'
2 2 2 3
Na S O + 2 NaI (e)
2 4 6
Reaction sequence
The series of reactions involves
five different operational steps in
b All added reagents are in excess
to improve contact possibilities
and to force the reaction toward
completion.
The first conversion, O
MnO(OH)2 (reactions a, 5) is an
oxygen transfer operation where
the dissolved oxygen in the water
combines with manganous
hydroxide to form an oxygenated
manganic hydroxide.
a The manganous salt can react
with oxygen only in a highly
alkaline media,
b The manganous salt and alkali
must be added separately with
addition below the surface of
the sample to minimize reaction
with atmospheric oxygen via
air bubbles or surface contact.
Reaction with sample dissolved
oxygen is intended to occur
upon mixing of the reagents and
sample after stoppering the
full bottle (care should be used
to allow entrained air bubbles
to rise to the surface before
adding reagents to prevent
high results due to including
entrained oxygen).
c Transfer of oxygen from the
dissolved state to the pre-
cipitate form involves a two
phase system of solution and
precipitate requiring effective
mixing for quantitative
transfer. Normally a gross
CH. O. do. 3ie. li.11
9-1
-------
Dissolved Oxygen Determination
f
excess of reagents is used to limit
mixing requirements. Mixing by
rapid inversion 25 to 35 times will
accomplish the purpose. Less energy-
is required by inversion 5 or 6 times,
allowing the floe to settle until there
is clear liquid above the floe, repeating
the inversion, & allowing the floe to
settle about two-thirds of the way down
in the bottle. The reaction is rapid;
contact is the principal problem in
the two phase system.
d If the alkaline floe is white,
no oxygen is present.
4 Acidification (reactions c and d)
changes the oxygenated.manganic
hydroxide to manganic sulfate
which in turn reacts with
potassium iodide to form elemental
iodine. Under acid conditions,
oxygen cannot react directly with
the excess manganous sulfate
remaining in solution.
5 Iodine (reaction e) may be titrated
with sodium thiosulfate standard
solution to indicate the amount of
dissolved oxygen originally
present in the sample.
a The blue color of the starch-
iodine complex commonly is
used as an indicator. This
blue color disappears when
elemental iodine has been
reacted with an equivalent
amount of thiosulfate.
b Phenylarsine oxide solutions are
more expensive to obtain but
have better keeping qualities
than thiosulfate solutions.
Occasional use, field operations
and situations where it is not
feasible to calibrate thio
solutions regularly ^ usually
encourage use of purchased
PAO reagents.
a Fill a 300 ml bottle* under
conditions minimizing DO
changes. This means that the
sample bottle must be flushed
with test solution to displace
the air in the bottle with water
characteristic of the tested
sample*
*DO test bottle volumes should
be checked - discard those
outside of the limits of 300 ml
+ or - 3 ml.
b To the filled bottle:
1) Add MnSO^ reagent (2 ml)
2) Add KOII, KI, NaN reagent
(2 ml)
Stopper, mix by inversion,
allow to settle until there is
clear liquid above the floe,
rejjeat the inversion, & allow
the floe to settle about two-thirds
of the way down in the bottle.
Highly saline & other test waters
may settle very slowly. In this
case, allow some reasonable time
(e. g. 2 min.) for completion of the
reaction.
c To the alkaline mix (settled
about half way) add 2 ml of
sulfuric acid, stopper and mix
until the precipitate dissolves,
d Transfer the contents of the
bottle to a 500 ml Erlenmeyer
flask and titrate with 0. 0375
normal thiosulfate. Each
ml of reagent used represents
1 mg of DO/liter of sample.
6
For practical purposes the DO
determination scheme involves the
following operations.
-------
Dissolved Oxygen Determination
The same thing applies for
other sample volumes when
using an appropriate titrant
normality; e. g.,
1) For a 200 ml sample, use
0. 025 N Thio
2) For a 100 ml sample, use
0.0125 N Thio
7 The addition of the first two DO
reagents, (MnSO^ and the KOH, KI
and NaNg solutions) displaces an
equal quantity of the sample. This
is not the case when acid is added
because the clear liquid above the
floe does not contain dissolved
oxygen as all of it should be con-
verted to the particulate MnOtOH^.
Some error is introduced by this
displacement of sample during
dosage of the first two reagents.
The error upon addition of 2 ml of
each reagent to a 300 ml sample
is i. X 100 or 1,33% loss in DO.
300
This may be corrected by an
appropriate factor or by adjust-
ment of reagent normality. It is
generally considered small in
relation to other errors in sampling,
manipulation and interference,
hence this error may be recognized
but not corrected.
8 Reagent preparation and pro-
cedural details can be found irj
reference 1.
IV The sequential reactions for the
Chemical DO determination provides
several situations where significant inter-
ference may occur in application on
polluted water, such as:
A Sampling errors may not be strictly
designated as interference but have the
same effect of changing sample DO.
Inadequate flushing of the bottle con-
tents or exposure to air may raise the
DO of low oxygen samples or lower the
DO of supersaturated samples.
B Entrained air may be trapped in a DO
bottle by:
1 Rapid filling of vigorously mixed
samples without allowing the
entrained air to escape before
closing the bottle and adding DO
reagents,
2 Filling a bottle with low temperature
water holding more DO than that in
equilibrium after the samples warm
to working temperature.
3 Aeration is likely to cool the sample
permitting more DO to be introduced
than can be held at the room or
incubator temperatures.
4 Samples warmer than working
incubator temperatures will be
only partially full at equilibrium
temperatures.
Addition of DO reagents results in
reaction with dissolved or entrained
oxygen. Results for DO are invalid
if there is any evidence of gas
bubbles in the sample bottle.
C The DO reagents respond to any oxidant
or reductant in the sample capable of
reacting within the time allotted. HOC1
or II2O2 may raise the DO titration
while IlgS, & SH may react with sample
oxygen to lower the sample titration.
The items mentioned react rapidly and
raise or lower the DO rjjpult promptly.
Other items such as Fe or SO„ may
or may not react completely within the
time allotted for reaction. Many
organic materials or complexes from
benthic deposits may have an effect upon
DO results that are difficult to predict.
They may have one effect during the
alkaline stage to release iodine from
Kl while favoring irreversible
absorption of iodine during the acid
stage. Degree of effect may increase
with reaction time. It is generally
inadvisable to use the iodomctric
titration on samples containing large
amounts of organic contaminants or c[~ ^
-------
Dissolved Oxygen Determination
benthic residues. It would be expected
that benthic residues would tend toward
low results because of the reduced iron
and sulfur content - they commonly
favor high results due to other factors
that react more rapidly, often giving
the same effect as in uncontrolled
nitrite interference during titration.
D Nitrite is present to some extent in
natural waters or partially oxidized
treatment plant samples. Nitrite is
associated with a cyclic reaction during
the acid stage of the DO determination .
that may lead to erroneous high results.
1 These reactions may be repre-
sented as follows;
2HN02 + 2 HI -I2 + 4II20 + N O^ (a)
t \
2HN02 + l/202+ HgO + N202 (b)
These reactions are time, mixing
and concentration dependent and
can be minimized by rapid
processing.
F Polluted water commonly contains
significant interferences such as C.
It is advisable to use a membrane
protected sensor of the electronic type
for DO determinations in the presence
of these types of interference.
G The order of reagent addition and prompt
completion of the DO determination is
critical. Stable waters may give valid
DO results after extended delay of
titration during the acidified stage. For
unstable water, undue delay at any stage
of processing accentuates interference
problems.
REFERENCE
1 Methods for Chemical Analysis of
Water & Wastes, U. S. Environmental
Protection Agency, Environment
Monitoring & Support Laboratory,
Cincinnati, Ohio 45268, 1974,
acknowledgments
2 Sodium azide (NaNg) reacts with This outline contains significant materials
nitrite under acid conditions to from previous outlines by J. W. Mandia,
form a combination of N + N20 Review and comments by C. R. Hirth and
which effectively blocks the R. L. Booth are greatly appreciated,
cyclic reaction by converting the
HNO to noninterfering compounds
of nitrogen.
3 Sodium azide added to fresh
alkaline KI reagent is adequate to
control interference up to about
20 mg of N00 N/liter of sample.
The azide is unstable and grad- This outline was prepared by F. J. Ludzac.k,
ually decomposes. If resuspended former Chemist, National Training Center,
benthic sediments are not detectable and revised by C. R. Feldmann, Chemist,
in a sample showing a returning National Training & Operational Technology
blue color, it is likely that the Center, MOTD, OWPO, USE I'A, Cincinnati,
azide has decomposed in the Ohio 45268.
alkaline KI azide reagent.
Descriptors; Chemical Analysis, Dissolved
E Surfactants, color and Fe+++ may Oxygen, Oxygen, Water Analysis
confuse endpoint detection if present
in significant quantities.
9-4
-------
laboratory procedure for dissolved oxygen
Winkler Method-Azide Modification
I APPLICABILITY
A The azide modification is used for
most wastewaters and streams
which contain nitrate nitrogen
and not more than 1 mg of ferrous
iron/1. If 1 ml 40% KF solution is
added before acidifying the sample and
there is no delay in titration, the method
is also applicable in the presence of
100-200 mg ferric iron/1.
B Reducing and oxidizing materials
should be absent.
C Other materials which interfere with
the azide modification are: sulfite,
thiosulfate, appreciable quantities of
free chlorine or hypochlorite, high
suspended solids, organic substances
readily oxidized in a highly alkaline
medium, organic substances readily
oxidized by iodine in an acid medium,
untreated domestic sewage,, biological
floes, and color which may interfere
with endpoint detection. A dissolved
oxygen meter should be used when
these materials are present in the
sample.
II REAGENTS
Distilled water is to be used for the
preparation of all solutions.
A Manganous Sulfate Solution
Dissolve 480 g MnSO/ 4H„0 (or 400 g
MnSO * 2H O, or 3644g MASO^ HgO) in
water and ailute to 1 liter.
B Alkaline-Iodide-Azide Solution
Dissolve 500 g sodium hydroxide (or
700 g potassium hydroxide) and 135 g
sodium iodide (or 150 g potassium iodide)
in water and dilute to 1 liter. To this
solution add 10 g of sodium azide
dissolved in 40 ml water.
C Sulfuric Acid, Cone.
The strength of this acid is 36 N.
D Starch Solution
Prepare an emulsion of 10 g of soluble
starch in a mortar or beaker with a
small quantity of water. Pour this
emulsion into 1 liter of boiling water,
allow to boil a few minutes, and let
settle overnight. Use the clear supernate.
This solution may be preserved by the
addition of 5 ml per liter of chloroform
and storage in a refrigerator at 10°C.
E Sodium Thiosulfate Stock Solution 0. 75 N
Dissolve 186. 15 g Na S O ¦ 5H O in boiled
£ Ci «
and cooled water and dilute to 1 liter.
Preserve by adding 5 ml chloroform.
F Sodium Thiosulfate Standard Titrant 0. 0375N
Dilute 50,0 ml of stock solution to 1 liter.
Preserve by adding 5 ml of chloroform.
G Potassium Biiodate Solution 0.0375N
Dry about 5 g of Kll (JO^ at 103°C for
two hours and cool in a desiccator.
Dissolve 4. 873 g of the solid in water and
dilute to 1 liter. Dilute 250 ml of this
solution to 1 liter.
H Sulfuric Acid Solution 10%
Add 10 ml of cone sulfuric acid to 90 ml
of water. Mix thoroughly and cool.
I Potassium Iodide Crystals
III STANDARDIZATION OF THE TITRANT
A Dissolve 1-3 g of potassium iodide in
100-150 ml of water.
B Add 10 ml of 10% sulfuric acid and mix.
C Pi pet in 20 ml of the 0. 0375N potassium
biiodate and mix. Place in the dark for
5 minutes.
D Titrate with the 0. 0375N sodium
thiosulfate standard titrant to the
appearance of a pale yellow color.
CH. O. do. lab. 3d. 8. 78
10-1
-------
Laboratory Procedure for Dissolved Oxygen
Mix the solution thoroughly during the
titration.
E Add 1-2 ml of starch solution and mix.
The solution is now blue in color.
F Continue the addition of the titrant,
with thorough mixing, until the
solution turns colorless.
G Record the ml of titrant used.
H Calculate the N of the sodium
thiosulfate standard titrant. It will be
approximately 0.0375.
N = (ml x N) of the biiodate
ml of titrant
= 20. 0 x 0.0375
ml of titrant
= 0.75
ml of titrant
IV PROCEDURE
A Addition of Reagents
1 Mariganous sulfate and alkaline
iodide-azide
To a full BOD bottle (300 ml f 3 ml),
add 2 ml manganous sulfate solution
and 2 ml alkaline-iodide azide reagent
with the tip of each pipette below the
surface of the liquid.
2 Stopper the bottle without causing
formation of an air bubble.
3 Rinse under running water.
4 Mix well by inverting 4-5 times,
5 Allow the precipitate to settle until
at least 200 nil of clear supernate
have been produced.
6 Repeat steps 4 and 5.
7 Add 2 ml cone, sulfuric acid with the
tip of the pipette above the surface of
¦ the liquid.
8 Stopper the bottle without causing
formation of an air bubble.
9 Rinse under running water.
10 Mix by inverting several times to
dissolve the precipitate.
11 Pour contents of bottle into a wide-
mouth 500 ml Erlenmeyer flask.
B TITRATION
1 Titrate with 0. 0375N thiosulfate to a
pale yellow color.
2 Add 1-2 ml starch solution and mix.
3 Continue the addition of the titrant,
with thorough mixing, until the
solution turns colorless.
4 Record the ml of titrant used.
C CALCULATION
mg DO /1 = ml titrant x N titrant x 8 x 1000
ml sample
If the N of the titrant exactly = 0. 0375,
mg DO/1 = ml titrant x 0. 0375 x 8 x 1000
300
=- ml titrant x 1
= ml titrant
REFERENCE
Methods for Chemical Analysis of Water
8i Wastes, U.S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, Cincinnati, Ohio 45268, 1974
This outline was prepared by C. R. Feldmann,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 452 68
Descriptors: Analytical Techniques,
Chemical Analysis, Dissolved Oxygen,
Laboratory Tests, Oxygen, Water Analysis
10-2
-------
Laboratory Procedure for Dissolved Oxygen
DATA SHEET
ml of titrant =
N of titrant =
ml of sample
t^/t.l ml titrant x N titrant x 8 x 1000
mg DO/liter =
ml sample
x 8 x 1000
10-3
-------
DISSOLVED OXYGEN
DETERMINATION BY ELECTRONIC MEASUREMENT
I INTRODUCTION
A Electronic measurement of DO is attractive
for several reasons:
1 Electronic methods are more readily
adaptable for automated analysis, con-
tinuous recording, remote sensing or
portability,
2 Application of electronic methods with
membrane protection of sensors affords
a high degree of interference control.
3 Versatility of the electronic system
permits design for a particular measure-
ment, situation or use.
4 Many more determinations per man-
hour are possible with a minor expend-
iture of time for calibration.
Electronic measurements or electro-
metric procedures - procedures using
the measurement of potential differences
as an indicator of reactions taking
place at an electrode or plate.
Reduction - any process in which one
or more electrons arc added to an atom
or an ion, such as + 2c -* 20
The oxygen has been reduced.
Oxidation - any process in which one
or more electrons are removed from
an atom or an ion, such as Zn° - 2e
—~ Zn+^, The zinc has been oxidized.
Oxidation - reduction reactions - in a
strictly chemical reaction, reduction
cannot occur unless an equivalent
amount of some oxidizable substance
has been oxidized. For example:
B Electronic methods of analysis impose
certain restrictions upon the analyst to
insure that the response does, in fact,
indicate the item sought.
1 The ease of reading the indicator tends
to produce a false sense of security.
Frequent and careful calibrations are
essential to establish workability of the
apparatus and validity of its response.
2H0 + O
2H
'2
4e
02 + 4e
2H2°
+1
4H hydrogen oxidized
-9.
20 ~ oxygen reduced
Chemical reduction of oxygen may also
be accomplished by electrons supplied
to a noble metal electrode by a battery
or other energizer.
The use of electronic devices requires
a greater degree of competence on the
part of the analyst. Understanding of
the behavior of oxygen must be supple-
mented by an understanding of the
particular instrument and its behavior
during use.
C Definitions
Electrochemistry - a branch of chemistry
dealing with relationships between
electrical and chemical changes.
6 Anode - an electrode at which oxidation
of some reactable substance occurs.
7 Cathode - an electrode at which
reduction of some reactable substance
occurs. For example in I. C. 3, the
reduction of oxygen occurs at the
cathode.
8 Electrochemical reaction - a reaction
involving simultaneous conversion of
chemical energy into electrical energy
or the reverse. These conversions are
Note: Mention of Commercial Products and Manufacturers Doe
Not Imply Endorsement by the Environmental Protection
Agency.
CH. O. do. 32a. 11.77
11-1
-------
Dissolved Oxygen Determination
equivalent in terms of chemical and
electrical energy and generally are
reversible.
9 Electrolyte a solution, gel, or mixture
capable of conducting electrical energy
and serving as a reacting media for
chemical changes. The electrolyte
commonly contains an appropriate
concentration of selected mobile ions
to promote the desired reactions,
10 Electrochemical cell - a device con-
sisting of an electrolyte in which 2
electrodes are immersed and connected
via an external metallic conductor,
The electrodes may be in separate
compartments connected by tube con-
taining electrolyte to complete the
internal circuit,
a Galvanic (or voltaic) cell - an
electrochemical cell operated in
such a way as to produce electrical
energy from a chemical change,
such as a battery (See Figure 1).
b Polarographic (electrolytic) cell -
an electrochemical cell operated in
such a way as to produce a chemical
change from electrical energy
(See Figure 2),
Cathode
POLAROGRAPHiC CELL
Figure 2
Cathode
GALVANIC CELL
Figure 1
D As indicated in I. C. 10 the sign of an
electrode may change as a result of the
operating mode. The conversion by the
reactant of primary interest at a given
electrode therefore designates terminology
for that electrode and operating mode.
In electronic oxygen analyzers, the
electrode at which oxygen reduction occurs
is designated the cathode.
E Each cell type has characteristic advantages
and limitations. Both may be used
effectively,
1 The galvanic cell depends upon
measurement of electrical energy
produced as a result of oxygen
2
-------
Dissolved Oxygen Determination
reduction. If the oxygen content of the
sample is negligible, the measured
current is very low and indicator driving
force is negligible, therefore response
time is longer.
2 The polarographic cell uses a standing
current to provide energy for oxygen
reduction. The indicator response
depends upon a change in the standing
current as a result of electrons
released during oxygen reduction.
Indicator response time therefore is
not dependent upon oxygen concentration.
3 Choice may depend upon availability,
habit, accessories, or the situation,
In each case it is necessary to use
care and judgment both in selection
and use for the objectives desired.
II ELECTRONIC MEASUREMENT OF DO
A Reduction of oxygen takes place in two
steps as shown in the following equations:
1 0„ + 2 HO + 2e -» HO + 20H~
Ct Ct Ci Ci
2 H_0 + 2e~ -» 20II~
£j t-l
Both equations require electron input to
activate reduction of oxygen. The first
reaction is more important for electronic
DO measurement because it occurs at a
potential (voltage) which is below that
required to activate reduction of most
interfering components (0.3 to 0.8 volts
relative to the saturated calomel electrode -
SCE). Interferences that maybe reduced
at or below that required for oxygen
usually are present at lower concentrations
in water or may be minimized by the use
of a selective membrane or other means.
When reduction occurs, a definite quantity
of electrical energy is produced that is
proportional to the quantity of reductant
entering the reaction. Resulting current
measurements thus are more specific for
oxygen reduction.
B Most electronic measurements of oxygen
are based upon one of two techniques for
evaluating oxygen reduction in line with
equation II.A. 1. Both require activating
energy, both produce a current propor-
tional to the quantity of reacting reductant.
The techniques differ in the means of
supplying the activating potential; one
employs a source of outside energy, the
other uses spontaneous energy produced
by the electrode pair.
1 The polarographic oxygen sensor
relies upon an outside source of
potential to activate oxygen reduction.
Electron gain by oxygen changes the
reference voltage.
a Traditionally, the dropping mercury
electrode (DME) has been used for
polarographic measurements. Good
results have been obtained for DO
using the DME but the difficulty of
maintaining a constant mercury drop
rate, temperature control, and
freedom from turbulence makes it
impractical for field use.
b Solid electrodes are attractive
because greater surface area
improves sensitivity. Poisoning
of the solid surface electrodes is
a recurrent problem. The use of
selective membranes over noble
metal electrodes has minimized
but not eliminated electrode con-
tamination, Feasibility has been
improved sufficiently to make this
type popular for regular use.
2 Galvanic oxygen electrodes consist of
a decomposable anode and a noble
metal cathode in a suitable electrolyte
to produce activating energy for oxygen
reduction (an air cell or battery). Lead
is commonly used as the anode because
its decomposition potential favors
spontaneous reduction of oxygen. The
process is continuous as long as lead
and <>>xygen are in contact in the electrolyte
and the electrical energy released at
the cathode may be dissipated by an
outside circuit. The anode may be
conserved by limiting oxygen availability.
Interrupting the outside circuit may
produce erratic behavior for a time
after reconnection. The resulting
11-3
-------
Dissolved Oxygen Determination
current produced by oxygen reduction
may be converted to oxygen concen-
tration by use of a sensitivity coefficient
obtained during calibration. Provision
of a pulsed or interrupted signal makes
it possible to amplify or control the
signal and adjust it for direct reading
in terms of oxygen concentration or to
compensate for temperature effects.
IH ELECTRONIC DO ANALYZER
APPLICATION FACTORS
A Polarographic or galvanic DO instruments
operate as a result of oxygen partial
pressure at the sensor surface to produce
a signal characteristic of oxygen reduced
at the cathode of some electrode pair.
This signal is conveyed to an indicating
device with or without modification for
sensitivity and temperature or other
influences depending upon the instrument
capabilities and intended use,
1 Many approaches and refinements have
been used to improve workability,
applicability, validity, stability and
control of variables. Developments
are continuing. It is possible to produce
a device capable of meeting any reasonable
situation, but situations differ.
2 Most commercial DO instruments are
designed for use tinder specified con-
ditions. Some are more versatile than
others. Benefits are commonly reflected
in the price. It is essential to deter-
mine the requirements of the measure-
ment situation and objectives for use.
Evaluation of a given instrument in
terms of sensitivity, response time,
portability, stability, service
characteristics, degree of automation,
and consistency are used for judgment
on a cost/benefit basis to select the
most acceptable unit.
B Variables Affecting Electronic DO
Measurement
1 Temperature affects the solubility of
oxygen, the magnitude of the resulting
signal and the permeability of the
protective membrane. A curve of
oxygen solubility in water versus
increasing temperature may be concave
downward while a similar curve of
sensor response versus temperature
is concave upward. Increasing
temperature decreases oxygen solubility
and increases probe sensitivity and
membrane permeability. Thermistor
actuated compensation of probe
response based upon a linear relation-
ship or average of oxygen solubility
and electrode sensitivity is not precisely
correct as the maximum spread in
curvature occurs at about 17° C with
lower deviations from linearity above
or below that temperature. If the
instrument is calibrated at a temperature
within + or - 5 o C of working temperature,
the compensated readout is likely to be
within 2% of the real value. Depending
upon probe geometry, the laboratory
sensor may require 4 to 6% correction
of signal per 0 C change in liquid
temperature.
2 Increasing pressure tends to increase
electrode response by compression
and contact effects upon the electrolyte,
dissolved gases and electrode surfaces.
As long as entrained gases are not
contained in the electrolyte or under
the membrane, these effects are
negligible.
Inclusion of entrained gases results in
erratic response that increases with
depth of immersion,
3 Electrode sensitivity changes occur as
a result of the nature and concentration
of contaminants at the electrode sur-
faces and possible physical chemical or
electronic side reactions produced.
These may take the form of a physical
barrier, internal short, high residual
current, or chemical changes in the
metal surface. The membrane is
intended to allow dissolved gas pene-
tration but to exclude passage of ions
or particulates. Apparently some ions
or materials producing extraneous ions
within the electrode vicinity are able
to pass in limited amounts which
11-4
-------
Dissolved Oxygen Determination
become significant in time. Dissolved
gases include 1) oxygen, 2) nitrogen,
3) carbon dioxide, 4) hydrogen sulfide,
and certain others. Item 4 is likely to
be a major problem. Item 3 may pro-
duce deposits in alkaline media; most
electrolytes are alkaline or tend to
become so in line with reaction II.A. 1,
The usable life of the sensor varies
with the type of electrode system,
surface area, amount of electrolyte
and type, membrane characteristics,
nature of the samples to which the
system is exposed and the length of
exposure. For example, galvanic
electrodes used in activated sludge
units showed that the time between
cleanup was 4 to 6 months for electrodes
used for intermittent daily checks of
effluent DO; continuous use in the mixed
liquor required electrode cleanup in 2
to 4 weeks. Each electrometric cell
configuration and operating mode has
its own response characteristics.
Some are more stable than others.
It is necessary to check calibration
frequency required under conditions
of use as none of them will maintain
uniform response indefinitely. Cali-
bration before and after daily use is
advisable.
4 Electrolytes may consist of solutions
or gels of ionizable materials such as
acids, alkalies or salts, Bicarbonates,
KC1 and KI are frequently used. The
electrolyte is the transfer and reaction
media, hence, it necessarily becomes
contaminated before damage to the
electrode surface may occur. Electro-
lyte concentration, nature, amount and
quality affect response time, sensitivity,
stability, and specificity of the sensor
system. Generally a small quantity of
electrolyte gives a shorter response
time and higher sensitivity but also may
be affected to a greater extent by a
given quantity of contaminating sub-
stances,
5 Membranes may consist of teflon,
polyethylene, rubber, and certain
other polymeric films. Thickness
may vary from 0.5 to 3 mils {inches X
1/1000). A thinner membrane will
decrease response time and increase
sensitivity but is less selective and
may be ruptured more easily. The
choice of material and its uniformity
affects response time, selectivity and
durability. The area of the membrane
and its permeability are directly
related to the quantity of transported
materials that may produce a signal.
The permeability of the membrane
material is related to temperature and
to residues accummulated on the
membrane surface or interior. A
cloudy membrane usually indicates
deposition and more or less loss of
signal,
6 Test media characteristics control the
interval of usable life between cleaning
and rejuvenation for any type of
electrode. More frequent cleanup is
essential in low quality waters than for
high quality waters. Reduced sulfur
compounds are among the more
troublesome contaminants. Salinity
affects the partial pressure of oxygen
at any given temperature. This effect
is small compared to most other
variables but is significant if salinity
changes by more than 500 mg/1.
7 Agitation of the sample in the vicinity
of the electrode is important because
DO is reduced at the cathode. Under
quiescent conditions a gradient in
dissolved oxygen content would be
established on the sample side of the
membrane as well as on the electrode
.side, resulting in atypical response.
The sample should be agitated
sufficiently to deliver a representative
portion of the main body of the liquid
to the outer face of the membrane.
It is commonly observed that no
agitation will result in a very low or
negigible response after a short period
of time. Increasing agitation will cause
the response to rise gradually until
some minimum liquid velocity is reached
that will not cause a further increase
in response with increased mixing
energy. It is important to check
mixing velocity to reach a stable high
signal that is independent of a reasonable
change in sample mixing. Excessive
11-5
-------
Dissolved Oxygen Determination
mixing may create a vortex and expose
the sensing surface to air rather than
sample liquid. This should be avoided,
A linear liquid velocity of about 1 ft/sec
at the sensing surface is usually
adequate,
8 DO sensor response represents a
potential or current signal in the
milli-volt or milli-amp range in a
high resistance system. A high quality
electronic instrument is essential to
maintain a usable signal-to-noise ratio.
Some of the more common difficulties
include:
a Variable line voltage or low batteries
in amplifier power circuits.
b Substandard or unsteady amplifier
or resistor components.
c ^Independable contacts or junctions
in the sensor, connecting cables, or
instrument control circuits,
d Inadequately shielded electronic
components,
e Excessive exposure to moisture,
fumes or chemicals in the wrong
places lead to stray currents,
internal shorts or other malfunction,
C Desirable Features in a Portable DO
Analyzer
1 The unit should include steady state
performance electronic and indicating
components in a convenient but sturdy
package that is small enough to carry,
2 There should be provisions for addition
of special accessories such as bottle
or field sensors, agitators, recorders,
line extensions, if needed for specific
requirements. Such additions should
be readily attachable and detachable
and maintain good working characteristics.
3 The instrument should include a
sensitivity adjustment which upon
calibration will provide for direct
reading in terms of mg of DO/liter.
4 Temperature compensation and temp-
erature readout should be incorporated.
5 Plug in contacts should be positive,
sturdy, readily cleanable and situated
to minimize contamination. Water
seals should be provided where
necessary.
6 The sensor should be suitably designed
for the purpose intended in terms of
sensitivity, response, stability, and
protection during use. It should be
easy to clean, and reassemble for use
with a minimum loss of service time.
7 Switches, connecting plugs, and con-
tacts preferably should be located on
or in the instrument box rather than
at the "wet" end of the line near the
sensor. Connecting cables should be
multiple strand to minimize separate
lines. Calibration controls should be
convenient but designed so that it is
not likely that they will be inadvertently
shifted during use.
8 Agitator accessories for bottle use
impose special problems because they
should be small, self contained, and
readily detachable but sturdy enough
to give positive agitation and electrical
continuity in a wet zone.
9 Major load batteries should be
rechargeable or readily replaceable.
Line operation should be feasible
wherever possible.
10 Service and replacement parts avail-
ability are a primary consideration.
Drawings, parts identification and
trouble shooting memos should be
incorporated with applicable operating
instructions in the instrument manual
in an informative organized form.
D Sensor and Instrument Calibration
The instrument box is likely to have some
form of check to verify electronics,
battery or other power supply conditions
for use. The sensor commonly is not
included in this check. A known reference
11-6
-------
Dissolved Oxygen Determination
sample used with the instrument in an
operating mode is the best available
method to compensate for sensor variables
under use conditions. It is advisable to
calibrate before and after daily use under
test conditions. Severe conditions,changes
in conditions, or possible damage call for
calibrations during the use period. The
readout scale is likely to be labeled -
calibration is the basis for this label.
The following procedure is recommended:
1 Turn the instrument on and allow it to
reach a stable condition. Perform the
recommended instrument check as
outlined in the operating manual.
2 The instrument check usually includes
an electronic zero correction. Check
each instrument against the readout
scale with the sensor immersed in an
agitated solution of sodium sulfite
containing sufficient cobalt chloride to
catalyze the reaction of sulfite and
oxygen. The indicator should stabilize
on the zero reading. If it does not, it
may be the result of residual or stray
currents, internal shorting in the
electrode, or membrane rupture.
Minor adjustments may be made using
the indicator rather than the electronic
controls. Serious imbalance requires
electrode reconditioning if the electronic
check is O.K. Sulfite must be carefully
rinsed from the sensor until the readout
stabilizes to prevent carry over to the
next sample,
3 Fill two DO bottles with replicate
samples of clarified water similar to
that to be tested. This water should
not contain significant test interferences.
4 Determine the DO in one by the azide
modification of the iodometric titration.
5 Insert a magnetic stirrer in the other
bottle or use a probe agitator. Start
agitation after insertion of the sensor
assembly and note the point of
stabilization.
a Adjust the instrument calibration
control if necessary to compare
with the titrated DO,
b If sensitivity adjustment is not
possible, note the instrument
stabilization point and designate
it as ua. A sensitivity coefficient,
Ucl
(j> is equal to where DO is the
titrated value for the sample on
which ua was obtained. An unknown
DO then becomes DO = —1 . This
factor is applicable as long as the
sensitivity does not change.
6 Objectives of the test program and the
type of instrument influence calibration
requirements. Precise work may
require calibration at 3 points in the
DO range of interest instead of at zero
and high range DO. One calibration
point frequently may be adequate.
Calibration of a DO sensor in air is a
quick test for possible changes in
sensor response. The difference in
oxygen content of air and of water is
too large for air calibration to be
satisfactory for precise calibration
for use in water.
V This section reviews characteristics of
several sample laboratory instruments.
Mention of a specific instrument does not
imply USE PA endorsement or recommendation.
No attempt has been made to include all the
available instruments; those described are
used to indicate the approach used at one
stage of development which may or may not
represent the current available model.
A The electrode described by Carrit and
Kanwisher (1) is illustrated in Figure 3.
This electrode was an early example of
those using a membrane. The anode was
a silver - silver oxide reference cell with
a platinum disc cathode (1-3 cm diameter).
The salt bridge consisted of N/2 KC1 and
11-7
-------
Dissolved Oxygen Determination
KOH. The polyethylene membrane was
held in place by a retaining ring. An
applied current was used in a polarographic
mode. Temperature effects were relatively
large. Thermistor correction was studied
but not integrated with early models.
Retaining Ring .
Silver Bio*
Platinum Disk ^ Electrolyte Layer
Figure 3
B The Beckman oxygen electrode is another
illustration of a polarographic DO sensor
(Figure 4), It consists of a gold cathode,
a silver anode, an electrolytic gel con-
taining KC1, covered by a teflon membrane.
The instrument has a temperature readout
and compensating thermistor, a source
polarizing current, amplifier with signal
adjustment and a readout DO scale with
recorder contacts.
SENSOR
ELECTRONICS
:MSU
T
mo
SUVf B ANCOt
—COLO CATMODf
Figure 4, THE BECKMAN OXYGEN
SENSOR
The YSI Model 51 (3) is illustrated in
Figure 5, This is another form of
polarographic DO analyzer. The cell
consists of a silver anode coil, a gold
ring cathode and a KC1 electrolyte with
a teflon membrane. The instrument has
a sensitivity adjustment, temperature and
DO readout. The model 51 A has temp-
erature compensation via manual preset
dial. A field probe and bottle probe are
available.
YSI Model 51 DO Sensor
O Ring
Membrane
KCL Solution
Anode Coil
Cathode Ring
oH-6
D
Figure 5
The Model 54 YSI DO analyzer (4) is based
upon the same electrode configuration but
modified to include automatic temperature
compensation, DO readout, and recorder
jacks. A motorized agitator bottle probe
is available for the Model 54 (Figure 6),
rsi Madol 54 Agitator Probe '
Membrane Xotain
Agitate* Drive Sotf
II- 3
-------
Dissolved Oxygen Determination
E The Galvanic Cell Oxygen Analyzer (7, 8)
employs an indicator for proportional DO
signal but does not include thermistor
compensation or signal adjustment.
Temperature readout is provided. The
sensor includes a lead anode ring, and
a silver cathode with KOH electrolyte
(4 molar) covered by a membrane film
(Figure 7).
Precision Galvanic Cell Oxygen Probe
Connector Leads
Thermistor Coble
' Retainer
Tapered Section
to Fit BOt> Bottles
Plastic Membrane
Retainer Ring
Silver Cathode
Lead Anode Ring
Polyethylene Membrane
'Figure ?
F The Weston and Stack Model 300 DO
Analyzer (8) has a galvanic type sensor
with a pulsed current amplifier adjustment
to provide for signal and temperature
compensation. DO and temperature
readout is provided. The main power
supply is a rechargeable battery. The
sensor (Figure 8) consists of a lead anode
coil recessed in the electrolyte cavity
(50% KI) with a platinum cathode in the tip.
The sensor is covered with a teflon mem-
brane. Membrane retention by rubber
band or by a plastic retention ring may be
used for the bottle agitator or depth
sampler respectively. The thermistor
and agitator are mounted, in a sleeve that
also provides protection for the membrane.
G The EIL, Model 15 A sensor is illustrated
in Figure 9. This is a galvanic cell with
thermistor activated temperature com-
pensation and readout. Signal adjustment
is provided. The illustration shows an
expanded scheme of the electrode which
when assembled compresses into a sensor
approximating 5/8 inch diameter and 4 inch
length exclusive of the enlargement at the
upper end. The anode consists of com-
pressed lead shot in a replaceable capsule
(later models used fine lead wire coils),
a perforated silver cathode sleeve around
the lead is covered by a membrane film.
The electrolyte is saturated potassium
bicarbonate. The large area of lead
surface, silver and membrane provides
a current response of 200 to 300 micro-
amperes in oxygen saturated water at
2 0° C for periods of up to 100 days use (8).
The larger electrode displacement favors
a scheme described by Eden (9) for
successive DO readings for BOD purposes,
V Table 1 summarizes major characteristics
of the sample DO analyzers described in
Section IV. It must be noted that an ingenious
analyst may adapt any one of these for special
purposes on a do-it-yourself program. The
sample instruments are mainly designed for
laboratory or portable field use. Those
designed for field monitoring purposes may
include similar designs or alternate designs
generally employing larger anode, cathode,
and electrolyte capacity to approach better
response stability with some sacrifice in
response time and sensitivity. The electronic
controls, recording, telemetering, and
accessory apparatus generally are semi-
permanent installations of a complex nature.
ACKNOWLEDGMENTS:
This outline contains certain materials from
previous outlines by D. G. Ballinger,
N. C. Malof, and J.W. Mandia. Additional
information was provided by C.R. Hirth,
C.N. Shadiz, D.F. Krawczyk, J. Woods,
and others.
H- 9
-------
Dissolved Oxygen Determination
WESTON & STACK
DO PROBE
CD-
CD—
©—
©—
¦r i jcjV
i jX hP
f
1^0
a.i
CORD
CORD RESTRAINER
SERVICE CAP
PROBF SERVICE CAP
ELECTROLYTE FILL SCREW \
PROBE BODY \
PLATINUM CATHODE \
CONNECTOR PINS
> 0<
PIN HOUSING
) o <
) o <
LEAD ANODE
> O c
REMOVABLE PROBE SHIELD
AND THERMISTOR HOUSING
Figure 8
11-10
-------
Model A15A ELECTRODE COMPONENT PARTS
Cable Sealing
Nut
A 15017
Cable
Connection
Cover
A15016A
'O' Ring
R524
'O' Ring
R389
Lead Anode
Complete
(A15024A)
'O' Ring
R3B5
Membrane— Securing
'O' Ring
R317
Anode
Contact
Holder
A15015A
Anode
Contact
A 150140
(With Sleeve S24)
'O' Ring
R622
Membrane Securing
'O' Ring
R317
Silver Cathode
A15013A
'O' Ring
R612
Note: Red wire of cable connects to Anode Contact Holder
Black wire of cable connects to Anode Contact
Membrane not shown E. I. L. part number T22.
Filler Screw
Z471
III!
1 mil iii —
» 1 1 1 llll III II 1 11 1
O R ing
R622
End Cap
A15011A
Figure 9
-------
Dissolved Oxygen Determination
TABLE 1
CHARACTERISTICS OF VARIOUS LABORATORY DO INSTRUMENTS
DO Temp.
Sig, Comp. Accessories for
Anode Cathode Elec Type Membr Adj. Temp, Rdg, which designed
Carrit &
silver -
Pt
KC1
pol*
polyeth
no
no
Recording temp
Kanwisher
silver ox.
ring
disc
KOH
N/2
& signal adj. se]
assembled
Beckman
Aq
ring
Au
disc
KC1
gel
pol
teflon
yes
yes
yes
recording
Yellow Springs
Ag
Au
KC1
pol
teflon
yes
no*
field and bottle
51
coil
ring
soln
sat.
yes
probe
Yellow Springs
54
n
H
n
It
ii
yes
yes
yes
recording field
bottle & agitatoi
probes
Precision
Pb
silver
KOH
galv"'t*polyeth
no
no
Sci
ring
disc
4N
yes
Weston &
Stack
300
Pb
coil
Pt
disc
KI
40%
galv
teflon
yes
yes
yes
agit. probe
depth sampler
EIL
Pb
Ag
KHCOg
galv
teflon
yes
^es
yes
recording
Delta
Lead
Silver
KOH
galv
teflon
yes
yes
field bottle &
75
disc
IN
no
agitator probe
Delta
Lead
. Silver
KOH
galv
teflon
yes
yes
field bottle &
85
disc
IN
yes
agitator probe
*Pol - Polarographic (or amperometric)
*-:
-------
Dissolved Oxygen Determination
9 Eden, R. E, BOD Determination Using
a Dissolved Oxygen Meter, Water
Pollution Control, pp. 537-539. 19S7.
10, Skoog, D, A. and West, D. M, Fundamentals
of Analytical Chemistry, Holt,
Rinehart & Winston, Inc. 1S66.
11 Methods for Chemical Analysis of
Water & Wastes, U.S. Environmental
Protection Agency, Environmental
Monitoring & Support Laboratory,
Cincinnati, Ohio 45268, 1974
This outline was prepared by F. J. Ludzack, former
Chemist, National Training Center, MOTI), OWPO,
USEFA, Cincinnati, OH 45268 and Nate
Malof, Chemist, USEPA, OWPO, National
Field Investigations Center, Cincinnati, OH
Descriptors ; Chemical Analysis, Dissolved
Oxygen, Dissolved Oxygen Analyzers,
Instrumentation, On-Site Tests, Water Analysis,
Analysis, Wastewater, Oxygen
11-13
-------
BIOCHEMICAL OXYGEN DEMAND TEST PROCEDURES
I OXYGEN DEMAND OF POLLUTED
WATERS
Established practice includes common use
of the BOD test as a tool for estimation of
the bio-oxidizable fraction of surface waters
or wastewaters discharged to them. Any
index including a quantity per unit time such
as the BODj. is a rate expression. The
ultimate demand is more important than any
one point on the progression. The results
of a bottle test with minimum seeding and
quiescent storage are not likely to be as
high as those on the same influent in a mix-
ing situation and abundant seed of secondary
treatment or receiving waters. The BOD
is "a" fraction of total oxygen requirements.
A The particular technique used for BOD
commonly is specified by State agencies
and/or supervisors. They are required
to interpret the results as obtained by
laboratory testing. It is essential that
the tester and the interpreters have a
common understanding of what was done
and how. It is highly advisable to main-
tain a given routine until all concerned
agree upon a change.
1 Each particular routine has many un-
definable factors. The particular
routine is not as important as the con-
sistency and capability with which the
result was obtained.
2 This outline and Standard Methods^ ^
discusses several valid approaches for
obtaining BOD results. Selection of
"method" is not intended in this outline
or in the EPA Methods Manual^.
B The common 5-day incubation period for
BOD testing is a result of tradition and
cost. Initial lags are likely to be over
and some unknown fraction of the total
oxidizable mass has been satisfied after
5 days.
C A series of observations over a period of
time makes it possible to estimate the
total oxidizable mass and the fraction
oxidized or remaining to be oxidized at
any given time. The problem is to define
the shape of the deoxygenation pattern and
its limits. A fair estimate of the shape of
the deoxygenation pattern is available by
observations at 1, 2, or 3 days, 7 days
and 14 days. Increased observations are
desirable for more valid estimates of
curve shape, rate of oxidation and total
oxidizable mass or ultimate BOD.
D Increasing impoundment of surface waters
and concurrent increases in complexity
and stability of wastewater components
emphasize the importance of long-term
observation of BOD. The 5-day observation
includes most of the readily oxidizable
materials but a very small fraction of the
stable components that are the main factors
in impoundment behavior.
II DIRECT METHOD
A With relatively clean surface waters, the
BOD may be determined by incubation of
the undiluted sample for the prescribed
time interval. This method is applicable
only to those waters whose BOD is less
than 8 mg/1 and assumes the sample
contains suitable organisms and accessory
nutrients for optimum biological
stabilization.
B Treated effluents, polluted surface waters,
household and industrial wastewaters
commonly require dilution to provide the
excess oxygen required for the oxygen
demand determination. General guidelines
for dilution requirements for a given BOD
range in terms of the percent of sample in
BOD dilution water are:
For a 5-day BOD of
5-20 mg/1, use 25 to 100% sample
For a BOD of
20-100 mg/1, use 5 to 25% sample
For a BOD of
100-500 mg/ 1, use 1 to 5% sample
For a BOD of
500-5000 mg/ 1, use 0. 1 to 1. 0% sample
IE PROCEDURES
A Cylinder Dilution Technique
CH, O.bod. 57g. 11. 77
12-1
-------
Biochemical Oxygen Demand Test Procedures
1 Using an assumed or estimated BOD
value as a guide, calculate the factors
for a range of dilutions to cover the
desired depletions. Those dilutions
ranging from a depletion of 2 mg/1
and a residual of 1 mg/1 are most
reliable. At least three dilutions in
duplicate should be used for an
unknown sample.
2 Into a one-liter graduate cylinder (or
larger container if necessary) measure
accurately the required amount of mixed
sample to give one liter of diluted waste.
Fill to the on? liter mark with dilution
water. Carefully mix. The initial DO by
calculation includes IDOD (VIII) a
determined initial does not. Both are
essential to estimate significance of
IDOD. Entrapment of air bubbles during
manipulation must be avoided.
3 Siphon the mixture from the cylinder into
three 300 ml glass stoppered bottles,
filling the bottles to overflowing.
4 Determine the DO concentration on one
of the bottles by the appropriate
Winkler modification and record as
"Initial EX)".
5 Incubate the two remaining bottles at
20°C in complete darkness. The
incubated bottles should be water-sealed
by immersion in a tray or by using a
special water-seal bottle.
6 After 5 days of incubation, or other
desired interval, determine the DO on
the bottles. Average the DO concentration
of the duplicates and report as "Final DO".
B Direct Dilution Technique
1 It may be more convenient to make the
dilution directly in sample bottles of
known capacity. A measured volume of
sample may be added (as indicated in
A-1) above, and the bottle filled with dilution
water to make the desired sample
concentration for incubation. In this
case, the sample must be precisely
measured, the bottle carefully filled,
but not overfilled, and the bottle volumes
comparable and known. Precision is
likely to be poorer than for cylinder
dilution.
2 Continue the procedure as in A-4, 5,
and 6 above.
C Seeded Cylinder Dilution Technique
1 Many wastewaters may be partially or
completely sterile as a result of
chlorination, effects of other toxic
chemicals, heat, unfavorable pH or
other factors detrimental to biological
activity. Validity of the BOD result
depends upon the presence of organisms
capable of prompt and effective bio-
degradation and favorable conditions
during the particular test. Correction
of the cause resulting in sterilization
must be corrected by adjustment,
dilution, etc. , prior to reinoculation to
achieve meaningful BOD data. Receiving
water, biologically treated effluents, and
soil suspensions are a good source of
organisms likely to be adapted for
stabilization of wastewaters. Untreated
wastewaters provide numerous organisms
but are likely to contain nutrients
contributing to excessive seed corrections
and may require appreciable time for
adaptation before test waste oxidation
becomes significant.
2 The amount of added inoculant must be
determined by trial. The concentration
added should initiate biochemical
activity promptly but should not exert
enough oxygen demand to unduly reduce
the oxygen available for sample
requirements.
3 Estimate the sample concentration
desired in accordance with A-l and
C-2 above and add the sample aliquot to the
dilution cylinder,
4 Add approximately half of the required
amount of dilution water to the sample
and mix. This is necessary to assure
that the concentrated waste does not
exert a toxic effect on the seed organisms.
5 Measure a suitable aliquot of seed into
the bottle or cylinder and fill with
dilution water. Mix the combined sample,
seed and dilution water without excessive
air entrainment.
6 Continue as in III-A steps 4, 5, and 6 above.
IV INTERPRETATION OF RESULTS
Standard Methods^ includes a calculation
section that is valid and concise. Preceding
it are details of reagent preparation and
12-2
-------
Biochemical Oxygen Demand Test Procedures
procedures for the test. These will not be
reprinted here. This section considers
certain items that may cause concern about
the validity of results unless they are care-
fully considered and controlled,
A The initial DO of the BOD test obviously
should be high. The method of attaining
a high DO can trap the analyst.
1 Aeration of dilution water is the most
commonly considered treatment.
This technique does produce a high DO
but it is a treacherous ally.
a Dirty air passing through clean
dilution water can produce clean
air and dirty water. This is a
simple air-washing operation.
Filtering the entering air stream
may remove brickbats and 2 x 4's,
but filters tend to pass organic
gases, fine aerosols, and partic-
ulates.
b A stream of air passing through
water tends to cool the water by
o
evaporation 1 to 3 C below ambient
temperature. The cooled liquid
picks up more DO than it can hold
at ambient temperature. The
physical loss of oxygen may produce
an erroneously high depletion value
for a determined initial DO, or a
low depletion on a calculated
initial DO. Erroneous blanks are
a particular concern. The dilution
water temperature/DO shift is
critical.
2 Raising DO by allowing the sample to
equilibrate in a cotton-plugged bottle
for 2 or 3 days permits oxygenation
with minimum air volume contact.
3 Shaking a partially filled bottle for a
few seconds also oxygenates with min-
imum opportunity of gas washing con-
tamination, super.saturation, or
temperature changes.
B Seeding always is a precarious procedure
but a very necessary one at times. Often
the application of seed corrections is a
" if you do, if you don't"
situation. Hopefully, seed corrections
are small because each individual
biological situation is a "universe" of
its own.
1 Unstable seeding materials such as
fresh wastewater have "seed" organisms
characteristic of their origin and
history. Saprophytes resulting in
surface water stabilization may be a
small fraction of the population. Re-
actable oxygen-demanding components
produce excessive demands upon test
oxygen resources,
2 A seed containing viable organisms at
a lower energy state because of limited
nutritional availability theoretically is
the best available seed source. An
organism population grown under
similar conditions should be most
effective for initiating biochemical
activity as soon as the nutrient situation
favors more activity. The population
should not be stored too long because
organism redistribution and die-out
become limiting. This type of seed
would most likely be found in a surface
water or a treatment plant effluent
with a history of receiving the particular
material under consideration.
3 Seed sources and amounts can only be
evaluated by trial. Different seed
sources and locations require checkout
to determine the best available material
from a standpoint of rapid initiation of
activity, low correction, and predictable
high oxygen depletion tinder test.
C Chlorination and BOD results fundamentally
are incompatible. Chlorination objectives
include disinfection as the number one
goal. Chlorine is notoriously non-specific
in organism effects. Chlorine acts like
an oxidant in the DO determination. Test
organisms are less suitable for activity
than they were before chlorination.
Nutrients may be less available after
chlorination. Certainly the conditions are
less suitable for biological response after
chlorination. Dechlorination is feasible
with respect to the oxidizing power of
12-3
-------
Biochemical Oxygen Demand Test P rose dure s
chlorine, but many organic chlorine com-
pounds that do not show strong oxidizing
action still have toxic effects on biologic
response.
Numbers are obtainable after dechlorination
and reseeding. The meaning of these
numbers is obscure. At least two states
(New York and New Jersey) specify BOD's
before chlorination only.
V PRECISION OF THE BOD TEST
A The DO test precision often has been used
to suggest precision of the BOD result,
DO precision is a relatively minor and
controllable factor contributing to BOD
results. Other factors such as organism
suitability, members, adaptation and
conditional variables are much more
difficult to control or to evaluate.
B The Analytical Reference Service report
on Water Oxygen Demand, July, 1960
(Sample type VII) included the results of
seeded samples of glucose-glutamic acid
BOD results from 34 agencies on 2, 3, 5
and 7 day incubations.
The relative geometric standard deviation
(average) was 19% on 2% sample and 24%
on 1% sample concentrations. Rate
coefficients ranged from 0. 10 to 0. 27 with
a median of 0, 16 from 21 different
laboratories that participated in rate studies.
VI ALTERNATE BOD TECHNIQUES
Reaeration methods are becoming increasingly
popular in order to approach more nearly the
actual waste concentration in the receiving
water. It is common to obtain "sliding" BOD
results related to the concentration of waste
in a series of dilutions of the same sample.
This may result from greater possibilities
for toxic effects at higher concentrations, or
to a different selection of organisms and change
in oxidation characteristics at low concentrations
of sample. The most reliable estimate of stream
behavior is likely to be from that dilution closest
to the wastewater dilution in the receiving water.
A Reaeration can be accomplished by the
usual series techniques by dumping all of
the remaining sealed bottles into a common
container when the residual DO reaches about
1. 0 mg/l. After reoxygenation, the remaining
bottles are refilled and a new initial DO
determined. Subsequent dissolved oxygen
depletions are added incrementally as a
summation of the total oxygen depletion
from the start of the test. If necessary,
the reaeration technique may be performed
several times but at a sacrifice of double
DO determinations for each day on which
reaeration occurs.
B Special methods of reaeration have evolved
to minimize the extra manipulation for
reaeration of individual sample dilutions.
1 Elmore Method
A relatively large volume of the sample
is stored in an unsealed bottle. Small
bottles are withdrawn in sets of 5 or
more, sealed, incubated, and the DO
determined at appropriate intervals.
When the DO concentration in the smaller
bottles reaches 1. 0 mg/1, a new set is
withdrawn from the large unsealed
bottle, after reoxygenation if necessary.
2 Orford Method
The deoxygenation is carried out in a
large sealed jug from which samples
for DO are withdrawn at appropriate
intervals. To maintain the waste level
and a sufficient DO in the jug, additional
waste is added from a second open
container. See diagram.
C Excess oxygen may be provided by
oxygenation with commercial oxygen
instead of with air to increase the initial
oxygen content for incubation while limiting
the number of dilutions or reaeration steps.
When oxygen is used in place of air the
oxygen saturation in water at 20°C is about
40 mg/l instead of 9 mg/l. Limited results
are available hence the analyst must verify
his technique. The DO tends to decrease
as soon as the bottle is opened hence, about
35 mg/l of oxygen content is the top of the
practical working concentration. There has
been no evidence that the biota is inhibited
by the higher oxygen content with respect
to BOD progression.
D Reaeration or Oxygenation Advantages and
Limitations,
1 Reaeration expands the range of BOD
results obtainable directly at field
concentrations, but is not advisable for
applications when the sample BOD
exceeds 50 mg/l.
12-4
-------
Biochemical Oxygen Demand Test Procedures
Dilution water problems are eliminated,
to the extent that the stream sample may
be tested without dilution.
Incubator storage space becomes a real
problem for multiple sample routine.
VII Dissolved oxygen electrodes, polarographic
and others, are feasible for use in BOD
determinations, often making it possible to
make an estimate of DO or BOD when sample
interference prevents a valid Winkler DO
determination.
Electronic probe DO makes it possible to
determine many successive DO1 s at different
time intervals on the same bottle with
negligible sample loss. Reaeration or
extended time series, therefore, are more
feasible.
Another outline in this series describes
response of reaerated BOD with electronic
DO probes.
VIII
It is the responsibility of the analyst to
evaluate:
1 Applicability of the specified technique
and sample.
2 To determine requirements for mixing
and possible thermal effects while
mixing in terms of instrument response
and biochemical reaction.
3 To evaluate long-term calibration or
standardization and their effects upon
precision and accuracy of the BOD
result.
IMMEDIATE DISSOLVED OXYGEN
DEMAND (IDOD)
Immediate dissolved oxygen demand includes
dissolved oxygen utilization requirements of
substances such as ferrous iron, sulfite
and sulfide which are susceptible to high
rate chemical oxidation.
REAERATION METHODS FOR B.O.D. DETERMINATION
reservoir
A A A A A
sealed bottles
ELMORE METHOD
reservoir
sealed jug
a a n a a a
d.o. samples
OR FOR0 METHOD
12-5
-------
Biochemical Oxygen Demand Test Procedures
A The IDOD is an apparent response as
indicated by a specified technique. Since
DO titration is based upon iodine titration,
any factor that causes I2 response different
from that produced by the reaction of KI
and molecular oxygen confuses the IDOD
determination.
B IDOD Determination
1 The IDOD determination includes the
determination of DO on a sample and
dilution water separately, A waste
likely to have a significant IDOD is
unlikely to show a DO.
2 According to mixing theory, it should
be possible to calculate the DO of any
definite mixture of the sample and
dilution water from the DO of component
parts and their proportion.
3 The same relative proportions of sample
and dilution water should be mixed
without air entrainment and the DO
determined after the arbitrarily selected
time olio minutes.
4 Any difference between the calculated
initial DO as obtained in 2 above, and
the DO determined in 3 above, may be
designated as IDOD,
5 Sample aeration, DO interference, and
other factors affect results for IDOD.
C Sample Calculation of IDOD
1 Sample DO checked and shown to
be 0. 0 mg/1
Dilution water DO found to be 8. 2 mg/1
Assume a mixture of 9 parts of dilution
water and 1 part (V/V) of sample.
Calculated DO =
1X0=0
9 X 8. 2 = 73. 8
10 parts of the mixture contain 73. 8/10
or 7. 4 mg DO/1. Note that mixing has
reduced the DO concentration because
the original amount is present in a
larger package.
2 The mixture described above was held
for 15 minutes and the DO determined
was 4. 3 mg/1.
IDOD = DO DO, , X _ 1QQ. ...
calc detm 7c sample
used
= 7.4 - 4.3 X 10
= 31 mg IDOD/ 1
REFERENCES
1 Standard Methods, 14th ed, 1975.
2 Methods for Chemical Analysis of
Water & Wastes, U.S. Environmental
Protection Agency, Environmental
Monitoring & Support Laboratory,
Cincinnati, Ohio, 45268, 1974.
This outline was prepared by F. J. Ludzack,
former Chemist, National Training Center,
MOTD. OWPO, USEEA, Cincinnati, Ohio
Descriptors; Biochemical Oxygen Demand,
Chemical Analysis, Dissolved Oxygen,
Water Analysis, Analysis, Wastewater
12-6
-------
BIOCHEMICAL OXYGEN DEMAND TEST
DILUTION TECHNIQUE
GENERAL
A Standard Methods (1) lists three ways
of diluting biochemical oxygen demand
(BOD) samples: in a 1 or 2 liter
graduated cylinder, in a bottle of known
capacity (e. g., the BOD bottle), or in
a volumetric flask for dilutions greater
than 1: 100, followed by final dilution
in the incubation bottle.
B The dissolved oxygen (DO) determina-
tions may be made using the azide
modification of the Winkler procedure,
or a DO meter.
D Calcium Chloride Solution - dissolve
27. 5g anhydrous calcium chloride,
CaCl„» in water and dilute to 1 liter.
E Ferric Chloride Solution - dissolve
0. 25g ferric chloride, FeCl^. in water
and dilute to 1 liter.
F Dilution water - add 1 ml each, of
solutions II B, II C, II D, and II E for
each liter of distilled water (11A). If the
dilution water is to be stored, add the
phosphate buffer (IIB) just before use.
11 REAGENTS
A Distilled water - obtained from a block
tin or all glass still; or use deionized
water. It must contain no more than
0.01 mg of copper/liter. It must be
free of chlorine, chloramines, caustic
alkalinity, organic material and acids.
Aerate the water in one of three ways:
loosely plug the container with cotton
and store at 20"C for about 48 hours;
shake 20 "C water in a partially filled
container; bubble clean compressed
through 20°C water. Use distilled
(but not necessarily aerated) water for
the preparation of all solutions,
B Phosphate Buffer Solution - dissolve
8.5g potassium dihydrogen phosphate,
KH^PO., 21. 75g dipotasium hydrogen
phosphate, K^HPO^, 33. 4g disodium
hydrogen phosphate heptahydrate,
Na HPO ' 7H20, and 1. 7g ammonium
chloride, NH^Cl, in about 500 ml of
water and dilute to 1 liter. The pH of
this solution is 7. 2. Discard it if any
biological growth appears in the bottle.
G Seeded Dilution Water - the standard
seed material is the supernatant
liquid from domestic wastewater which
has been allowed to settle for 24-36 hours
at 20°C. Use an amount which will produce
a seed correction of at least 0. 6 mg/'liter.
Add the seed to the dilution water (II F)
on the day the dilution water is to be used.
H Sodium Sulfite Solution, 0, 025N - dissolve
1. 575g anhydrous sodium sulfite, Na^SO ,
in water and dilute to 1 liter. Prepare this
solution daily; it is unstable.
I Acetic Acid Solution 50% - slowly pour 50 ml
acetic acid, HC^H^O^, into 50 ml of water.
J Potassium Iodide Solution, 10% - dissolve
lOg potassium iodide, KI, in 90 ml water.
K Sodium Hydroxide Solution, IN -dissolve 4g
sodium hydroxide, NaOH, in water and dilute
to 100 ml.
L Sulfuric Acid Solution, IN - slowly pour
2. 8 ml of conc. sulfuric acid, H SO ,
into 98 ml of water.
Caution; heat will be generated.
C Magnesium Sulfate Solution - dissolve
22. 5g magnesium sulfate heptahydrate,
MgSO ' 7H O, in water and dilute to
1 liter.
M Powdered Starch Indicator - Thyodene is
one brand name.
N Bromthymol Blue Indicator - or a pH meter.
CH. O. bod. lab. 3c. 2.79
13-1
-------
Biochemical Oxygen Demand Test Dilution Technique
III INTERFERENCES/PRETREATMENT
A Caustic Alkalinity or Acidity - this
must be neutralized to a pH of about 7
with 1 N sulfuric acid or sodium hydroxide.
Use a pH meter or bromthymol blue as
an external indicator,
B Residual Chlorine Compounds - some
residual chlorine will dissipate if the
sample is allowed to stand for 1 or 2
hours. Higher residuals must be
determined, and then neutralized. To
a known volume of sample between 100
and 1000 ml, add 10 ml of acetic acid
solution, 10 ml of potassium iodide solu-
tion, mix, and titrate to the disappearance
of blue color with 0.025N sodium sulfite
and using powdered starch indicator (or
starch solution). Use a proportionate
amount of the 0. 025N sodium sulfite to
dechlorinate the entire sample. (The
portion of sample used above to
determine the chlorine content of the
sample should be discarded, and is not
to be used for the BOD determination.)
After 10-20 minutes, check a portion of
the dechlorinated sample to make sure
the dechlorination is complete.
C Other Toxic Substances - samples contain-
ing other toxic substances, e. g. metals in
plating wastes, require special study and
treatment.
D Super saturation - if you suspect that the
sample contains more than 9 mg of
oxygen/liter at 20°C, shake it vigorously
in a large bottle or flask, or pass clean
compressed air through the sample.
IV SUGGESTED SAMPLE DILUTIONS
Standard Methods (1) suggests the following
sample dilutions. However, actual dilutions
should be determined on the basis of experience,
or information supplied with the sample.
Type of Waste % Dilution
Strong Trade 0. 1 - 1.0
Raw & Settled Sewage 1-5
Oxidized Effluents 5-25
Polluted River Waters 25 - 100
During the 5-day incubation period, at
least 2 mg of oxygen/liter must be
consumed, and at least 1 mg of oxygen/
liter must remain at the end of the
incubation period.
V PROCEDURE
The steps below represent one of
several ways in which the BOD can be set up.
For example purposes, assume the dilution
water does not have to be seeded.
A Siphon 20 "C high quality distilled
water to the 1000 ml line in a
graduated cylinder. Tilt the cylinder
slightly and allow the water to run
down the sides of the cylinder. If the
siphon was "primed", with other water,
"waste" about 100 ml before filling the
cylinder.
B Add 1 ml of the calcium solution and
mix with a plunger-type mixer.
C Add 1 ml of the magnesium solution and
mix with a plunger-type mixer.
D Add 1 ml of the ferric solution and mix
with a plunger-type mixer.
E Add 1 ml of the buffer solution and mix
with a plunger-type mixer. (If the
dilution water were to be seeded, it
would be done at this point).
F Siphon about 250 ml of the dilution water
into a 1 liter graduated cylinder. If more
than 750 ml of sample are to be used, less
than 250 ml of dilution water would, of
course, be siphoned in initially. Use the
same technique as In A above.
G Measure the amount of well mixed sample
to be used. Use a graduated pipet for
smaller sample volumes. If solids "are
present in the sample, the tip of the
pipet may be cut off below the bottom
graduation line. For larger sample
volumes, use the appropriate size
graduated cylinder.
13-2
-------
Biochemical Oxygen Demand Test Dilution Technique
H Add the sample to the cylinder containing
the 250 ml of water. Allow the sample
to run down the sides of the cylinder.
I Siphon in additional dilution water to the
1000 ml line, and mix with a plunger-
type mixer. If other dilutions of the
same sample, or other smaples, are
being set up, be sure to rinse the
mixer between uses.
J Siphon the dilution water-sample mixture
into two BOD bottles. Hold the end of the
siphon close to the bottom of the bottle,
open the siphon slowly, and keep the tip
of the siphon just above the surface of
the surface of the liquid as the bottle
fills. Allow a small amount of the
mixture to overflow the bottle. If the
siphon was "primed", "waste" about
100 ml before filling the bottles.
K Insert the stoppers into the BOD
bottles with a slight twisting motion.
Do not use so much force that an air
bubble is created.
I. Determine the initial DO (DOi) on one of
the bottles within 15 minutes. Use the
Winkler procedure, azide modification,
or a DO meter.
M Water-seal the second bottle and incubate
in the dark, at 20°C + 1DC, for five days.
N Determine the final DO (DOf) on the
second bottle. Use the same method as
in T, above. (Recall the restrictions noted
at the end of section IV).
VI EXAMPLE CALCULATIONS
DO initial = DO i = 7.5 mg/1
DO final = DO f = 2.5 mg/1
100 ml = sample volume diluted in the
1 liter graduated cylinder =
10% dilution (0. 1 as a
decimal fraction)
mg five-day BOD/1 = DOi - DOf
% sample dilution
expressed as a
decimal
mg five-day BOD/1 = 7. 5 - 2. 5
0. 1
- 50
VII SEED CORRECTION
A If you d^ seed the dilution water, a
correction must be applied to the
calculation in VI above,
B Do this by setting up another five-day
BOD exactly as described above, except,
use seed material instead of sample.
C In this case however, the five-day oxygen
depletion must be 40-70%. (In the case of
the sample it was a depletion of at least
2 mg/1 with at least 1 mg/1 remaining).
Consequently, it may be necessary to set
up several dilutions of the seed in order to
get one with a 40-70% depletion.
D Example Seed Correction Calculation
Two hundred fifty ml of seed material
are diluted to 1000 ml with dilution water.
250 x 100 = 25% seed material
Tooo
DO i = 7.0 mg/1
DO f = 3.0 mg/1
Depletion = 7. 0 mg/1 -3.0 mg/1
= 4. 0 mg/1
% depletion = 4. 0 mg/1 x 100
7. 0 mg/1
= 56
Since the 25% seed dilution gave an oxygen
depletion in the desired 40-70% range (56%),
it can be used to calculate the seed correction,
E Example Seed Correction Calculation
(Continued)
Assume that in preparing the dilution water
(V A through V E), you added 2 ml of seed
material to the graduated cylinder before
adding dilution water to the 1000 ml line.
13-3
-------
Biochemical Oxygen Demand Test Dilution Technique
2 x 100 = 0. 2% seed material in the
1000 dilution water
F Example Seed Correction Calculation
(Continued)
In the example calculation in VI, a 10%
sample dilution was assumed.
If the BOD bottles contained 10% sample,
they therefore contained 90% dilution
water.
3 00 ml (volume of BOD bottles)
0. 90 (% dilution water in the BOD
bottles expressed as a decimal)
270.00 ml (volume of dilution water in the
BOD bottles)
G Example Seed Correction Calculation
(Continued)
270 ml (volume of dilution water in the
BOD bottles)
0. 002 (% seed material in the dilution
water expressed as a decimal)
0. 540 ml (volume of seed material in the
BOD bottles)
H Example Seed Correction Calculation
(Continued)
0. 54 x 100 = 0. 18% seed material in the
3 00 BOD bottles
I You now have all the data you need to
calculate the seed correction.
mg five-day BOD/l =
(DOi - DOf) of sample -
[(DOi - DOf) of seed material x
factor]
% of sample expressed
as a decimal
DOi of sample -7.5 mg/1 (from VI above)
DOf of sample = 2.5 mg/1 (from VI above)
DOi of seed material = 7.0 mg/1 (from VII D
above)
DOf of seed material = 3. 0 mg/1 (from VII D
above)
% of seed in the sample BOD bottles =
0. 18 (from VII H above)
% of seed in the seed BOD bottles = 25
(from VII D above)
% of sample expressed as a decimal
fraction = 0. 1 (from VI above)
factor = % of seed in the sample BOD bottles
% of seed in the seed BOD bottles
= 0. 18
25
= 0.0072
Finally, mg five-day BOD/l = (7. 5 - 2.5) -
[ (7.0 - 3.0) x 0. 0072]
_ 5.0 - [ 4.0 x 0.00721
0. 1
A five-day BOD on unseeded dilution water
must not be greater than 0. 2 mg/1 (and
preferably not more than 0. 1 mg/1.) If it is
greater than 0.2 mg/1, check for contamination
in the distilled water and, or, dirty BOD bottles.
Do not use the value as a correction on the BOD.
REFERENCES
1 Standard Methods for the Examination of
Wastewater, 14th ed, APHA, AWWA,
WPCF, New York, pg 543, 1975,
This outline was preparedby Charles R. Feldmann,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, TJSFPA,
Cincinnati, Ohio 45288
Descriptors: Analytical Techniques,
Biochemical Oxygen Demand, Chemical
Analysis, Laboratory Tests, Water Analysis
5.0-0.03
0. 1
= 49.7
VIII Dilution Water Check
13-4
-------
Biochemical Oxygen Demand Test Dilution Technique
DATA SHEET
Initial DO in mg/1, DOi ~
Final DO in mg/1, DOf =
% sample dilution expressed
as a decimal (e. g., 18% = 0. 18) =
mg 5-day unseeded BOD in rng/liter = ^^ecimail
13-5
-------
SOURCES AND ANALYSIS OF ORGANIC NITROGEN
I INTRODUCTION
A Organic nitrogen refers to the nitrogen in
combination with any organic radical.
For sanitary engineering the main interest
is the nitrogen contained in proteins,
peptides, amines, amino acids, amides
and related compounds of animal or
vegetable origin.
II SOURCES OF ORGANIC NITROGEN
A Natural Origin
1 Dead animal and plant residues
2 Animal wastes - urea, feces
3 Autotrophic organisms - algae, s.bact.
4 Heterotrophic organisms
B Industrial Origin
1 Food processing wastewater - meat,
milk, vegetables
2 Pharmaceutical wastes, antibiologicals
3 Plastics - polyamides, nit riles
4 Chemical intermediates or products
5 Dye industry - azo, nitro
III TOTAL KJELDAHL NITROGEN
PROCEDURE
A Organic nitrogen is determined using the
Total Kjeldahl Nitrogen (TKN) method.
This determination includes both organic
nitrogen and free ammonia. By dis-
tilling the free ammonia off the sample
before the determination, organic nitrogen
can be determined directly.
B Scope
The procedure converts nitrogen compon-
ents of biological origin such as amino
acids, proteins and peptides to ammonia
but it may not convert the nitrogenous
compounds of some industrial wastes
such as amines, nitro compounds,
hydrazones, oximes, semi-carbazones
and some refractory tertiary amines.
C Method Summary^'
The sample is heated in the presence of
a concentrated sulfuric acid-potassium
sulfate-mercuric sulfate'mixture and
evaporated until sulfur trioxide fumes are
obtained and the solution becomes color-
less or pale yellow. The residue is
cooled and diluted, then treated and made
alkaline with a hydroxide-thiosulfate
solution. The ammonia is distilled off
and then determined either by nessler-
ization or by titration.
1 Nessleri zation (colorimetric) is the
method used when the ammonia-
nitrogen concentration is less than 1.0
mg N/liter.
2 For ammonia-nitrogen concentrations
above 1. 0 mg N/liter, the ammonia is
determined by titration with 0. 02 N
H9 SO in the presence of a mixed
indicator.
For a detailed description of the
procedure and reagent preparation,
consult the EPA methods manual.
D Precision and Accuracy
1 Thirty - one analysts in 20 laboratories^
used the Total Kjeldahl Nitrogen
procedure to analyze natural water
samples containing the following increments
of organic nitrogen; 0.20, 0.31, 4, 10
and 4,61 mg N/liter.
a Precision results for a standard
deviation were 0. 197, 0. 247, 1. 056
and 1. 191 mg N/liter, respectively.
b Accuracy as bias was +0. 03, +0. 02,
+0. 04 and -0. 08 mg N/liter, respect-
ively.
CH.N. 8a. 11. 77
14-1
-------
Sources and Analysis of Organic Nitrogen
2 The nature and composition of ex-
traneous materials affect analytical
recovery. High salt concentrations
may raise digestion temperature.
High concentrations of organic sample
components may require excessive acid
during digestion tending to low nitrogen
yield.
3 The digestion temperature is critical.
380 to 390°C usually gives high
analytical recovery on the more re-
fractory nitrogen compounds of natural
origin. Nitrogen losses occur above
420 C.
E Optimum temperature is associated with
a digestion mix containing 1 gram of
potassium sulfate for each ml of sulfuric
acid. However, the digestion mixture
usually contains a fraction of
greater than this.
During heating, the water boils off first,
followed by fuming as the HgSO^ becomes
concentrated. Fuming and oxidation of
the sample components lead to loss of
HgSO^ and a progressive decrease in acid
content during heating. As the acid de-
creases, the temperature of the heated
mixture rises.
Valid nitrogen determinations require a
slight excess of acid to retain NH„ as
NH.HSO . rather than the more volatile
(NH ) Su Concurrently, excess acid
will tend foward incomplete oxidation of
sample components as a result of lowering
digestion temperature.
Sodium nitroprusside, which increases
the intensity of the color, is added to
obtain necessary sensitivity for measure-
ment of low level nitrogen.
V PRESERVATION OF SAMPLES
A Most nitrogen compounds are characterized
by rapid conversion from one form to
another by biological and chemical action.
Hydrolysis, deamination, peptide formation,
and other reactions may appreciably alter
the original form of sample nitrogen
within a short time. Organic nitrogen
tends to hydrolyze to free NH^ during
storage. Therefore, valid results require
prompt analysis.
B Addition of 2ml of concentrated sulfuric
acid or 40 mg HgO^/liter of sample and
storage at 4°C are preservation measures
for samples. Even when so preserved,
samples are unstable and should be
analyzed as soon as possible.
REFERENCE
^ Methods for Chemical Analysis of
Water & Wastes, U.S. Environmental
Protection Agency, Environment
Monitoring & Support Laboratory,
Cincinnati, Ohio, 45268, 1974.
IV AUTOMATED PHENATE METHOD
The EPA manual* ^ also presents an
automated (phenate) method for organic
nitrogen. The sample is automatically
digested with a sulfuric acid solution con-
taining potassium sulfate and mercuric
sulfate as a catalyst, then neutralized with
sodium hydroxide solution and treated with
alkaline phenol reagent and sodium hypoch-
lorite reagent. This treatment forms a
blue color designated as indophenol.
This outline was prepared by Audrey E.
Kroner, Chemist. National Training and
Operational Technology Center, MOTD,
OWPO, US EPA, Cincinnati, Ohio 45268
Descriptors: Chemical Analysis, Nitrogen,
Nutrients, Water Analysis, Water Pollution
Sources
14-2
-------
AMMONIA, NITRITES AND NITRATES
I SOURCES AND SIGNIFICANCE OF
AMMONIA, NITRITES AND NITRATES
IN WATER
The natural occurrence of nitrogen com-
pounds is best demonstrated by the nitrogen
cycle (Figure 1),
A Ammonia
1 Occurrence
Ammonia is a product of the micro-
biological decay of animal and plant
protein, In turn, it can b e used
directly to produce plant protein.
Many fertilizers contain ammonia,
2 Significance
The presence of ammonia nitrogen
in raw surface waters might indicate
domestic pollution. Its presence in
waters used for drinking purposes
may require the addition of large
amounts of chlorine in order to
produce a free chlorine residual.
The chlorine will first react with
ammonia to form chloramines be-
fore it exerts its full bactericidal
effects (free chlorine residual),
B Nitrites
1 Occurrence
Nitrite nitrogen occurs in water
as a n intermediate stage in the
biological decomposition of organic
nitrogen. Nitrite formers (nitro-
somorias) convert ammonia under
aerobic conditions to nitrites. The
bacterial reduction of nitrates can
also produce nitrites under anaero-
bic conditions. Nitrite is used as
a corrosion inhibitor in industrial
process water,
2 Significance
Nitrites are usually not found in
surface water to a great extent.
CH. N. 6g. 8.80
The presence of large quantities
indicates a source of wastewater
pollution.
C Nitrates
1 Occurrence
Nitrate formers convert nitrites
under aerobic conditions to nitrates
(nitrobacter). During electrical
storms, large amounts of nitrogen
(N2) are oxidized to form nitrates.
Finally, nitrates can be found in
fertilizers.
2 Significance
Nitrates in water usually indicate
the final stages of biological sta-
bilization. Nitrate rich effluents
discharging into receiving waters
can, under proper environmental
conditions, cause stress to stream
quality by producing algal blooms.
Drinking water containing exces-
sive amounts of nitrates can cause
infant meth omo u'obinemia.
IT PRESERVATION OF AMMONIA, NITRATE
AND NITRITE SAMPLES (8)
A If the sample is to be analyzed for Ammonia,
Nitrate or Nitrite, cool to 4rC and analyze
within 24 hours.
B For Ammonia and Nitrate, the storage time
can be extended by lowering the sample pH to
less than 2 by the addition of concentrated
sulfuric acid and storing at 4QC. (2 ml of acid
per liter is usually sufficient. Check with
pH paper).
C Mercuric chloride is effective as a preservative,
but its use is discouraged because:
1 The Hg ion interferes with some of the
nitrogen tests.
2 The Hg presents a disposal problem.
D Even when "preserved", conversion from one
nitrogen form to another may occur. Samples
should be analyzed as soon as possible.
15-1
-------
Ammonia* Nitrites and Nitrates
NITRITE
NO,
ATMOSP^$> <2^
v
Ql,
^aV^g,
FECAL
MATTER
ORG.
URINE
UREA
^/NITRATE
no3
ANIMAL
PROTEIN
ORGANIC
PLANT
PROTEIN
RGANIC N
foodstuff
THE NITROGEN CYCLE
Figure 1
15-2
-------
Ammonia, Nitrites and Nitrates
III DETERMINATION OF AMMONIA
A Nesslerization
1 Reaction
Nessler's reagent, a strong
alkaline solution of potassium
mercuric iodide, combines
with NH3 in alkaline solution
to form a yellowish brown
colloidal dispersion.
2K2HgI4 + NH3 + 3 KOH
I
Hg/
^O + 7KI - 2HoO
\nh2
Yellow-Brown
The intensity of the color
follows the Beer- Lambert Law
and exhibits maximum absorp-
tion at 425 nm,
2 Interferences
^ a Nessler's reagent forms
a precipitate with some
ions (e.g., Ca++, Mg++,
Fe+++, and S=). These
ions can be eliminated in a
pretreatment flocculation
step with zinc sulfate and
alkali. Also, EDTA or
Roche lie salt solution pre-
vents precipitation with
Ca++ or Mg++.
b Residual chlorine indicates
ammonia maybe present
in the form of chloramines.
The addition of sodium thio -
sulfate will convert these
chloramines to ammonia.
c Certain o r gani c s may
produce an off color with
Nessler's reagent. If
these compounds are not
steam di^tillable, the interference
may be eliminated in the distillation
method.
d Many organic and inorganic compounds
cause a turbidity interference in this
colorimetric test. Therefore direct
nesslerization is not a recognized
method. The following distillation
procedure is a required, preliminary
treatment.
B Distillation
(6, 7, 8)
Reaction
The sample is distilled in
the presence of a borate
buffer at pH 9. 5
nh4 +•
H+ + Na2 B407
Buffer
NH3 + H+
pH 9.5 maintained
The ammonia in the dis-
tillate is then measured by
either of two techniques.
1)
2)
Nesslerization is used
for samples containing
less than 1 mg/1 of
ammonia nitrogen.
Absorption of NH3 by
boric acid and back ti-
tration with a standard
strong acid is more
suitable for samples
containing more than
1 mg NH3-N/l(6. 7, 8)
nh3 + hbo2 ¦
NH.+ + BOo"
BO,
+ H
+ Methyl Red
HBO,
Methylene Blue
pH 7. 8 - 8. 0
Green
pH 6. 8 - 7.0
Purple
15-3
-------
Ammonia, Nitrites and Nitrates
2 Interferences
a Cynate may hydrolyze, even at
, pH 9. 5.
b Volatile organics may come over in
the distillate, causing an off-color
for Nesslerization. Aliphatic and
aromatic amines cause positive
interference by reacting in the acid
titration. Some of these can be
boiled off at pH 2 to 3 prior to
distillation.
c Residual chlorine must be removed
by pretreatment with sodium
thiosulfate.
d If a mercury salt was used for
preservation, the mercury ion
must be complexed with sodium
thiosulfate (0. 2 g) prior to distil-
lation.
3 Precision and Accuracy^-
Twenty-four analysts in sixteen
laboratories analyzed natural water
samples containing the following
amounts of ammonia nitrogen:
0.21, 0.26, 1.71, and 1,92 mg
NH3-N/liter.
membrane and alters the pH of the
internal solution, which is sensed by
a pH electrode. The constant level of
chloride in the internal solution is
sensed by a chloride selective ion
electrode which acts as the reference
electrode.
2 Interferences
a Volatile amines are a positive
interference.
b Mercury forms a complex with
ammonia so it should not be used
as a preservative.
3 Precision and Accuracy
In a single laboratory (EPA) four surface
water'samples were analyzed containing
the following amounts of ammonia
nitrogen: 1.00, 0.77, 0.19, and 0.13 mg
NH3-N/ liter.
a Precision
The standard deviations were 0. 038,
0.017, 0.007, and 0.003 mg
NHg-N/liter, respectively.
b Accuracy
a Precision
The standard deviation was: 0. 122,
0.070, 0.244, and 0,279 mg
NHg-N/liter, respectively,
b Accuracy
The bias was: -0.01, -0,05,
+ 0. 01, and -0. 04 mg NIIg-N/liter,
respectively.
C Selective Ion Electrode^
1 Principle
A hydrophobic, gas-permeable mem-
brane is used to separate the sample
solution from an ammonium chloride
internal solution. The ammonia in
the sample diffuses through the
15-4
The % recovery on the 0. 19 and 0, 13
concentrations was 96% and 91%
respectively.
D NPDES Ammonia Methodology
Manual distillation is not required if compara-
bility data on representative effluent samples
are on company file to show that this prelimin-
ary distillation is not necessary. However,
manual distillation will be required to resolve
any controversies. '
Nesslerization, titration, and the selective ion
electrode are all recognized methods. The
automated phenolate method is also cited.
IV DETERMINATION OF NITRITE
A Diazotization^' ^
1 Reaction
a Under acid conditions, nitrite ions
rear* with sulfanilic acid to form
a diazo compound.
-------
Ammonia, Nitrites and Nitrates
b The diazo compound then couples
with a-naphthylamine to form an
intense red azo dye which exhibits
maximum absorption at 540 nm.
c Diazotization of Sulfanilamide
B NPDES Nitrite Methodology
The colorimctric diazotization method,
either manual or automated, is the only
one cited in the Federal Register.
NIT
+
N:
N
SO., H
+ NO, +2H-
pH 1.
+ 2H O
a
S°3H
SULFANIUC ACID DIAZONIUM COMPOUND
d Coupling
SO„H
so;h
intense red azo dye
2 Interferences
a There are very few known
interferences at concentrations
•less than 1000 times that of
nitrite.
b High alkalinity (greater than
600 mg/liter) will give low results
due to a shift in pH.
c Strong oxidants or reductants in
the sample also give low results.
3 Precision
On a synthetic sample containing
0. 25 mg nitrite nitrogen/ 1, the ARS
Water Minerals Study (1.961) re-
ported 125 results with a standard
deviation of + 0. 029 mg/1.
V DETERMINATION OF NITRATE
A Brucine Sulfate^6' "• ^
1 Reaction
Brucine sulfate reacts with nitrate
in a i:-iN sulfuric acid solution to
form a yellow complex which ex-
hibits maximum absorption at 410 nm.
Temperature control of the color
reaction is extremely critical.
The reaction does not always follow
Beer's Law. However, a modifica-
tion by Jenkins and Medsker^ has
been developed. Conditions are
controlled in the reaction so that
Beer's Law is followed for concen-
trations from 0. 1 to 2 mg nitrate
N/liter. The ideal range is from
0. 1 to 1 nig NO3-N/liter.
2 Interferences
a Nitrite may react the same as
nitrate but can be eliminated
by the addition of sulfanilic acid
to the brucine reagent.
b Organic nitrogen compounds may
hydrolyze and give positive inter-
ference at low I less than 1
hie
fl)
nitrate nitrogen concentrations.
A correction factor can be deter-
mined by running a duplicate of
the sample with all the reagents
except the brucine-sulfanilic acid.
c Residual chlorine may be eliminated
by the addition of sodium arsenite.
d Strong oxidizing or reducing agents
interfere,
e The effect of salinity is eliminated
by addition of sodium chloride to
the blanks, standards, and samples.
15-5
-------
Ammonia, Nitrites rind NiL ates
3 Precision and Accuracy'®^
a Twenty-seven analysts in 15 lab-
oratories analyzed natural
. water samples containing the
following increments of inorganic
nitrate; 0. 16, 0. 19, 1.08 and
1. 24 mg N/liter.
b Precision results as standard
deviation were 0.092, 0. 083,
0.245, and 0. 214 mg N/liter
respectively.
c Accuracy expressed as bias was
-0.01, + 0.02, + 0. 04 and+ 0.04
mg N/liter, respectively.
B Cadmium Reduction'®'
1 Reaction
A non-turbid sample is passed through
a column containing granulated
copper-cadmium to reduce nitrate
to nitrite. The nitrite (that originally
present plus reduced nitrate) is deter-
mined by diazotizing with sulfanilamide
and coupling with N-( 1-naphthyl)-
ethylenediamine dihydrochloride to
form an intensd.y colored azo dye
which is measured spectrophotometrically.
To obtain the value for only nitrate,
more of the non-turbid sample is tested
using the same colorimetric reaction
but without passing it through the re-
duction column. The resulting value
represents the nitrite originally present
in the sample. Subtracting this nitrite
value for the non-reduced sample from
the nitrate + nitrite value for the re-
duced sample gives the value for nitrate
originally present in the sample.
2 Interferences
a Build-up of suspended matter in
the reduction column will restrict
sample flow. Filtration or floc-
culation with zinc sulfate should
remove turbidity.
b High concentrations of iron,
copper or other metals may
interfere. EDTA is used to
complex these.
c Lui-ge concentrations of oil and
grease in a sample can coat the
surface of the cadmium. Pre-
extracting the sample with an
organic solvent removes oil and
grease.
3 Precision and accuracy'®'
In 11 laboratories, three samples
were analyzed containing the follow-
ing amounts of nitrate nitrogen:
0. 05, 0. 5, and 5 mg
NO 3-N/liter.
a Precision
The relative standard deviation
was 96.4%, 25.6%, and 9.2%,
respectively.
b Accuracy
The relative error was 47. 3%,
6.4%, and 1.0%, respectively.
4 Automated cadmium reduction
Standard Methods'®1 and the EPA Methods
Manual'®' contain details for the auto-
mated procedure.
C Hydrazine Reduction
A method using hydrazine to reduce
nitrate to nitrite followed by subsequent
measurement of nitrite by diazotization
/1 \
was reported by Fishman, et al.v '
The means to determine nitrate is the
same as above in the Cadmium Reduction
Method. Subtraction of nitrite (deter-
mined from non-reduced sample) from
the total nitrite (reduced nitrate +
original nitrite) will give the original
nitrate nitrogen concentration.
Tho procedure was adapted to the Auto
Analyzer by Kamphake, et al.
It is available from Environmental
Monitoring and Support Laboratory,
U.S. EPA, Cincinnati, Ohio, 45268.
15-6
-------
Ammonia, Nitrites and Nitrates
D Compliance Nitrate Methodology
1 NPDES/Certifications
The Federal Register lists the brucine
sulfate, cadmium reduction and auto-
mated cadmium reduction or hydrazine
reduction methods.
2 Drinking Water
The National Interim Primary
Drinking Water Regulations
list one brucine sulfate
method (13th ed. of reference 6)
and one codmium
reduction method (reference
8). Amendments to the NIP DWR,
August,27, 1980, p. 57 344 added
the automated hydrazine
reduction method
(1971 ed. of reference 8). ,
REFERENCES
1 Fishman, Marvin J., Skougstad, Marvin
W., and Scarbio, George, Jr. Diazotiza-
tion Method for Nitrate and Nitrite. JAWWA
. 56:633-638 May, 1960.
2 Jenkins, David and Medsker, Lloyd L.
Brucine Method for Determination of
Nitrate in Ocean, Estuarine and Fresh
Waters. Anal. Chem. 36:610-612.
March, 1964.
3 Kamphake, L. J. , Hannah, S. and Cohen, J.
Automated Analysis for Nitrate by Hydrazine
Reduction. Water Research, 1, 205. 1967.
4 Lishka, R. J,, Lederer, L. A., and
MeFarren, E. F. Water Nutrients No. 1,
Analytical Reference Service 1966.
5 Sawyer, Clair N. Chemistry for Sanitary
Engineers. McGraw-Hill Book Co.,
New York. 1960.
6 Standard Methods for the Examination
of Water and Waste Water. APHA,
A WW A. WPCF. 14th Ed. 1976.
7 Annual Book of Standards, Part 31,
Water, 1975.
8 Methods for Chemical Analysis of
Water & Wastes, U. S. Environmental
Protection Agency, Environment
Monitoring & Support Laboratory,
Cincinnati, Ohio, 45268, 1974.
This outline was prepared by B. A.
Punghorst, former Chemist, and C. R.
Feldmann, Chemist, and updated by A. D.
Kroner, Chemist, National Training ai d
Operational Technology Center,
OWPO, USEPA, Cincinnati, Ohio 45268
Descriptors: Ammonia, Chemical Analysis,
Nitrates, Nitrites, Nitrogen, Nitrogen
Compounds, Nitrogen Cycle, Nutrients,
Water Analysis, Water Pollution Sources
15-7
-------
DETERMINATION OF KJELDAHL NITROGEN
(MICRO APPARATUS-NESSLERIZATION)
I REAGENTS
A Distilled Water
This should be ammonia-free. Pass
distilled water through an ion exchange
column with strongly acidic cation resin
mixed with a strongly basic anion resin.
B Sulfuric Acid (20%)
20 ml acid/100 ml distilled water
C Mercuric Sulfate Solution
Dissolve 8g mercuric oxide in 50 ml of
20% sulfuric acid. Dilute to 100 ml with
distilled water.
D Digestion Reagent
Dissolve 134g potassium sulfate in about
650 ml distilled water. Add 200 ml
concentrated sulfuric acid. Add 25 ml of
mercuric sulfate solution (C above) and
dilute to 1 liter.
E Sodium Hydroxide - Sodium Thiosulfate
Solution
Dissolve 500g sodium hydroxide and 25g
sodium thiosulfate pentahydrate in distilled
water and dilute to 1 liter.
F Boric Acid Solution, 2%
G Ammonium Chloride Stock Solution
Dissolve 3. 819g NFI^Cl in distilled water
and dilute to 1 liter. 1. 0 ml " 1,0 mg
NH -N.
H Ammonium Chloride Standard Solution
Dilute 10. 0 ml of the stock solution with
distilled water to 1 liter.
1. 0 ml = O.OlmgNHg-N
I Sodium Hydroxide Solution
Dissolve 160 g sodium hydroxide in 500 ml
distilled water.
J Nessler Reagent
Dissolve 100g mercuric iodide and 70g
potassium iodide in a small volume of
water. Add this mixture slowly to the
sodium hydroxide solution (J above), then
dilute to 1 liter.
II EQUIPMENT PREPARATION
A This procedure should be used if the
apparatus has been out of service for 4
hours or more.
1 Add about 50 ml of a 1 • 1 mixture of
ammonia-free distilled water and
sodium hydroxide-sodium thiosulfate
solution to each of the micro Kjeldahl
flasks to be used.
2 Add glass beads to each flask.
3 Attach a flask to the steam distillation
apparatus and distill about half the
mixture.
4 Add 1 ml of the Nessler reagent to the
distillate to check for ammonia.
a If the distillate is colorless, the
equipment is ammonia-free and the
procedure can be repeated with the
next flask to be used.
b If the distillate is yellow, discard it,
distill another half of the mixture and
check this distillate with 1 ml Nessler
reagent. Repeat the process until the
distillate is colorless.
CH.N. lab, 8b. 8.80
16-1
-------
Determination of K jeldahl Nitrogen
HI DIGESTION OF SAMPLE
A Preparation of Digestion Mixture
1 Shake the sample.
2 Measure 50, 0 ml sample into a 100 ml
Kjeldahl flask.
3 Add 10 glass beads.
4 Add 10 ml of the digestion reagent,
B Digestion
1 Place the flask in a properly ventilated
Kjeldahl digestion apparatus.
2 Turn on the heat source.
6 Carefully add 10 ml of the sodium hydroxide-
sodium thiosulfate solution from the
dropping funnel.
B Distillation
1 Turn on the heat source.
2 Distill at a rate of 6-10 ml/minute up
to the 35 ml mark on the Nessler tube.
3 Remove the receiving flask.
4 Put a small beaker under the condenser
tip to receive any additional distillate.
5 Turn off the heat source if there are no
more digestion residues to distill.
3 Evaporate the mixture until sulfur
trioxide (SO^) fumes are given off.
(SOg) fumes are white and TOXIC.
Also, the solution will be pale yellow,)
4 Continue heating for an additional 30
minutes.
5 Turn off the heat source.
6 Cool the residue In the flask.
IV STEAM DISTILLATION
A Preparation of the Digestion Residue
1 Add 30 ml of ammonia-free distilled
water to the digested residue in the
Kjeldahl flask.
2 Connect the flask to the ground glass
joint of the micro steam distillation
apparatus.
3 Measure 35 ml of water in a graduate,
pour it into a 50 ml Nessler tube and
mark the tube at the 35 ml meniscus.
Empty the tube.
4 Add 5 ml of 2% boric acid to the 50 ml
Nessler tube.
5 Position the Nessler tube so that the
tip of the condesnser is below the
level of the boric acid solution in
the tube.
V COLORIMETRY FOR AMMONIA-
NESSLERIZATION
If the ammonia content is found to be greater
than 1 mg/liter, a titration procedure should
be used1 ^ rather than Nesslerization.
A Preparation of Standards and Sample
1 Label five 50. 0 ml Nessler tubes with
the following: 0, 2, 5, 8, and 10.
2 Pipet the following volumes of ammonium
chloride standard solution into the corre-
spondingly labeled tubes; 2. 0 ml, 5. 0 ml,
8.0 ml, and 10.0 ml.
3 Mark "S" on the Nessler tube
containing 35 ml of distillate.
4 Pour ammonia-free distilled water into
the tube labeled "0", bringing the volume
to the 50. 0 ml line.
5 Add ammonia-free distilled water to each
of the remaining tubes, bringing the
volume of each to the 50. 0 ml line.
16-2
-------
Determination of Kjeldahl Nitrogen
6 Add 1. 0 ml of Nessler Reagent to each
of the 9 tubes.
7 Mix the solution in the "0" tube using a
cap or a rubber stopper on the top, then
inverting the tube three times.
8 Remove, rinse, and dry the cap or
stopper.
9 Repeat steps 7 and 8 to mix the contents
of the other 8 tubes.
10 After mixing, let the tubes stand in a
rack for 20 minutes before getting
absorbance readings.
B Spectrophotometry Readings
1 Turn the instrument on.
2 Set the wavelength at 425 nm.
3 After the 20 minute time span, use the
contents of the "0" tube to adjust to zero
absorbance on the spectrophotometer.
4 Using the contents of the tube labeled
0. 5, rinse, then fill an instrument cell.
5 Place the cell in the holder and record
the absorbance value from the instrument.
6 Discard the contents from the cell.
7 Use ammonia-free distilled water to
rinse the cell three times.
8 Repeat steps 4 through 7 to obtain
absorbance values for the rest of the
standards and for the sample.
9 Turn off the instrument.
VI CALIBRATION CURVE
A Constructing the Curve
1 Calculate the concentration of each
standard by multiplying the ml of
working standard used times 0. 01 mg/ml,
which is the concentration of the
standard solution. This was diluted to
50.0 ml in the Nessler tube, so the
result is mg NH3-N/5O.O ml. For
example, if 0. 5 ml of standard was used,
the concentration is
(0. 5)(0. 01) = 0. 005 mg NH3-N/ 50. 0 ml.
2 Plot the absorbance values for the
standards against these calculated
concentrations.
3 Draw the best straight line from zero
through all the points.
B Using the Curve
1 To find the NHg-N concentration in the
sample, locate its absorbance value on
the curve.
2 Find the corresponding mg NH3-N/5O.O ml
by dropping a vertical line to the
concentration axis.
3 Record this result.
VII FINAL CALCULATIONS
A Use this formula to calculate Total Kjeldahl
Nitrogen:
n AX 1000 B
TKN, mg/1 = — 7 X —
ml sample C
Where:
A = mg NH,,-N/50. 0 ml from curve
B = ml total distillate including boric acid
C = ml distillate taken for Nesslerization
ml sample = ml of original sample taken
B An example calculation using the value from
the calibration curve would be:
TKN, mg/1 = AX 1000 B_
ml sample'' C
If:
A = 0.045
B = 35 ml (30 ml distillate + 5 ml boric acid)
C = 35 ml
ml sample =50 ml
1 (?_¦?
-------
Determination of Kjeldahl Nitrogen
Then:
TKN, mg/1
20
.045 X
1
v n
i
This outline was prepared by Audrey D. Kroner,
Chemist, National Training and Operational
Technology Center, MOTD, QWPO, USEPA,
Cincinnati, Ohio 45268.
TKN
= 0.045 X 20 X 1
= 0.045 X 20
= 0. 90
0. 90 mg/1
REFERENCE
1 Methods for Chemical Analysis of
Water & Wastes, U, S. Environmental
Protection Agency, Environment
Monitoring & Support Laboratory,
Cincinnati, Ohio, 45268, 1974.
Descriptors; Ammonia, Analytical Techniques,
Chemical Analysis, Laboratory Tests, Nitrogen
Nitrogen Compounds, Nutrients, Water Analysis
¦a i*"> A
-------
Determination of Kjcldahl Nitrogen
DATA SHEET
ml of ammonium
Chloride standard
solution used - mg of NH^ - N/50 ml Absorbance
0.0 _0.
0.5 _0.
1. o __0.
2.0 _£¦
4.0 _0-
5.0 __0.
8. 0 _0.
10. 0 0.
Absorbance of sample = 0.
A = mg NHg - N/50 ml from curve =
B - ml total distillate including boric acid =
C = ml distillate taken for nesslerization =
ml sample = ml of original sample taken =
• itx Ax 1000 B
TKN. in mg/liter = — r x —
& ml sample C
x 1000 x
16-5
-------
16-6
-------
TOTAL CARBON ANALYSIS
I INTRODUCTION
A History of Carbon Analyses
In the wake of a rapid population growth,
and the increasing heavy use of our
natural waterways, the nation, and indeed
the world, is presented with the acute
problem of increased pollutional loads on
streams, rivers and other receiving
bodies. This has resulted in a growing
awareness of the need to prevent the
pollution of streams, rivers, lakes and
even the oceans. Along with this aware-
ness has developed a desire for a more
rapid and precise method of detecting and
measuring pollution due to organic
materials.
B The Methods
In the past, two general approaches have
been used in evaluating the degree of
organic water pollution.
1 The determination of the amount of
oxygen or other oxidants required to
react with organic impurities,
2 The determination of the amount of
total carbon present in these impurities.
C Oxygen Demand Analyses
The first approach is represented by
conventional laboratory tests for deter-
mining Chemical Oxygen Demand (COD)
and Biochemical Oxygen Demand (BOD).
One of the principal disadvantages of these
tests is that they are limited primarily
to historical significance, that is, they
tell what a treatment plant had been doing,
since they require anywhere from two
hours to five days to complete. Since up
to now no faster method has been
available, traditional BOD and COD
determinations have become accepted
standards of measure in water pollution
control work even though they are
essentially ineffective for process
control purposes.
Until the introduction of the Carbonaceous
Analyzer, all methods taking the second
approach, the total carbon method of
evaluating water quality, also proved
too slow.
II THE ANALYSIS OF CARBON
A Pollution Indicator
Now the carbonaceous analyzer provides
a means to determine the total carbon
content of a dilute water sample in
approximately two minutes. With proper
sample preparation to remove inorganic
carbonates, the instrument determines
the total organic carbon content in the
sample.
B Relationship of Carbon Analysis to BOD
and COD
This ratio varies with the structure
from 27 percent for oxalic acid through
40 percent for glucose to 75 percent for
methane. The ratio of COD to mg carbon
also varies widely from 0. 67 for oxalic
acid through 2.67 for glucose to 5, 33 for
methane. Representative secondary
sewage effluents have given a ratio of
COD to carbon content of between 2.5 and
3. 5 with the general average being 3. 0.
The BOD, COD and carbon contents of
these and some other representative
compounds are summarized in the follow-
ing table.
CH. MET. 24c. 1. 78
17-1
-------
Total Carbon Analysis
Sample
Stearic Acid - C^gH^O^
Glucose - C,H. „Or
6 11 6
Oxalic Acid -
Benzoic Acid
C2H2°4
" C7H6°2
Phenol
C6H6°
5-Day
BOD-mg/mg
. 786
. 73
. 14
1. 38
, 05 to 2. 1 de-
pending upon
concentration
COD-
mg/mg
2, 91
1.07
. 18
1,97
2.36
% Carbon
76
40
27
69
77
Potassium Acid Phthalate
KHC8H4°4
Salicylic Acid - C^II^O^
Secondary Effluent, Clarified
. 95
1. 25
13*
23*
4*
1. 15
1. 60
75*
67*
36*
47
61
21 =
12:
1-
* In units of mg/l
III THE INFRA-RED TYPE C All BON
ANALYZER
A Principle of Operation
Basically the infra-red carbonaceous analyzer
made by Bcckman, consists of three
sections - a sampling and oxidizing system,
a Beckman Model 315 Infrared Analyzer
and a strip-chart recorder.
Infra-red Carbonaceous Analyzer Schematic
temp,
controller
manual or '
slide J
valve •
injection &
of sample *
h
meter
condenser
f
W
Beckman
nfrared
analyzer
¦xygen carrier from cylinder
A micro sample (20-40 ^ 1) of the water to
be analyzed is injected into a catalytic com-
bustion tube which is enclosed by an electric
furnace thermostated at 950°C. The water
is vaporized and the carbonaceous material
is oxidized to carbon dioxide (COg) and steam
in a carrier stream of pure oxygen or COg-
free air. The oxygen flow carries the steam
and COg out of the furnace where the steam is
condensed and the condensate removed. The
COg* Oxygen or air, and remaining water
vapor enter an infrared analyzer sensitized
to provide a measure of COg. The output
of the infrared analyzer is recorded on a
strip chart, after which, the curve produced
can be evaluated by comparing peak height
with a calibration curve based upon standard
solutions. Results are obtained directly in
milligrams of carbon per liter.
Application
Results show that the method is applicable
for most, if not all, water-soluble organic
compounds -- including those that contain
sulfur, nitrogen, and volatiles.
Nonvolatile organic substances can be
differentiated from volatiles, such as
carbon dioxide or light hydrocarbons by
17-2
-------
Total Carbon Analysis
determination of carbon both before and
after the sample solution has been blown
with a carbon-free gas.
C Sample Preparation
The Carbonaceous Analyzer is often
referred to as a total carbon analyzer
because it provides a measure of all the
carbonaceous material in a sample, both
organic and inorganic. However, if a
measure of organic carbon alone is de-
sired, the inorganic carbon content of
the sample can be removed during sample
preparation.
1 Removal of inorganic carbon
The simplest procedure for removing
inorganic carbon from the sample is
one of acidifying and blowing. A few
drops of HC1 per 100 ml of sample
will normally reduce pH to 2 or less,
releasing all the inorganic carbon as
COg. Five minutes of blowing with
a gas free of carbon sweeps out the COg
formed by the inorganic carbon. Only
the organic carbon remains in the
sample and may be analyzed without
the inorganic interference.
2 Volatile carbonaceous material
Volatile carbonaceous material that
may be lost by blowing is accounted
for by using a dual channel carbon
analyzer. Beckman's new analyzer
has the previously detailed high
temperature (950°C) furnace plus a
low temperature (150°C) one. Using
quartz chips wetted with phosphoric
acid, the low temperature channel
senses only the CO2 (freed by the
acid) in the original sample. The
remaining organics and water are
retained in the condenser connected
to this low temperature furnace.
None of the organics are oxidized by
the 150°C furnace.
By injecting a sample into the low
temperature furnace, a peak repre-
senting the inorganic carbon is ob-
tained on the strip chart. Injecting
a nonacidified sample into the high
temperature furnace yields a peak
representing the total carbon. The
difference between the values deter-
mined for the two peaks is the total
organic carbon,
3 Dilute samples
If the sample is dilute {less than 100
mg/liter carbon) and is a true solution
(no suspended particles) no further
preparation is required,
4 Samples containing solids
If the sample contains solids and/or
fibers which are to be included in the
determination, these must be reduced
in size so that they will be able to pass
through the needle which has an opening
of 170 microns (needles having larger
openings may be obtained if necessary),
In most cases, mixing the sample in a
Waring Blender will reduce the particle
size sufficiently for sampling,
rV PROCEDURE FOR ANALYSIS
A Interferences
Water vapor, resulting from vaporization of
the sample, causes a slight interference in
the method. Most of the water is trapped
out by the air cooled condenser positioned
immediately after the combustion furnace.
However, a portion of the water vapor
passes through the system into the infrared
detector and appears on the strip chart as
carbon. The water blank also appears on
the standard calibration curve, and is
therefore removed from the final calcu-
lation, In tests of solutions containing the
following anions: NO3, CI , SO"2, PO4 ,
no interference was encountered with con-
centrations up to one percent.
B Precision and Accuracy
The recovery of carbon from standard
solutions is 98,5 - 100.0 percent. The
minimum detectable concentration using
the prescribed operating instructions is 1
mg/1 carbon. Generally, the data are
reproducible to + 1 mg/1 with a standard
deviation of 0,7 mg/1 at the 100 mg/1 level.
17-3
-------
Total Carbon Analysis
V THE FLAME IONIZATION TYPE
CARBON ANALYZER
An example of a flame ionization
carbonaceous analyzer is the one
produced by Dohrmann.
To determine TOTAL ORGANIC CARBON
a 30 }il acidified water sample is
injected into a sample boat containing a
cobalt oxide oxidizer at room temper-
ature. The boat is then advanced to the
90° C vaporization zone where H^O,
CO„ (from dissolved CO^. carbonates
ancfbicarbonates) and organic carbon
materials which are volatile at .90° C
are swept into the bypass column. Here
volatile organic carbon (VOC) is trapped
on a Porapak Q column at 60° C while
the h O and C'O are swept through the
switching valve and vented to atmosphere.
After sample vaporization, the valve is
automatically switched to the pyrolyze
position and the boat is then advanced to
the pyolysis zone. Residual organic
carbon (ROC) materials left in the boat
react with the Co^O^ at 850° C to
produce CO^* At the same time the by-
pass column is backflushed at 120 °C thus
sweeping the VOC material through the
pyrolysis zone. Both the VOC and the
CO (from the ROC) are swept by helium
Li
into the hydrogen enriched nickel
catalyst reduction zone where all carbon
is converted to methane at 350° C.
The reduction product is swept through
the switching valve, the water detention
column and into the flame ionization
detector which responds linearly to
methane. The detector output is
integrated and displayed in milligrams
per liter (mg/1) or parts per million
(ppm) on the digital meter.
To determine total carbon, simply set
the function switch to TOTAL CARBON,
and cycle an unacidified sample through
the vaporize and pyrolyze steps. The
switching valve remains in the pyrolyze
position, directing ALL carbonaceous
matter to the detector.
VAPORIZATION STEP
VDC TflAPPEO
sSMSSSJ
E3TPASS COLUMN GO
WATER DETENTION
FLAME
IONIZATION
DETECT0H
JOt'C
stmrn
SWITCHING
PYROLYSIS
ZONE
REDUGTI08
ZONE
|15D°CJ
"2
(850 °C
SO3O4
SAMPLE BOAT
HiCT BOOM TEMP.
VOLATILE ORGANIC CARBON
RESIDUAL ORGANIC CARBON
17-4
-------
Total Carbon Analysis
VI THE AUTOCLAVE TYPE OF
CARBON ANALYZER
Oceanography International makes a TOC
apparatus in which the sample is placed
in an ampule that contains phosphoric acid
and potassuum persulfate. The ampule is
flame sealed and autoclaved. The tip of
the ampule is then broken and the CO^
removed by a gas stream that carries the
CO from the sample to an infrared de-
tector. The autoclave digestion approxi-
mates a COD in which the COD's HLSO^
is replaced with HPO^ and K^Cr^O^
is replaced by K Sv>0 . This instrument
does not have difficulty with salts coating
a catalyst, but has a comparatively high
time requirement.
VII LOW ORGANIC CARBON LEVELS
The organic carbon in solution can be
measured down to levels as low as 50 ppb
using an instrument manufactured by
Barnstead. Samples up to 100 ml in
volume are introduced into the instrument.
The sample is introduced into the system
with a syringe or pipet. Inorganic carbon
in the form of dissolved C.Q is first
stripped out of the sample by a stream of
air (acidification is used to bring the pH
to 4.2). The air carries the inorganic
carbon (CO„) to a measuring chamber
containing 18 megohm-cm, zero-organic
water. The COg dissolves in the pure
water and the resultant change in specific
resistance is measured and stored in
memory. See the figure on page 17-6.
Following the determination of inorganic
carbon, the ultra-violet lamp within the
irradiation chamber is energized. All
dissolved organic compunds - both volatile
and non-volatile - are completely oxidized.
The resulting CO^ is carried by the air
stream to the measuring chamber where
it also dissolves in the water. The unit
again measures the resistivity of the water
within the measuring chamber to determine
the total carbon of the sample. The system
now automatically subtracts the stored
inorganic carbon value from the total carbon
* value and displays the difference, which
is the level of organic carbon in the
sample.
After the result has been displayed, the
unit automatically begins a clean-up
cycle in which all COg and other contami-
nants in the system are removed by an
ion-exchange cartridge. Once all water
within the system has returned to an 18
megohm-cm, zero-organic condition, the
system is ready for the next sample.
Dohrmann has a ultra-violet lamp system
for low organic carbon levels that connects
along side of their regular analyzer. The
COa from the ultra-violet lamp component
passes to the regular analyzer and its
flame ionization detector.
Ultra-violet systems will give low results
if the organic carbon is not in solution.
VIII APPLICATIONS
Several of the many research and industrial
applications of the Carbonaceous Analyzer
are listed below;
A Determine the efficiency of various waste-
water renovation processes, both in the
laboratory and in the field.
B Compare a plant's waste outlet with its
water inlet to determine the degree of
contamination contributed.
C Monitoring a waste stream to check for
product loss.
D Follow the rate of utilization of organic
nutrients by micro-organisms,
E To detect organic impurities in inorganic
compounds.
17-5
-------
Total Carbon Analysis
Measuring Chamber
Irradiation Chamber
Ultra—Violet Lamp
Cei
Sample Septum
Eductor \
/ Micro—
^process otj
Aspirating Valve
Pump
LED Display
Valve Ion—exchange Valve
Cartridge
Ultra-violet low organic carbon Barnstead apparatus.
A summary of some TOC laboratory instrument:
Instrument Manufacturer
Beckman
Oceanography International
Astro, sold by Curtin Matheson
Dohrmann-Envirotech
Dohrmann-Envirotech
Barnstead
Mode of Oxidation
950°C furnace and
catalyst of Co oxides
H3P°4-K2S2°8 P1US
autoclave digestion
850 °C combustion
chamber
850° furnace plus
reduction to methane
(Low Organic Carbon Levels)
Ultra-violet lamp,
H3P°4
Detector
Ultra-violet lamp,
H3P°4
Note that there may be other manufacturers of TOC equipment.
Infra-red
Infra-red
Infra-red
Flame ionization
Returned to unit with
flame ionization
Conductivity
17-6
-------
Total Carbon Analysis
IX ADVANTAGES OF CARBON ANLYZER
A Speed
The Carbonaceous Analyzer's most
important advantage is its speed of
analysis. One analysis can be performed
in 2 - 3 minutes for a channel on the
Beckman instrument or double that on the
Dohrman. This speed of analysis brings
about economy of operation. This is
probably more than the number of COD or
BOD tests that can even be started, much
less completed, in the same period of time.
B Total Carbon
Another advantage is that the measure of
carbon is a total one. The oxidizing
system of the analyzer brings about com-
plete oxidation of any form of carbon. No
compound has been found to which the
method is inapplicable.
REFERENCES
1 Van Hall, C. E. , Safranko, John and
Stenger, V. A. Anal. Chem. 35,
315-9. 1963.
2 Van Hall, C. E. , and Stenger, V, A,
Draft of Final Report - Phase I - Con-
tract PH 86-63-94, Analytical Research
Toward Application of the Dow Total
Carbon Determination Apparatus to the
Measurement of "Water Pollution.
3 Van Hall, C. E. , Stenger, V. A,
Beckman Reprint - R6 215. Taken from
Paper Presented at the Symposium on
Water Renovation, Sponsored by the
Division of Water and Waste Chemistry,
ACS in Cincinnati. Jan. 14-16, 1963.
4 Dobbs, 11. A. , R. II. Wise and R. B. Dean,
Anal. Chem. 39, 1255-58. 1967.
5 Various manufacturers' literature.
X CONCLUSIONS
The Carbonaceous Analyzer provides a
rapid and precise measurement of organic
carbon in both liquid and air samples. It
should be found useful for many research
and industrial applications, a few of which
have been mentioned.
This outline was prepared by Robert T.
Williams, Chief, and revised by Charles J.
Moench, Jr., Waste Identification and
Analysis Section, MERL, USE PA, Cincinnati,
Ohio 45268.
Because of its rapidity it may be found more
useful than the more time-consuming BOD
and COD measurements for monitoring
industrial waste streams or waste treatment
processes.
Descriptors: . Biochemical Oxygen Demand,
Carbon, Chemical Analysis, Chemical Oxygen
Demand, Organic Matter, Organic Wastes,
Water Analysis, Instrumentation, Nutrients
17-7
-------
CHEMICAL OXYGEN DEMAND AND COD/BOD RELATIONSHIPS
I DEFINITION
A The Chemical Oxygen Demand (COD) test
is a measure of the oxygen equivalent of
that portion of the organic matter in a
sample that is susceptible to oxidation
under specific conditions of oxidizing
agent, temperature and time.
B A variety of terms have been and are used
for the test described here as COD:
1 Oxygen absorbed (OA) primarily in
British practice.
2 Oxygen consumed (OC) preferred by
some, but unpopular.
3 Chemical oxygen demand (COD) current
preference.
4 Complete oxygen demand (COD)
misnomer.
5 Dichromate oxygen demand (DOC)
earlier distinction of the current pre-
ference for COD by dichromate or a
specified analysis such as Standard
Methods.
6 Others have been and are being used.
Since 1960, terms have been generally
agreed upon within most professional
groups as indicated in I-A and B-3 and
the explanation in B-5,
C The concept of the COD is almost as old
as the BOD. Many oxidants and varia-
tions in procedure have been proposed,
but none have been completely
satisfactory.
1 Ceric sulfate has been investigated,
but in general it is not a strong
oxidant.
2 Potassium permanganate was one of
the earliest oxidants proposed and
until recently appeared in Standard
Methods (9th ed,) as a standard pro-
cedure. It is currently used in
British practice as a 4-hr. test at
room temperature.
a The results obtained with perman-
ganate were dependent upon concen-
tration of reagent, time of oxidation,
temperature, etc., so that results
were not reproducible.
3 Potassium iodate or iodic acid is an
excellent oxidant but methods employing
this reaction are time-consuming and
require a very close control.
4 A number of investigators have used
potassium dichromate under a variety
of conditions. The method proposed
by Moore at SEC is the basis of the
standard procedur e. (2) Statistical
comparisons with other methods are
described, ^
5 Effective determination of elemental
carbon in wastewater was sought by
Buswell as a water quality criteria.
a Van Slyke^ described a carbon
determination based on anhydrous
samples and mixed oxidizing agents
including sulfuric, chromic, iodic
and phosphoric acids to obtain a
yield comparable to the theoretical
on a wide spectrum of components.
b Van Hall, et al., ^ used a heated
combustion tube with infrared
detection to determine carbon quickly
and effectively by wet sample
injection.
6 Current development shows a trend to
instrumental methods automating
CH.Q.oc. lOe. 11.77
18-1
-------
Chemical Oxygen Demand and COD/BOD Relationships
conventional procedures or to seek
elemental or more specific group
determination.
II RELATIONSHIP OF THE COD TEST WITH
OTHER OXIDATION CRITERIA IS
INDICATED IN TABLE 1.
A Table 1
Test
Test
Temp. °C
Reaction
time
Oxidation
system
Variables
BOD
20
days
Biol, prod.
Enz. Oxidn.
Compound, environ-
ment, biota, time,
numbers. Metabolic
acceptability, etc.
COD
145
2 hrs.
50% H2S04
K2Cr2°7
May be cata-
lyzed
Susceptibility of
the test sample to
the specified
oxidation
IDOD
20
15'
Diss. oxyg.
Includes materials
rapidly oxidized by
direct action,
Fe , SH.
Van Slyke
Carbon detn.
400+
1 hr.
H3P04
hio3
h2so4
K2Cr2°7
Anhydrous
Excellent approach
to theoretical oxi-
dation for most
compounds (N-nil)
Carbon by
combustion
+IR
950
minutes
Oxygen atrn.
catalyzed
Comparable to
theoretical for
carbon only.
Chlorine
Demand
20
20 min.
HOC1 soln.
Good NH3 oxidn.
Variable for other
compounds.
B From Table 1 it is apparent that oxidation
is the only common item of this series of
separate tests.
1 Any relationships among COD & BOD
or any other tests are fortuitous be-
cause the tests measure
the oxidizability of a given sample
under specified conditions, which are
different for each test.
2 If the sample is primarily composed
of compounds that are oxidized by
both procedures (BOD and COD) a
relationship may be established.
18-2
-------
Chemical Oxygen Demand and COD/BOD Relationships
a The COD procedure may be sub-
stituted (with proper qualifications)
for BOD or the COD may be used
as an indication of the dilution
required for setting up BOD -
analysis.
b If the sample is characterized by a
predominance of material that can
be chemically, but not biochemi-
cally oxidized, the COD will be
greater than the BOD. Textile
wastes, paper mill wastes, and
other wastes containing high con-
centrations of cellulose have a
high COD, low BOD.
c If the situation in item b is reversed
the BOD will be higher than the
COD. Distillery wastes or refinery
wastes may have a high BOD, low
COD, unless catalyzed by silver
sulfate.
d Any relationship established as in
2a will change in response to
sample history and environment.
The BOD tends to decrease more
rapidly than the COD. Biological
cell, mass or detritus produced by
biological action has a low BOD
but a relatively high COD. The
COD/BOD ratio tends to increase
with time, treatment, or conditions
favoring stabilization,
III ADVANTAGES AND LIMITATIONS OF
THE COD TEST(2) AS RELATED TO BOD
A Advantages
1 Time, manipulation, and equipment
costs are lower for the COD test.
2 COD oxidation conditions are effective
for a wider spectrum of chemical
compounds.
3 COD test conditions can be standardized
more readily to give more precise
results.
4 COD results are available while the
waste is in the plant, not several
days later, hence, plant control is
facilitated.
5 COD results are useful to indicate
downstream damage potential in the
form of sludge deposition.
6 The COD result plus the oxygen equiva-
lent for ammonia and organic nitrogen
is a good estimate of the ultimate BOD
for many municipal wastewaters.
B Limitations
1 Results are not applicable for estimating
BOD except as a result of experimental
evidence by both methods on a given
sample type.
2 Certain compounds are not susceptible
to oxidation under COD conditions or
are too volatile to remain in the oxida-
tion flask long enough to be oxidized.
Ammonia, aromatic hydrocarbons,
saturated hydrocarbons, pyridine, and
toluene are examples of materials with
a low analytical response in the COD
test.
3 Dichromate in hot 50% sulfuric acid
requires close control to maintain
safety during manipulation.
4 Oxidation of chloride to chlorine is not
closely related to BOD but may affect
COD results.
5 It is not advisable to expect precise
COD results on saline water.
IV BACKGROUND OF THE STANDARD
METHODS COD PROCEDURE
A The COD procedure^ ^ considered dichro-
mate oxidation in 33 and 50 percent sul-
furic acid. Results indicated preference
of the 50 percent acid concentration for
oxidation of sample components. This is
the basis for the present standard
procedure.
18-3
-------
Chemical Oxygen Demand and COD/BOD Relationships
B Muers^ ^ suggested addition of silver
sulfate to catalyze oxidation of certain
low molecular weight aliphatic acids and
alcohols. The catalyst also improves
oxidation of most other organic components
to some extent but does not make the COD
test universally applicable for all chemical
pollutants.
C The unmodified COD test result (A) includes
oxidation of chloride to chlorine. Each mg
of chloride will have a COD equivalent of
0. 23 mg. Chlorides must be determined
in the sample and the COD result corrected
accordingly.
1 For example, if a sample shows 300
mg of COD per liter and 200 mg CI"
per liter the corrected COD result will
be 300 -(200 x 0.23)or 300 - 46 = 254
mg COD/1 on a chloride corrected basis.
2 Silver sulfate addition as a catalyst
tends to cause partial precipitation of
silver chloride even in the hot acid-solu-
tion. Chloride corrections are ques-
tionable unless the chloride is oxidized
before addition of silver sulfate, i.e.,
.reflux for 15 minutes for chloride ox-
idation, add Ag SO , and continue the
reflux or use of HgSO^(D).
(rj)
D Dobbs and Williams ' proposed prior
complexation of chlorides with HgS04 to
prevent chloride oxidation during the test.
A ratio of about 10 of Hg+_r to 1 of CI" (wt.
basis) appears essential. The CI" must
be complexed in acid solution before addi-
tion of dichromate and silver sulfate.
1 For unexplained reasons the HgSO^
complexation does not completely
prevent chloride oxidation in the
presence of high chloride concentrations.
2 Factors have been developed to provide
some estimate of error in the result
due to incomplete control of chloride
behavior. These tend to vary with the
sample and technique employed.
E It is not likely that COD results will be
precise for samples containing high
chlorides. Sea water contains 18000 to
21000 mgCl~/l normally. Equivalent
chloride correction for COD exceeds
4000 mg/1. The error in chloride
determination may give negative COD
results upon application of the correction..
Incomplete control of chloride oxidation
with IIgS04 may give equally confusing
results.
HgS04 appears to give precise results
for COD when chlorides do not exceed
about 2000 mg/1. Interference in-
creases with increasing chlorides at
higher levels.
F The 12th edition of Standard Methods re-
duced the amount of sample and reagents
to 40% of amounts utilized in previous
editions. There has been no change in
the relative proportions in the test. This
step was taken to reduce the cost of pro-
viding expensive mercury and silver sul-
fates required. Results are comparable
as long as the proportions are identical.
Smaller aliquots of sample and reagents
require more care during manipulation
to promote precision.
G The EPA Methods for COD
1 For routine level COD (samples having
an organic carbon concentration
greater than 15 mg/liter and a chloride
concentration less than 2000 mg/liter),
the EPA specifies the procedures found
in Standard Methods ^and in ASTM^,
2 For low level COD (samples with less
than 15 mg/liter organic carbon and
chloride concentration less than 2000
mg/liter), EPA provides an analytical
procedure The difference from
the routine procedure primarily in-
volves a greater sample volume and
more dilute solutions of dichromate
and ferrous ammonium sulfate,
3 For saline samples (chloride level
exceeds 2000 mg/liter), EPA provides
an analytical procedure^ ' involving
preparation of a standard curve of COD
versus mg/liter chloride to correct
the calculations. Volumes and concen-
trations for the sample and reagents
are adjusted for this type of determination.
18-4
-------
Chemical Oxygen Demand and COD/BOD Relationships
V PRECISION AMD ACCURACY*9*
Eighty six analysts in 58 laboratories
analyzed a distilled water solution contain-
ing oxidizable organic material equivalent
to 270 mg/l COD, The standard deviation
was i 17. 76 mg/l COD with an accuracy
as percent relative error (bias) of —4. 7%.
For a solution equivalent to 12. 3 mg/l
COD (low level), the standard deviation
was t 4. 15 mg/l with an accuracy as percent
relative error (bias) of 0. 3%. (EPA Method
Research Study 3)
VI
REMARKS PERTINENT TO EFFECTIVE
COD DETERMINATIONS INCLUDE:
Sample size and COD limits for 0.250 N
reagents are approximately as given.
For 0. 025 N reagents multiply COD by
0.1. Use the weak reagent for COD's
in the range of 5-50 mg/l, (low level).
Sample Size
20 ml
10 ml
5 ml
mg COD/1
2000
4000
8000
B Most organic materials oxidize rela-
tively "rapidly under COD test condi-
tions. A significant fraction of
oxidation occurs during the heating upon
addition of acid but the orange color of
dichromate should remain. If the
sample color changes from orange to
green after acid addition the sample was
too large. Discard without reflux and
repeat with a smaller aliquot until the
color after mixing does not go beyond
a brownish hue. The dichromate color
change is less rapid with sample com-
ponents that are slowly oxidized under
COD reaction conditions.
C Chloride concentrations should be known
for all test samples so appropriate
analytical techniques can be used.
D Special precautions advisable for the
regular COD procdure and essential
when using 0. 025 N reagents include:
Keep the apparatus assembled
when not in use.
Plug the condenser breather tube
with glass wool to minimze dust
entrance.
Wipe the upper part of the flask
and lower part of the condenser
with a wet towel before disassembly
to minimize sample contamination.
Steam out the condenser after use
for high concentration samples and
periodically for regular samples.
Use the regular blank reagent mix
and heat, without use of condenser
water, to clean the apparatus of
residual oxidizable components.
Distilled water and sulfuric acid
must be of very high quality to
maintain low blanks on the refluxed
samples for the 0. 025 N oxidant.
VII NPDES METHODOLOGY
Under the National Pollutant Discharge
Elimination System, the accepted method
(Federal Register, vol. 41, no. 232, part
II, Wednesday, Dec. 1, 1976) for doing
the chemical oxygen demand test is given
in Standard Methods (2), p. 550; ASTM (8),
p, 472; & the EPA manual (9), p. 20.
ACKNOWLEDGEMENT:
Certain portions of this outline contain
training material from prior outlines by
R. C. Kroner, R. J, Lishka, and
J, W. Mandia,
REFERENCES:
1 Moore, W, A., Kroner, R. C, and
Ruchhoft, C. C. Anal. Chem.
21:953, 1949.
| f.f
-------
Chemical Oxygen Demand and COD/BOD Relationships
2 Standard Methods, 14th ed, 1975
3 Moore, W. A., Ludzack, F. J. and
Ruchhoft, C. C. Anal. Chem.
23:1297, 1951,
4 Van Slyke, D, D, and Folch, J, J.
Biol. Chem. 136:509, 1940.
5 Van Hall, C. E„ Safranko, J. and
Stcnger, V. A., Anal. Chem.
35:315, 1963.
8 Annual Book of Standards, Part 31,
Water, 1975.
9 Methods for Chemical Analysis of Water
& Wastes, U.S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, Cincinnati, Ohio
45268, 1974
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, MOTD,
OWPO, USE PA, Cincinnati, Ohio 45268
6 Muers, M. M. J. Soc. Chem.
Ind. (London) 55:711, 1936.
7 Dobhs, R. A. and Williams, R. T.,
Anal. Chem. 35:1064, 1963.
Descriptors: Analysis, Biochemical
Oxygen Demand, Chemical Analysis,
Chemical Oxygen Demand, Chlorides,
Oxygen Demand, Wastewater, Water
Analysis
18-6
-------
LABORATORY PROCEDURE FOR ROUTINE
LEVEL CHEMICAL OXYGEN DEMAND
I REAGENTS
A Standard Potassium Bichromate (0. 250 N):
Dissolve 12. 259 g of primary standard grade
KgCr^O , which has been dried at 103° C
for two nours, in distilled water and dilute
to one liter. Ill
B Ferrous Ammonium Sulfate (0. IN):
Dissolve 39 g of Fe(NH .)„(SO.) • 6H„0 in
distilled water.
Carefully add 20 ml of concentrated HgSO^.
Cool and dilute to one liter.
C Ferroin Indicator:
Dissolve 1.485 g 1, 10-phenanthroline
monohydrate and 0. 695 g FeSO * 7IUO in
water and dilute to 100 ml. This indica-
tor may be purchased already prepared.
D Concentrated Sulfuric Acid (36 N):
E Mercuric Sulfate: Analytical Grade
F Silver Sulfate: Analytical Grade
G Concentrated Sulfuric Acid - Silver Sulfate:
Dissolve 22 g of silver sulfate in a 9 lb
bottle of concentrated sulfuric acid. (4-5
hours, with stirring, for dissolution)
(1)
II EQUIPMENT PREPARATION
Before use, the Erlenmeyer flask (500 ml,
24/40 standard taper joint) and reflux condenser
(300 mm jacket Liebig, West or equivalent)
with 24/40 standard taper joint, should be
steamed out to remove trace organic contami-
nants. Add 10 ml of 0. 250 N K^Cr^O^, 50 ml
of distilled water, and several boiling stones
to the flask. Carefully add 20 ml of concen-
trated H2SO . and mix thoroughly. Connect
the flask ana condenser, but do not turn on
the water to the condenser. Boil the mixture
so that steam emerges from the top of the con-
denser for several minutes. Cool the mixture,
carefully discard the acid, and rinse the con-
denser and flask with distilled water. In order
to prevent contamination from air-borne particles,
re-assemble the apparatus. The top of the con- •
denser should be lightly plugged with glass wool
during storage and use.
(2)
STANDARDIZATION OF FERROUS
AMMONIUM SULFATE
A Measure 10.0 ml of the standard potassium
dichromate sol. into a 500 ml, wide mouth
Erlenmeyer flask.
B Add about 90 ml distilled water and mix.
C Add 30 ml of concentrated H_SO, and cool
2 4
the mixture to room temperature.
D Add 2-3 drops of ferroin indicator and titrate
to a reddish-brown end point with the ferrous
ammonium sulfate sol..
E Calculate the normality, N, of the ferrous
ammonium sulfate sol. :
N of Fe(NH4)2 (SO^ -
ml K2Cr20? x N of K^r^
ml Fe(NH ) (SO )
4 * 4 &
PROCEDURE
A Pipet 20 ml of sample (or pipet a smaller
portion of sample and add enough distilled
water to make a 20 ml volume) into a 500
ml 24/40 standard taper Erlenmeyer flask.
B Add 0.4 g mercuric sulfate.
C Add 6-10 boiling chips.
D Slowly add 3 ml of concentrated sulfuric
acid and swirl to dissolve the mercuric
sulfate. (Some solid may remain). Cool
while mixing.
E Add 10. 0 ml of the 0. 250 N potassium
dichromate sol. and mix.
IV
CH. O. oc. lab. 3e. 8. 78
19-1
-------
Laboratory Procedure For Routine Level Chemical Oxygen Demand
F Attach the flask to the condenser and start
the cooling water.
G Slowly add 27 ml of the sulfuric acid-silver
sulfate reagent through the open end of the
condenser. Try to mix the contents while
adding the acid,
H CAUTION: After adding the acid, swirl the
flask to thoroughly mix the contents. Other-
wise;local heating can occur in the bottom of
the flask and the mixture may be blown out
of the condenser.
I Plug the open end of the condenser with glass
wool or cover with a small beaker (depending
on construction) to prevent intrusion of
contaminants.
J Turn on the heat source and reflux the mix-
ture for 2 hours after boiling begins.
K Turn off the heat and allow the solution to
cool. Then turn off cooling water.
L Wash down the inside walls of the condenser
with distilled water.
M With a twisting motion, disconnect the con-
denser from the flask, then rinse the joint
so the rinses go into the flask.
COD = chemical oxygen demand
A = ml Fe (NIl^gfSQ^)^ used for blank
B = ml Fe(NH.)„(SO.)„ used for sample
4 o 4 £
N = N of Fe(NH4)2(S04)2
8 = equivalent weight of oxygen
ACKNOWLEDGEMENT;
Portions of this outline were taken from out-
lines prepared by R. J. Lishka and Charles
R. Feldmann,
REFERENCES
1 Methods for Chemical Analysis of Water
and Wastes, USEPA, MDQARL, Cincinnati,
Ohio 1974.
2 Standard Methods, APHA-AWWA-WPCF,
14th Edition, 1975.
This outline was prepared by Audrey D. Kroner,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268.
N Add distilled water to the flask for a total
volume of about 130 ml,
O If the mixture is still warm, cool to room
temperature.
P Add 2-3 drops of ferroin indicator and mix.
Q Titrate the excess dichromate with standard
ferrous ammonium sulfate sol. to a reddish
brown end point.
Descriptors: Chemical Analysis, Chemical
Oxygen Demand, Organic Compounds,
Oxidation, Oxygen, Oxygen Demand, Oxygen
Requirements, Water Analysis
R A blank consisting of 20 ml of distilled water
and containing all the test reagents is to
be refluxed and titrated in the same manner
as the sample.
V CALCULATION
(A-B) N x 8 x 1000
mg COD/ 1
ml of sample
19-2
-------
Laboratory Procedure For Routine Level Chemical Oxygen Demand
DATA SHEET
ml of K Cr O used to standardize the ferrous ammonium sulfate (FAS)
a Ci 7
= 10.0
N of K2Cr207 = 0.25
ml of FAS used to titrate the K^Cr^O^
N FAS = 10-° x °-25
ml FAS
2.5
ml FAS used for blank, a =
ml FAS used for sample, b
ml sample =
, ... (a - b) x N FAS x 8 x 1000
COD, in mg liter = - - -
ml sample
( - ) x x 8 x 1000
19-3
-------
DETERMINATION OF SURFACTANTS
I NATURE OF SURFACTANTS AND
SYNTHETIC DETERGENTS
A Definitions
1 Detergents
The dictionary states that the verb,
deterge, means to wash off or to
cleanse. Detergency denotes a
cleansing power or quality.
Larson (5) characterizes a good de-
tergent as a material which is water
soluble, permits the water solution
to penetrate capillaries by lowering
interfacial tension (wetting action),
breaks up or separates particles
having agglomerated (dispersing
action), and links the dirt or oil
particles with the water (emulsify-
ing action).
2 Surface active material (Surfactant)
This term is reserved for those
organic compounds which exhibit
detergent properties plus stability
toward hardness. They are able
to alter the surface or interfacial
properties of their solutions to an
unusual extent, even when present
in low concentrations.
3 Synthetic detergents
The,term, synthetic detergent, is
not rigorously defined. In general,
however, it is a material which
contains a surfactant plus one or
more builders,
4 Builders
A builder is a compound which is
used to enhance the cleansing char-
acteristics of a synthetic detergent.
Sodium sulfate, sodium silicate,
sodium chloride, sodium tripoly-
phosphate, and carboxymethyl
cellulose are examples of builders
which might be found in household
or industrial synthetic detergents.
B Chemical Behavior of Surfactants
On the basis of their ionization in water,
surfactants maybe classified as anionic,
cationic and nonionic,
1 Anionic surfactants
These are the most widely used in
the manufacture of synthetic deter-
gents and are therefore the greatest
contributors to pollution. They
are characterized by the fact that
they ionize in water to give an an-
ion of large size and mass and a
cation of small size and mass.
a Soap exhibits detergent prop-
ties and ionizes as described
above, but has become less
popular for detergent use be-
cause of its lack of stability
toward hardness.
CiiH23C02Na - CnH23C02- + Na+
(Soap) (Anion) (Cation)
b The alkyl aryl sulfonates
represent a second type of
anionic surfactant.
R-C6H4-S03Na — R-C6H4-S03" + Na+
(Alkyl aryl sulfonate) (Anion) (Cation)
The CgH4 grouping represents
a benzene ring; the R represents
a chain of carbon atoms. If the
chain is "straight" the alkyl
aryl sulfonate is referred to as
LAS. If the chain is "branched"
the term ABS is applied.
c Alkyl sulfates comprise the
third type of anionic surfactant.
R-OSOsNa — R-OSO3- + Na+
(Alkyl sulfate) (Anion) (Cation)
DH. DS. 18e. 11.77
20-1
-------
Determination of Surfactants
It again represents a chain of
about twelve carbon atoms con-
nected in straight line fashion.
Cationic surfactants
Compared to the anionic surfact-
ants, the production of the cationics
is small. They are noted for their
germicidal properties and are used
as sanitizing agents in connection
with laundering and dishwashing.
They ionize in w at e r to give a
cation of large size and mass and
an anion of small size and mass.
L
CH„
C12H25- N ~ CH2
C6H5
CH,,
CH„
CI
1 C12H25~ N ~ CH2
! CiT
L_ 3
Cation
c6h5 ; + C1
J
was stated to be its ability to link dirt
or oil particles with the water (emulsi-
fying action,) This linking ability can
be considered by using an alkyl aryl
sulfonate anion as an example.
r-c6ii4-so3"
(Alkyl Aryl Sulfonate Anion)
The R-CgH^ portion of the anion is
referred to as being hydrophobic (water
repelling); i.e., it is insoluble in water,
but soluble in grease and oil. The SOg
portion of the anion is hydroph i 1 i c
(water attracting); i.e., it is soluble in
water, but is insoluble in grease and
oil. In Figure 1 below, the rod repre-
sents the hydrophobic portion of the
alkyl aryl sulfonate anion and the ball
represents the hydrophilic part of the
anion. In dilute water solution the an-
ion orient themselves around the dirt
and grease particles as shown in Fig-
ure 2. The dirt and grease particles
thus coated (emulsified) show little
tendency to coagulate and settle.
Anion
Interaction of cationic and anionic
surfactants yields compounds
which have neither germicidal
nor detergent properties.
Nonionic surfactants
r-c6h4- so
These do not ionize in water.
They show little tendency to foam
and are probably constituents of
the so-called "controlled suds"
detergents. Compounds, such as
those illustrated, may have from
5 to 12 "ethoxy'' or ether groups.
C«H17-C6H4-0C!H4,0SB)V0H
(A Nonionic Ether)
C Physical Behavior of Anionic Surfactants
One of the functions of a good detergent
Figure 1.
Dirt and Grease
Figure 2. ACTION OF SURFACTANT
ON DIRT PARTICLES
20-2
-------
Determination of Surfactants
II WASTES FROM THE USE OF
SYNTHETIC DETERGENTS
A Sources of Wastes
1 Domestic wastes
The concentration of synthetic de-
tergents (reported as MBAS) in raw
sewage varies as shown in Table 1.
Table 1. MBAS CONTENT OP RAW SEWAGE <6)
Table 3, MBAS CONTENT OF OHIO RIVER
WATER (1954 - 1959)
City
Oakland/ California
Ponca City, Oklahoma
Cincinnati, Ohio
West New York, New Jersey
MBAS in raw
sewage (mg/1)
4, 4
11. 8
3. 1
. 13,8
2 Laundry wastes
The average characteristics of a
composite waste from a laundro-
mat or small laundry operation,
are presented in Table 2,
Table 2. LAUNDROMAT EFFLUENT (5)
COD
(mg/1)
MBAS
(mg/1)
pH
(mg/1)
Suspended
Solids
(mg/1)
344 - 445
50 - 90
7.0- 8.1
140 - 163
B Effects of Detergent Wastes on Water
Quality
1 Foaming
MBAS levels in raw water at or
below the USPHS Standards of 0, 5
mg/1, do not cause foaming. At
levels of 1 mg/1 or above, foaming
can occur.
Table 3 presents a summary of
MBAS concentrations found in the
Ohio River from 1954 to 1959, and
reported by ORSANCO, ^
Value
MBAS (mg/1)
Median
0. 12
Average
0. 16
Weekly High
0.59
Weekly Low
o
o
s—'
2 Persistence in biological treatment
a Tctrapropylene ABS
Studies have indicated that
branched ABS compounds made
by a 1 ky la t ing benzene with
tetrapropylene (see Figure 3)
are only 40-70%biodegradable
in conventional activated sludge
treatment. Consequently, they
are known as biologically
"hard" compounds.
It has been hypothesized ^)
that the structure of the C j 2^2 5
benzene sulfonate isomer,
which would be most difficult
to degrade, would be:
CH
CH„
CH„
SOgNa
CH„
Linear alkyl sulfonate (LAS)
On the contrary, compounds
made by alkylating* benzene
with n-paraffins (see Figure 4)
have shown up to 98% biode-
gradability in conventional
activated sludge treatment.
These biologically "soft" sur-
factants known as LAS (linear
alkyl sulfonate) compounds,
20-3
-------
Determination of Surfactants
Tetrapropylene (C12)
A lkylation
\ ,
Branched Alkylbenzene
(C^ a Isomers)
Sulfonation and
Neutralization
\l
Branched Alkylbenzene
Sulfonates
Figure 3. HOW ABS IS MADE
have been developed commer-
cially to replace biologically
hard ABS compounds.
c Nonionics
Ethoxylated alkyl phoenol com-
pounds containing less than 5
ethoxy groups have shown 90%
to 95% biogradability. Larger
• numbers of ethoxy groups tend
to increase resistance to bio -
logical treatment,
3 Eutrophication
The phosphate content of detergent
wastes adds to the nutrient content
of raw water. When the other es-
sential nutrients are present,
nuisance algal growth is promoted.
Compounds derived from amino
carboxylic and hydroxycarboxylie
acids are being considered as sub-
stitutes for the phosphate builders
in order to decrease the nutrient
load to streams and lakes.
Ill ANALYTICAL METHODS
A Sample Collection
If a sample cannot be analyzed promptly,
several procedures may be followed in
order to preserve the sample.
1 Freezing will retard biological
activity.
2 The addition of 0. 8 mg concentrated
H2SO4 /1 of sample will also retard
biological activity and thus preserve
the sample.
It should be noted that losses of MRAS1
have been found to occur when samples
are stored in polyethylene containers. (2)
It is believed that these losses are due
- to adsorption of MBAS1 on the sides of
the containers.
n-Paraffins
\
Chlorination
\
Mono - Chloroparaffins
\
A lkylation
\»
Straight-Chain Sec-
Alky lb en zenes
Figure 4.
\
Sulfonation and
Neutralization
Linear Alkylbenzene
Sulfonates
HOW LAS IS MADE
20-4
-------
Determination of Surfactants
N
(h3c)2n
N (CH3)2
cr + k-
/>
SOq
Na+
—7>
METHYLENE BLUE
R-
(H3C)2N
-so,
I ^
0
1
N
+ Na+ CI"
N(CH3)2
Figure 5. METHYLENE BLUE METHOD FOR ANIONIC SURFACTANTS
B Anionics - Methylene Blue Method
1 Principle
Methylene blue reacts with anionic
surfactants (and other chemical species)
to form a blue-colored, slightly ionized
salt which is soluable in chloroform.
The color intensity of this product in
this solvent is measured at a wave-
length of 652 nm in a spectrophotometer.
Range of application is 0.025-100 mg/liter
for LAS.
Surfactants and other chemical species
which react with methylene blue are
classed as methylene blue active
substances (MBAS).
2 This is the method of analysis for
MBAS listed in the current EPA
Methods Manual (10). Reagent
preparation and procedural details
can be found in Standard Methods (11)
and ASTM Book of Standards (12).
3 Interferences
a Glassware used in this method must
be acid cleaned so that it is free of
even a trace of surfactant material.
b Organic sulfates, sulfonates,
carboxylates, phosphates and
phenols will complex methylene
blue, causing high results.
c Inorganic cyanates, chlorides,
nitrates and thiocyanates form
ion pairs with methylene blue,
again causing high results.
20-5
-------
Determination of Surfactants
d Organic compounds, especially
amines, can compete with the
methylene blue for the surfactant,
causing low results, (e.g.
proteins in sewage).
4 Precision and Accuracy
An Analytical Reference Service
study in 1968^"^ obtained the
following data;
a On a sample of filtered river
water, spiked with 2.94 mg
LAS/liter, 110 analysts obtained
a mean of 2.98 mg/liter with a
standard deviation of 0. 272,
b On a sample of tap water spiked
with 0.48 mg LAS/liter, 110
analysts obtained a mean of 0. 49
mg/1 with a standard deviation
of 0.048.
c On a sample of distilled water
spiked with 0.27 mg LAS/liter,
110 analysts obtained a mean of
0. 24 mg/1 with a standard
deviation of 0. 036.
. C Nonionics
1 Methods for nonionics have been based
on formation of a Cobaltha-thiocyanate
complex and subsequent colorLmetric
measurement. BurttschelP3' proposed
a more sensitive method (0. lmg/1) in
which a complex with the heterpoly acid
of tungsten is formed, hydrolyzed, and
colorimetrically measured as WO4
dithiol,
2 Infrared method
The Soap and Detergent Association
has developed a referee method which
measures "true" ABS or LAS, as
opposed to "apparent" ABS or LAS,
with the methylene blue method. This
method consists of extensive clean-up
procedures, followed by IR
identification,
ACKNOWLEDGMENT
This outline contains certain portions of a
previous outline by Betty Ann Punghorst,
former Chemist, National Training Center,
REFERENCES
1 Abbott, D, C. The Determination of
Traces of Anionic Surface-Active
Materials in Water. Analyst 87:
286. 1962.
2 Analytical Reference Service, Water
Surfactant No, 2, Public Health
Service, Robert A, Taft Sanitary
Engineering Center. May 1964,
3 Burttschell, R. H. Determination of
Ethylene Oxide Based Nonionic
Detergents in Sewage, American
Oil Chemists Society 43:366-370.
1966.
4 Hatch, L, F, , Scott, K, A, and
Weaver, P. V, Biodegradable
Detergents: A Special Report,
Hydrocarbon-Processing and
Petroleum Refiner 43:91-104,
March 1964.
5 Larson, T, E, Synthetic Detergents.
J A WW A 41:315. April 1949.
6 OHSA NCO Detergent Subcommittee.
Components of Household Detergents
in Water and Sewage,
JAWWA 55:369-402, March 1963,
7 Nemerow, N. L. Theories and Practices
of Industrial Waste Treatment.
Addison-Wesley Publishing Co., Inc.,
London. 1963,
8 Task Group Report: Characteristics
and Effects of Synthetic Detergents.
JAWWA 46:751-774. August 1964.
9 Task Group Report: Determination of
Synthetic Detergent Content of Raw-
Water Supplies. JAWWA SO: 1343-1352.
October 1958.
10 Methods for Chemical Analysis of Water &
Wastes, U. S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, Cincinnati, Ohio,
45268, 1974.
11 Standard Methods, 14th ed, 19 75
20-6
-------
Determination of Surfactants
REFERENCES (continued)
12 Annual Book of Standards, Part 31,
Water, 1975.
13 Analytical Reference Service, Water
Surfactant No, 3, Study No. 32, Public
Health Service, Robert A. Taft
Sanitary Engineering Center, 196 8.
This outline was prepared by C. R. Feldmann,
Chemist, National Training and Operational
Technology Center, and revised by Audrey
D. Kroner, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45260
Descriptors: Chemical Analysis, Surfactants,
Linear Alkylate, Sulfonates, Water Analysis
20-7
-------
LABORATORY DETERMINATION OF SURFACTANTS
(Methylene Blue Active Substances, MB AS)
I REAGENTS
A Stock Linear Alkylate Sulfonate (LAS)
Weigh an amount of LAS (order from
US EPA, EMSL. Quality Assurance
Branch, Cincinnati. Ohio 45260)
that will give a concentration of 1.00 mg
LAS per 1. 00 ml when dissolved in
distilled water and diluted to 1 liter.
Store the solution in a refrigerator.
Prepare fresh weekly.
B Standard Linear Alkylate Sulfonate (LAS)
Dilute 10. 0 ml of the stock LAS solution
to 1 liter; 1. 00 ml = 10. 0 ^g LAS. Prepare
fresh daily.
C Phenolphthalein Indicator
D Sodium Hydroxide, 1 N
Dissolve 40. 0 g of NaOH in water and dilute
to 1 liter.
E Sulfuric Acid, 1 N
Slowly add 28.0 ml of concentrated II2SO4
to water and dilute to 1 liter.
F Chloroform, CHClg
G Methylene Blue
Dissolve 100 mg of methylene blue in
100 ml of distilled water. Transfer 30 ml
of the solution to a 1 liter volumetric flask.
Add 500 ml distilled water, 6, 8 ml concen-
trated HgSO^, and 50 g NaH^PO^'HgO.
Shake the flask to dissolve the solid.
Dilute to the 1 liter mark.
H Wash Solution
Add 6. 8 ml concentrated H2SO4 to 500 ml
of distilled water in a 1 liter volumetric
flask. Add 50 g Nal^FO^" HgO and shake
until the solid dissolves. Dilute to the
1 liter mark.
II PROCEDURE
A Pipct the following volumes of the standard
LAS into 200 ml separatery funnels (with
Teflon stopcocks): 0.0, 1.0, 3.0, 5.0,
7.0, 9.0, 11.0, 13.0, 15.0 and 20.0. Add
sufficient water (graduated cylinder) to bring
the volume in each flask to 100 ml.
H Add 100 ml of sample (graduated cylinder)
to another separaterv funnel.
C Add 3 drops of phenolphthalein indicator to
the sample and standards, and mix.
D Add 1 N NaOH dropwise to the sample and
standards until a permanent pink color is
present.
E Discharge the pink color with 1 N H^SOj.
F Add 10 ml C.'HCl.j and 25 ml of the methylene
blue reagent (graduated cylinders) to the
separatory funnels containing the sample
and standards,
G Rock the funnels gently for a few seconds
and relieve the pressure in the funnels.
H Rock the funnels vigorously for 30 seconds.
I Allow the layers to separate. The upper
aqueous layer is dark blue in color while
the lower CIICl^ layer is light blue in color.
J Remove the funnel stoppers and drain the
lower CHClg layer into 125 ml Erlenmeyer
flasks.
K Add 10 ml CIICI3 to each of the funnels
(sample and standards).
L Repeat steps G, H, I and J, using the same
125 ml Erlenmeyer flasks as in step J.
M Repeat the extraction process 2 more times
(4 total) with 10 ml portions of CHClg.
Note that in the case of the sample funnel,
if the blue color disappears from the water
layer during any of the 4 extractions, a
smaller sample size must be used.
CH. DS. lab. 2b. 8. 80
21-1
-------
Laboratory Determination of Surfactants
N Discard the water layers left in the sample
and standards funnels.
O Rinse all of the separatory funnels vigor-
ously with tap water, and then rinse with
distilled water.
P Pour the CHCI3 from the Erlenmeyer flasks
back into the separatory funnels.
Q Add 50 ml of wash solution to the sample
and standards funnels.
R Repeat steps G and H.
S Allow the layers to separate.
T Place a small piece of glass wool in a
small filtering funnel.
U Place the filtering funnel in a 100 ml
volumetric flask.
V Drain the lower CHClg layer from the
separatory funnel, through the glass wool
in the filtering funnel, and into the 100 ml
volumetric flask.
W Add 10 ml CHCln to the separatory funnel
containing the wash solution.
X Repeat steps G and H.
Y Allow the layers to separate.
Z Repeat step V.
AA Repeat the extraction with a second 10 ml
portion of CHClg and drain it into the
100 ml volumetric flask.
BB Rinse the filtering funnel and glass wool
with a small amount of CHCI3 . Collect the
rinsings in the 100 ml volumetric flask.
CC Bring the CHClg in the volumetric flask to
the 100 ml mark with CHClg .
DD Using a CHCL blank and 1 cm cell, read the
absorbance or the sample and standards at
652 nm in a Spectronic 20 spectrophotometer.
Ill CALCULATIONS
A Prepare a calibration graph for the standards
of LAS concentration vs. absorbancy.
Example:
1 Concentration of standard LAS =
10. 0^g LAS per 1. 0 ml
^ X ° ml standarcJ ~ 50* 0 Vg LAS
3 The 50. 0 ^g may be used as a "mass"
value on the X axis of the graph, or
4 The 50. 0 is in a 100 ml volumetric
flask.
50. 0 ng _ 500. 0 fj,g _ 0. 5 mg
100 ml 1000 ml liter
The 0. 5 mg per liter may be used as a
"concentration" value on the X axis of
the graph.
REFERENCE
Standard Methods for the Examination of Water
and Wastewater, 13th ed., page 339,
Method 159 A. American Public Health
Association, American Water Works
Association, Water Pollution Control
Federation. Washington, D. C. 1971.
Standard Methods, 14th ed. Method 512A,
pg 600, 1975.
This outline was prepared by C. R. Feldmann,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268.
Descriptors; Chemical Analysis, Surfactants,
Laboratory Tests, Linear Alkylate, Sulfonates,
Water Analysis
21-2
-------
Laboratory Determination of Surfactants
ml of LAS standard
solution used
0. 0
1,0
3.0
5. 0
7.0
9.0
11. 0
13.0
15. 0
20.0
DATA SHEET
nig LAS/1 Absorbance
0.
0.
o.
_0.
__0.
_0.
0.
0.
Absorbance of sample = 0.
Concentration of LAS in sample in mg/litcr from curve =
-------
21-4
-------
OIL AND GREASE
I DEFINITION
A Definition
In the field of wastewater treatment,
the terms oil and grease are not
clearly defined. They arc partially
characterized by the analytical
method used for their determination.
A sample is extracted with an organic
solvent which is then separated from
the water and evaporated. The
residue is termed oil and grease,
regardless of its composition. Low
boiling components, such as lubricating
oil and kerosine, are lost, to some
extent, during the solvent removal step.
The determination of gasoline by the
solvent extraction method is completely
unreliable. Thus, the term oil and
grease is operationally defined.
B National Pollutant Discharge
Elimination System (NPDES)
Under provisions of the 1972 Amend-
ments to the Federal Water Pollution
Control Act (Public Law 92-500), the
NPDES places limitations on the
concentrations of pollutants which may
be discharged to receiving bodies of
water. One such pollutant is termed
oil and grease. Thus, although the
term is not clearly defined in the area
of wastewater treatment, it does have
significance under the NPDES,
C Components
In wastewater, the term grease
includes such classes of compounds as
waxes, fatty acids, fats, and oils.
Classes of compounds referred to as
oils are low to high molecular weight
hydrocarbons, such as gasoline, heavy
fuel oils and lubricating oils, and
animal and vegetable glycerides which
are liquid at ordinary temperatures.
II OCCURRENCE
Materials classified as oils and greases
enter receiving bodies of water and
wastewater treatment plants from
households and industries which either
manufacture or use the groups of
compounds mentioned above. Examples of
such industries are meat processing
plants, petroleum refineries, petro-
chemicals, trucking, laundry, machine
tool and steel.
Ill TREATMENT PROBLEMS
A Oil and grease cause special problems
in the handling of household and certain
industrial wastes, because they have a
low solubility in water and therefore
tend to separate from the water phase,
B They form scum layers in primary
settling tanks, sludge digestion units,
and final clarifiers. They coat
particles, producing floating masses
which arc unsightly and odorous.
When oil and grease coat organic
particles, oxygen transfer and bio-
degredation are inhibited. Such
interference can occur in the activated
sludge process, as well as in trickling
filters. In activated sludge plants, high
oil/grease concentrations can result in
significant carryover of biological solids
during final clarification by entrapment
of biofloc in the floating scum layer.
Oil and grease are resistant to
both aerobic biodegredation and
anaerobic digestion. These materials
cling to equipment surfaces such as
pipelines, pumps, screens, and filters,
thus reducing their operating efficiency.
Also, they are a safety hazard in waste-
water treatment plants, coating walkways
and ladders. Grease particles are often
present in an emulsified form. The
emulsifying coating is sometimes not
broken until the grease enters secondary
treatment units or the receiving stream.
IV WATER SUPPLY PROBLEMS
Even small quantities of oil and grease
can produce an objectional odor and
appearance in public water supplies. If
these materials are found in water
contemplated for use as a public water
CH.OG. 1. 11. 77
22-1
-------
Oil and Grease
supply, the source may be rejected,
even before a health problem is shown to
exist.
V ANALYTICAL METHODOLOGY
A NPDES
The analyses for pollutants performed
under the NPDES (see I. B. above) are
to be performed according to specified
methodology. This methodology is
spelled out in the Federal Register,
Wed., Dec., 1, 1976, vol. 41, no. 232, pt II,
pgs 52780-52786. The Federal Register cites
the Freon extraction on pg 515 of the 14th ed. of
Standard Methods (1), & an almost identical pro-
cedure on pg 229 of the EPA methods manual(2)
as tie procedures to be used when analyzing a
wastewater sample for oil and grease.
Steps in the analysis include; acidification
of the sample with 5 ml of 50% by volume
sulfuric acid per liter of sample,
extraction of the sample with several
portions of Freon (trichlorotrifluoroethane,
boiling point 47°C; Dupont Freon
precision cleaning agent or equivalent),
combining the Freon portions in a tared
distilling flask, distilling off all but about
10 ml of the Freon, boiling off the
remaining Freon, drying the flask, cooling
and weighing. The milligram increase in
weight, multiplied by 1000, and divided by
the milliliters of sample, gives the
milligrams of oil and grease per liter of
sample.
B Other Analytical Procedures, non NPDES
1. Standard Methods (1)
2. U.S. EPA Methods Manual
In addition to the above "approved
methods the U.S. EPA methods
manual (2) carries two other procedures.
In the first, the acidified sample is
filtered through a muslin cloth disc
overlaid with filter paper; filter aid is
also used. The filter paper any any
solids clinging to the muslin are then
extracted in a Soxhlet apparatus with
hexane. The solvent is evaporated and
the increase in flask weight is used to
calculate the mg of oil/grease per liter
of sample. The second is an infrared
method very similar to that given in
14th Standard Methods (1).
3 ASTM (3) lists no parameter specifically
referred to as oil and grease.
C Sample Collection and Storage
The method referred to in V. A. above,
directs that the sample must be
representative. However, since oil and
grease will be found on the surface of a
body of water, the sample will not be
.representative of the body of water as a
whole. The glass stoppered sample bottle
should be washed with solvent and air dried.
It should also be marked on the outside, to
indicate the desired sample volume. None
of the oil and grease should be lost by
clinging to the glass stopper. Therefore,
the bottle should not be filled to the top.
Preservation is accomplished by adding
5 ml of 50% by volume sulfuric acid per
liter of sample. No holding time is
specified, but it is generally good procedure
to begin the analysis as soon as possible.
In addition to the above "approved"
method, 14th Standard Methods (1) also
carries two other procedures applicable
to wastewater. The first is tentative, and
involves an extraction identical to that
described above, followed by infrared 2
detection. The second utilizes Freon
in a Soxhlet extractor. An eighty cycle
extraction is followed by evaporation of
the solvent and weighing of the residue.
REFERENCES
1 Standard Methods, 14th ed, 1975.
Methods for Chemical Analysis of Water &
Wastes, U. S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, Cincinnati, Ohio,
45268, 1974.
Annual Book of Standards, Part 31,
Water, 1975.
22-2
-------
REFERENCES (CONTINUED)
4 Sawyer, Chemistry for Sanitary Engineers,
McGraw-Hill Book Company, Inc., 1968.
5 Water Quality Criteria 197 2, a report of the
Committee on Water Quality Criteria,
Environmental Studies Board, National
Academy of Sciences, National Academy
of Engineering, 1972.
This outline was prepared by C. R. Feldmann,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268
Descriptors: Chemical Analysis, Oil, Oil
Pollution, Oil Wastes, Oily Water, Water
Analysis
-------
LABORATORY DETERMINATION OF OIL AND GREASE
I REAGENTS
A Freon TF (E. I. DuPont de Nemours) or
Genosolv D (Allied Chemical Co.); the
symbol TF/D will be used throughout this
procedure to mean this particular solvent.
B Hydrochloric or Sulfuric Acid
Fifty percent by volume.
C Sodium Sulfate
II PROCEDURE
A Weigh a 125 ml distilling flask on an
analytical balance. Handle the flask
throughout the entire procedure with a
tissue or crucible tongs. Set the flask
aside until it is needed.
B Shake the sample container,
C Measure 1 liter of sample in a graduated
cylinder.
D Measure 5 ml of 50% by volume HC1 or
H2SO4 in a small graduated cylinder,
E Add it to the 1 liter graduated cylinder.
Note: Do not do steps D and E if the sample
was acidified at the time of collection. The
sample pH should be 2, before doing step F.
Check the sample with pH sensitive paper.
F Empty the. 1 liter cylinder into a 1 liter
separatory funnel (Teflon stopcock).
G Stopper the funnel and shake gently so as
to mix the acid and sample.
H Check the pH of the sample with pH
sensitive paper. If it is not 2 or less,
add a few more drops of acid, mix, and
recheck the pH.
I Rinse the 1 liter cylinder with 30 ml of
TF ID (measured in a small graduated
cylinder).
J Add it to the separatory funnel. The TF/D
will form a separate layer beneath the
water.
K Replace the funnel stopper, invert the
funnel, and open the stopcock to relieve
the pressure.
L Close the stopcock and shake the funnel
gently for a few seconds.
M Open the stopcock to relieve the pressure.
N Repeat steps L and M.
O Close the stopcock and shake the funnel
more vigorously for 2 minutes. Thorough
mixing without excessive foaming is the
objective.
P Repeat step M.
Q Place the funnel back in the ring stand and
remove the stopper.
R Allow the water and TF/D layers to separate.
S Drain the lower TF/D layer into the
previously weighed 125 ml distilling
flask. About 1 drop of the TF/D should
remain in the separatory funnel. If the
TF / D layer is not clear, filter it into the
flask through a small funnel containing
filter paper and about 1 g (estimate) of
anhydrous Na2SO,j.
T Measure 30 ml of TF/D in a small graduated
cylinder and use it to rinse the 1 liter graduated
cylinder.
U Add the rinsings to the separatory funnel.
V Repeat the two gentle shakings, pressure
relief, 2 minute vigorous shaking, and layer
separation as above.
W Repeat step S, using the same distilling
flask. The flask now contains about 60 ml
of TF/D.
CH. OG.lab. lb. 8.80
23-1
-------
Laboratory Determination of Oil and Grease
X Repeat steps T, U, V. and W. The flask
now contains about 90 ml of TF/D. If the
small funnel and Na2S04 were used, wash
them with a few ml of TF/D. Collect the
washings in the distilling flask.
Y Evaporate the TF/D at 70"C on a water
bath, (Heat the lower third of the flask
only.)
Z Raise the temperature of the bath to 80°C
for 15 minutes.
A A Apply suction to the warm flask for 1
minute. (See figure below).
BB Wipe the outside of the flask thoroughly
with tissues and cool it in a desiccator for
about '20 minutes.
CC Weigh the flask on the same balance as
before.
1000 = Conversion factor, grams to
mg
1000 = Conversion factor, ml to
liter
REFERENCE
Methods for Chemical Analysis of Water &
Wastes, U. S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, Cincinnati, Ohio
45268, 1974.
III BLANK DETERMINATION
A To a second, weighed, 125 ml dis-
tilling flask, add about the same
volume of TF/D which was used
for the sample. This will be 90 ml,
plus rinsings. The volume used
for rinses will have to be estimated.
Handle the flask in the same
manner as described in II A.
B Repeat steps Y through CC, using
the second flask.
IV CALCULATIONS
nig of oil and grease/liter =
[ A - Bj - [ C - D] x 1000 x 1000/ml of sample
A = weight of sample flask + oil and
grease, in grams
B = weight of empty sample flask,
in grams.
C = weight of "blank'' flask + residue,
in grams.
D = weight of empty "'blank 1 flask,
in grams.
Tliis outline was prepared by C, R. Feldmann,
National Training and Operational Technology
Center, MOTD, OWPO, USEPA, Cincinnati,
Ohio 45260.
Descriptors: Chemical Analysis, Laboratory
Tests, Oil, Oil Pollution, Water Analysis
GLASS
TUBING
RUBBER
STOPPER
DISTILLING
FLASK
125ml
TO
VACUUM
23-2
-------
Laboratory Determination of Oil and Grease
DATA SHEET
sample identification =
ml of sample =
A = grams
B = grams
C = grams
D = grams
23-3
-------
DETERMINATION OF PHENOLICS
I DEFINITION AND SIGNIFICANCE
A Definition
The phenolic compounds in water
chemistry are defined as hydroxy de-
rivatives of benzene and its condensed
nuclei. These occur in domestic and
industrial wastewaters and in drinking
water supplies.
B Phenol and chlorinated derivatives in
water affect fish and water quality,
1 Fish
The threshold limit of toxicity at
infinite time for certain species of
fish is of the order of a few milli-
grams per liter. Some chlorinated
phenols exhibit toxicity in concen-
trations as low as 0.2 mg/1.
2 Fish flesh tainting
C The chlorine-to-phenol ratio at maximum
intensity of taste and odor is 2:1. The
proportion of 2, 6-DCF was greatest at
the 2:1 chlorine-to-phenol ratio,
III PRESERVATION AND STORAGE
OF SAMPLES
A Since phenolics are subject to oxidation,
samples should be analyzed within 4
hours of collection.
B Samples can be preserved and stored
up to 24 hours as follows;
1 Adjustment of pH to less than 4.0 with
H3P°4
2 Aeration, if sulfides are present
3 Addition of 1.0 g CuSO^* SH^O/liter
4 Storage at 4°C
II
A
Fish living in waters of lesser
phenolic concentrations can acquire
an unpleasant and obnoxious taste.
Water quality
The presence of as little as 14
Mg/1 of the chlorinated phenols can
impart a taste to drinking water.
CHLORINE DERIVATIVES OF PHENOL
CAUSING TASTE AND ODOR(1)
All chlorination products may contribute
to the intensity of taste and odor.
At maximum taste and odor intensity,
the major contributor is 2, 6-dichloro-
phenol.
CH. PHEN. 32f,5. 78
IV DETERMINATION OF PHENOLICS
A NPDES Methodology
The 1976 Federal Register Guidelines for
National Pollutant Discharge Elimination
System (NPDES) requirements specify
distillation to separate out interferences,
followed by the 4-amionoantipyrine (4AAP)
colorimetric determination. Phenol is to be
used as a standard.
Comments on the procedure can be found
in the EPA Methods Manual. ^ The step-
wise procedure can be found in Standard
Methods^'3) and ASTM1'4).
B Other Analytical Procedures
For purposes other than NPDES requirements,
Standard Methods(3) lists a 4AAP method for
halogenated phenols which employs 2, 4-di-
chlorophenol as a standard. It also presents
a gas-liquid chromatographic method for
samples containing certain phenolics present
in concentrations greater than 1 mg/liter.
Thin-layer chromatography has also been
utilized for phenol and certain substituted
phenols in raw surface water^).
24-1
-------
Determination of Phenolics
v pretreatment and distillation
OF SAMPLES*3, 4)
Depending on interferences present,
samples must be treated prior to the
color determination,
A Pretreatment of Samples
1 Oxidizing agents, as detected by
the odor of chlorine or by the
starch-iodide test, are removed
immediately after sampling by
adding an excess of ferrous sulfate
or sodium arsenite.
1 Phenols are distilled from non-
volatile impurities.
2 Addition of copper sulfate to sample
forms CuS, thus preventing the
formation of H S or SO^ which
interfere with the determination.
CuSO^ also prevents biochemical
degradation of phenolics,
3 Acidification of the sample with
phosphoric acid assures the presence
of the copper ion and prevents the
formation of CuCOH)^, an oxidizer
of phenolics.
Oils and tars in a sample may, con-
tain phenolics. An alkaline extrac-
tion to remove these is required
prior to adding CuSO and dis-
tillation.
VI
A
3 If sulfur compounds are present, e.g.
or SO , and the sample has not been pre-
serves, acidify the sample to less than 4
with HgPO , and aerate it briefly. (These
treatments'^are part of the preservation pro-
cedure if the presence of sulfur compounds
is known).
B Distillation
NPDES specifies a preliminary distillation B
to remove common interferences. The rate
of volatilization of phenols is gradual so the
volume of the distillate should equal that of lite
sample being distilled. If the sample was not
preserved, acidify it with 1 + 9 HgP04
and add copper sulfate solution.
4- AMES'OANTJPYRINE DETERMINATION
(q 4)
Applicability 1
This method determines phenol, ortho
and meta-substituted phenols, and para-
substituted phenols in which the substitution
is a carboxyl, halogen, methoxyl, or sulfonic
acid group. It does not determine those para-
substituted phenols in which the substitution
is an alkvl, aryl, nitro, benzoyl, nitroso or
aldehyde group. Paracresol is an example
of a common phenolic that is not sensitive
to this determination.
Method
(2)
After pretreatment and distillation, the
sample is reacted with 4-aminoantipyrine
at pH 10. 0 + 0. 2 in the presence of
potassium ferricyanide {an oxidant) to
produce colored antipyrine dyes. (See
Figure 1).
pH 10
K^Fe(CN)£
4-aminoantlpyrine + phenol *"* N antipyrine dye ;
CHg-N C-O
ch3-c = c~nh2
OH
CH,-N C=0
PH - 10 ~ 3 | I
K Kc (CM' I I
3 6 CHg-C = C-N
O
Figure 1
24-2
-------
Determination of Phenolics
An absorbance measurement is made and
the phenolic concentration is estimated
using a calibration curve with phenol as
a standard,
1 For original phenolic concentrations
of 5ngll to 1000/j g/1, the reaction
product dyes are concentrated using
the Chloroform Extraction Method.
Cell path lengths of 5cm or more are
required for very low levels. Absorb-
ance is measured at 460 nm, and the
results expressed as n g/liter phenol.
2 For original phenolic concentrations
greater than 5Op g./liter, the reaction
product dyes are kept in the water
solution for a Direct Photometric
determination. Absorbance is meas-
ured at 5 lOnm., and the results are
expressed as mg/liter phenol. This
method is applicable to original phe-
nolic concentrations up to 50 mg/liter.
3 Details of reagent preparation and
the stepwise procedures can be found
in the current editions of Standard
Methods^ and ASTM Standards
C Variables
1 Sensitivity varies with pH, A
buffer is used to maintain pH at
10.0 + 0,2 to prevent the form-
ation of antipyrine red and to
minimize interference from
aniline and undesirable enol-keto
systems.'6, * ^
The EPA manual notes that the ammonium
hydroxide-ammonium chloride buffer used
in the water hardness test is an alternative
to the chemicals used in the other write-
ups to raise the pH to 10 + 0. 2.
2 The amounts of 4-aminoantipyrine
and potassium ferricyanide used
have a definite bearing on the
amount of color developed. ^
4 Direct sunlight or strong artificial
light may have a bleaching effect
on the colored materials.^)
5 Filtration of the chloroform
extracts removes water and in-
creases their color stability to 3
hours. The mixtures measured in
water solutions are not too stable
and should be read within 30
minutes.
VII PHENOL STANDARD
1 Because phenol is extremely
sensitive to the 4-aminoantipyrine
determination, the calibration
curve used in the procedure is
derived using phenol standards.
These standards are prepared on
the day of use by diluting a more
concentrated stock solution of phenol.
The stock solution of phenol can be
prepared by direct weighing. If
extreme accuracy is required, this
stock solution can be standardized
using a bromate-bromide solution. ^
2 Phenolics (substituted phenols)
respond with various sensitivities
to this test and produce colors of
various densities. An example is
this comparison of the absorbance
values of the cresols to that of
phenol;
Absorbance Values
Compound Compared to Phenol(%)
phenol 100
orthocresol 74
metacresol 69
paracresol 3
3 Most phenolics sensitive to the test
produce dyes with absorbancy maxima
at or near the same wavelength so a
photometric determination can be
made.
3 Temperature affects the rate of 7)
color change of the product dyes ACCURACY '
and of the blank. All materials
used should be at the same Results are an approximation and rep-
temperature. (6. 8) resent the minimum amount of phenol
and phenolics present. J
-------
Determination of Phenolics
Only phenol is used as the color
standard since it is impractical
to prepare a standard containing
a mixture of phenol and phenolics
corresponding to each sample.
Different phenolics exhibit
different sensitivities to the test.
Different phenolics produce differ-
ing shades of color which affect the
final absnrbance reading for the
mixture.
K
PRECISION
,(2)
2 Methods for Chemical Analysis of Water
and Wastes, EPA-EMSL, Cincinnati,
Ohio, 45268, 1974.
3 Standard Methods for the Examination of
Water and Wastewater, 14th Edition, 1975.
4 ASTM Book of Standards, Part 31, 1975
Method D1783-70.
5 Smith, D. and Lichetenberg, J., Deter-
mination of Phenols in Surface Waters
by Thin-Layer Chromatography,
Microorganic Matter in V\ ater, ASTM
STP 448, 1969.
Reproducibility of results depends on
on the interferences present in samples
and on the skill of the analyst,
1 Using the Chloroform Extraction
Method to concentrate color, six
laboratories analyzed samples at
concentrations of 9.6, 48,3 and 93.5
Mg/liter. Standard deviation res-
pectively, was 0,99, 3. 1 and 4,2
vgf liter.
2 Using the Direct Photometric
Method, six laboratories analyzed
samples at concentrations of 4.7,
48,2 and 97,0 mg/liter. Standard
deviation, respectively, was 0. 18,
0. 48, and 1.58 mg/liter.
REFERENCES
1 Burttschell, R., Rosen, A., Middleton,
F,, and Ettinger, M. Chlorine De-
rivatives of Phenol Causing Taste and
Odor, Jour. American Waterworks
Association, 51:2, 1959.
6 Ettinger, M. 13., Ruchhoft, C. C. and
Lishka, R. J. Sensitive 4-Amino -
antipyrine Method for Phenolic Com-
pounds. Anal. Chem, 23:1783. 1951.
7 Dannis, M. Determination of Phenols by
the 4-Aminoantipyrine Method. Sew.
and Ind. Wastes 23:1516. 1951.
8 Mohler, E. F, and Jacob, L.N.
Comparison of Analytical Methods for
Determination of Phenolic-Type Com-
pounds in Water and Industrial Wastes
Water, Anal. Chem. 29:1369, 1957.
9 Martin, R. Anal. Chem. 21:1419, 1949.
This outline was prepared by A. D. Kroner,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268
Descriptors: Chemical Analysis, Phenols,
Water Analysis
24-4
-------
LABORATORY DETERMINATION OF PHENOL
DIRECT PHOTOMETRIC METHOD
I REAGENTS
A Methyl Orange Indicator
Dissolve 0. 5 g of the indicator in 1 liter
of distilled water.
B Phosphoric Acid Solution
Dilute 10 ml of 85% II3PO4 to 100 ml with
distilled water.
C Copper Sulfate Solution
Dissolve 100 g of C11SO4 " 511^0 in distilled
water and dilute to 1 liter.
D Stock Phenol Solution
Dissolve 1, 00 g of reagent grade phenol
in freshly boiled and cooled distilled water
and dilute to 1 liter. Ordinarily, this direct
weighing of phenol constitutes a standard
solution. However, if extreme accuracy
is needed, the solution must be standardized
[>ee section 510 B. 3. a. 1) o» page 578 of
reference 2].
E Intermediate Phenol Solution
Dilute 10. 0 ml of the stock phenol solution
to 1 liter with freshly boiled and cooled
distilled water;
1 ml = 10. 0 n g phenol
F Ammonium Chloride Solution
Dissolve 50 g of NH4CI in distilled water
and dilute to 1 liter.
G Aminoantipyrine Solution
Dissolve 2. 0 g of 4-aminoantipyrine in
distilled water and dilute to 100 ml.
Prepare this solution on the day of use.
H Potassium Ferricyanide Solution
Dissolve 8.0 g of K3Fe{CN)g in distilled
water and dilute to 100 ml. Prepare this
solution fresh weekly.
I Concentrated Ammonium Hydroxide
J pH 9, 10, or 11 buffer
II PROCEDURE
A Measure 500 ml of sample (graduated cylinder)
and pour it into a large beaker,
B Add 3 drops of methyl orange indicator to
the sample and mix.
C Add a drop of II3PO4 solution (eyedropper)
to the sample and mix. A pink/red color
should be present (pH approximately 4).
If it is not, add a second drop of H3PO4 and
mix,
D Pipet 5. 0 ml of CuSO^. solution into the sample
and mix.
E Remove the small rubber stopper from the
distilling apparatus, insert a small funnel
in the hole, and pour the sample through the
funnel into the distilling flask. Glass beads
are already in the flask.
F Distill 450 ml of sample into a 1 liter
Erlenmeyer flask.
G While the sample is distilling (ckeck it every
5-10 minutes), pipet the following amounts of
intermediate phenol solution into 100 ml
volumetric flasks and dilute to the mark
with distilled water; 0.0, 0.5, 2.0, 5.0,
10,0, 20.0, and 35.0 ml. Use these ml
values as markings on the volumetric flasks.
CH. PHEN.lab. lb. 8. 80
2 5-1
-------
Laboratory Determination of Phenol, Direct Photometric Method
H Remove the source of heat from under the
distilling flask.
1 When boiling ceases, add 50 ml of phenol
free distilled water (graduated cylinder) to
the distilling flask,
J Begin the distillation again, collecting a
total of 500 ml in the 1 liter Erlenmeyer
flask. (If the distillate is turbid, additional
treatment is required. See Method 510 A,
par. 4c and 4d in Standard Methods2).
K Pipet 25. 0 ml of the distillate into a 100 ml
volumetric flask and dilute to the mark with
distilled water. Mark the flask 25.
I, Fill a 100 ml volumetric flask to the mark
with the distillate. Mark the flask 100.
M Put the same numbers on nine 125 ml
Erlenmeyer flasks as were used on the
nine volumetric flasks.
N Pipet 2. 0 ml of ammonium chloride solution
into each of the nine Erlenmeyer flasks.
O Pour the contents of each volumetric flask
into the correspondingly marked Erlenmeyer
flask. Mix by swirling the flasks.
p Calibrate a pH meter using pH 9, 10, or 11
buffer.
Q Using an eyedropper, add concentrated
ammonium hydroxide to each Erlenmeyer
flask until a pH of 10. 0 -t 0. 2 is obtained.
Be careful to rinse the electrode thoroughly
after each use.
R Turn on the Spectronic 20 (warm up).
S Pipet 2. 0 ml of 4-aminoantipyrine into all
of the Erlenmeyer flasks. Mix well by
swirling.
U After 15 minutes measure the absorbance
of all nine of the solutions at 510 nm.
"Zero" the instrument against the solution
in the "0" flask, i.e., the reagent blank.
Ill CALCULATIONS
A Prepare a calibration graph with absorbancies
along the vertical axis and mg of phenol along
the horizontal axis. For example*
Concentration of intermediate phenol
solution = 10. 0 gjml. If 35. 0 ml of this
solution is used, then 0. 350 mg (350.0 y, g)
is the value on the horizontal axis.
B Determine the mg of phenol present in the
distillate (sample) and "scale it up" to a per
liter basis. For example:
If there were 0, 10 mg in 100 ml of distillate,
then the final answer is 1. 0 mg of phenol/liter.
Note: The name of the parameter as
listed in Table 1 of the Federal Register,
Wednesday, December 1, 1976, vol. 41,
number 232 is "Phenols" (item 96).
However, positive results are also given
by certain substituted phenols.
REFERENCES
1 Methods for Chemical Analysis of Water
& Wastes, U. S. Environmental Protection
Agency, Environmental Monitoring &
Support Laboratory, 1974.
2 Standard Methods for the Examination
of Water and Wastewater, 14th edition,
APHA, AWWA, WPCF, 1975.
Pipet 2. 0 ml of potassium ferricyanide into
all of the Erlenmeyer flasks. Mix well by
swirling.
This outline was prepared by C. R. Feldmann,
Chemist, National Training and Operational
Technology Center, MOTD, OWPO, USEPA,
Cincinnati, Ohio 45268.
Descriptors: Chemical Analysis, Laboratory
Tests, Water Analysis, Phenols
25-2
-------
Laboratory Determination of Phenol - Direct Photometric Method
DATA SHEET
ml of intermediate
phenol solution used mg phenol/1 Absorbanee
0.0 0.
0.5 0.
2. 0 _0.
5.0 0.
10.0 o.
20.0 0.
35.0 0.
Absorbanee of sample = 0.
Concentration of phenol in sample in mg /Liter from curve -
25-3
-------
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25-4
-------
INTRODUCTION TO GAS-LIQUID CHROMATOGRAPHY
Part I
I INTRODUCTION
A Definition
Gas-liquid chromatography is an analytical
method for the separation and identification
of a mixture of volatile (usually organic)
components in a sample. As with any
chromatographic technique the column
consists of two phases, the immobile or
stationary phase (a liquid on an inert solid
support), and the mobile phase (an inert
gas). The column functions to separate
the sample components because they have
varying vapor pressures and affinities for
the stationary phase. In many ways the
column behavior resembles that of
fractional distillation. The partition which
occurs between the mobile and immobile
phases will thus cause the components to
proceed through the column at varying
rates. The separation is recorded and
quantitated by the detector system.
B Advantages
1 GLC can be used to separate compounds
of similar boiling points which cannot
easily be separated by distillation.
(See Table 1)
2 GLC can be extremely sensitive; for
example, using the electron capture
detector it is possible to "see"
picogram (lCT^2) quantities.
Table 1. SEPARATIONS BY GLC
Compounds
Reference
3 -Methylcyclohexene
(B.P. 104° C) and
4-Methylcyclohexan
(B.P. 103° C)
Cyclohexane (B.P,
80, 80 C) and Benzene
(B.P. 80.2OC)
Aerograph Research
Notes, (Spring 1964)
Chromosorb News-
letter (FF-104)
C Disadvantages
1 Due to the extreme sensitivity
possible it is often necessary to
apply extensive cleanup techniques.
2 The many variables of the technique
require a skilled analyst.
II COMPONENTS OF A GAS
CHROMATOGRAPH (See Figure 1)
A Gas Supply
The mobile phase (carrier gas) transports
the sample components through the
column into the detector. The type of
gas used varies with the detector .(See
Table 2)
B Injector
Liquid samples are manually introduced
into the heated injector block through a
rubber septum by means of a syringe.
Automatic liquid injectors as well as
injection systems for solid and gaseous
samples are commercially available.
C Column
The vaporized sample enters the column
which can be glass or metal and of varying
length (11 - 20') and diameter (1/8" - 1/4").
The column is packed with the stationary
(immobile) phase and contained within a
constant temperature oven.
1 Solid support
The solid support should have a large
surface area yet be inert so that active
sites will not cause adsorption of
sample components. Diatomaceous
earths, teflon and glass beads have
been used, (See Table 3)
CH. MET. cr. 5b. 1. 74 ' 26-1
-------
Introduction to Gas-Liquid Chromatography
SAMPLE
PRESSURE
REGULATOR
~1
jer]
GLASS
WOOL
AUTOMATIC STRIP
CHART RECORDER*
z
o
z
CL
SILICONE RUBBER
STOPPERS
1/16" DIAMETER
S.S. TUBING
Figure 1. COMPONENTS OF A GAS CHROMATOGRAPH*
Table 2. CARRIER GASES
Detector
Carrier gas
Thermal conductivity
Helium (Purified,
Grade A)
Microcoulometric
Helium (Purified,
Grade A)
Flame ionization
Hydrogen (Purified)
Electron capture
Nitrogen or a mixture
of 95% argon and 5%
methane (Purified)
Table 3. SOLID SUPPORTS
Support
Surface
area (m^/gm)
Chromosorb P
(Diatomaceous Earth)
4.8
Chromosorb W
(Diatomaceous Earth)
1.2
Chromosorb G
(Diatomaceous Earth)
0.5
Chromosorb T
(Teflon)
7.
0-8.0
2 Stationary liquid
The separation and partition occurring
in the column is directly affected by the
choice of stationary liquid. For ex-
ample, in the separation of benzene
(B, P, 80. 1°C) and cyclohexane (B, P.
80, 8°C), the choice of a non-polar phase
such as hexadecane results in benzene
preceding cyclohexane off the column.
However, if a more polar phase such as
benzylbiphenyl is chosen cyclohexane
precedes benzene. Table 4 shows some
typical stationary liquids and their uses.
(NOTE: One requirement for any liquid
is that it have a high boiling point so that
it will not boil off the column)
D Detector
The detector or brain of the gas chroma-
tograph senses and measures the quantity
of sample component coming off the column.
The detector should be maintained at a
temperature higher than the column so that
condensation does not occur in the detector
block. Several types of detectors are in
use today.
~Reproduced (with permission) from Chemistry.
(37:11, p. 13. November 1964).
26-2
-------
Introduction to Gas-Liqaid Chromatography
Table 4. STATIONARY LIQUIDS
Stationary liquid
Silicone oils QF-1, Dow Corning 200,
and Dow 11, OV-1, OV-3, OV-17,
OY-ZIO, OV-225
Silicone oil SE-3 0
Benzyl-Cyanide-Silver Nitrate
Polyethylene Glycol
Cyano Silicone
1 Thermal conductivity
This detector consists of a Wheatstone
bridge two arms of which are thermal
conductivity cells each containing a
small heated element. When only carrier
gas is flowing through both the sample
cell and reference cell, the resistance
of the heated element is constant in both
cells. The bridge remains balanced and
baseline is recorded. However, when
carrier gas plus sample component enter
the sample cell, the thermal conductivity
in that cell changes thus also producing
a change in the resistance of the heated
element. The bridge becomes unbalanced
and a peak is recorded. The main dis-
advantage of the TC cell in water pollution
work is its lack of sensitivity.
2 Ionization detectors
a Flame
This detector consists of a flame
situated between a cathode and anode.
As carrier gas alone burns, some
electrons and negative ions are pro-
duced which are collected at the
anode and recorded as baseline.
When carrier gas plus sample com-
ponent are burned, more electrons
and negative ions are produced which
result in a peak on the recorder.
The detector is capable of "seeing"
nanogram quantities of organic com-
pounds; however, the detector is
sensitive to all organic compounds.
This lack of specificity produces dis-
advantages in the analysis of water
Used to separate
Chlorinated hydrocarbons
pesticides
Homologous series of n-alkanes
Homologous series of olefins
Amines
Steroids
extracts which contain a variety of
naturally occurring organics.
b Electron capture (See Figure 2)
This detector consists of a radiation
source (e.g., tritium) capable of
producing slow electrons in a carrier
gas such as nitrogen. The electrons
collected at the anode are recorded
as baseline. When sample com-
ponents which have an electron
affinity (e.g., chlorinated hydro-
carbons) enter the detector, electrons
are "captured. " The subsequent de-
crease in current is recorded as a
peak. The detector has the advantage
that it is extremely sensitive (pico-
gram range) and is somewhat selective.
c Thermionic
A recent adaptation of the flame
ionization detector shows promise
for the specific analysis of organic
phosphorus compounds. An alkali
salt is incorporated into the design
of a conventional flame ionization
detector so that the salt heated by
the flame produces an ion current.
When compounds containing phos-
phorus emerge from the column,
they give 600X the response with this
detector as with the conventional
flame,
3 Microcoulometric
Although less sensitive (by approximately
a factor of 10) than electron capture.
26-3
-------
Introduction to Gas-Liquid Chromatography
GAS EXHAUST
TO
ELECTROMETER
-•-ELECTROMETER
ANODE
COLUMN
RADIOACTI VE
TRITIUM
FOIL
N2+ e SLOW
COLUMN
GAS —
FLOW
SLOW +X~* *
LOSS OF f"
REDUCESCURRENT
Figure 2. ELECTRON CAPTURE DETECTOR (Wilkens Instrument Company)
this detector is finding wide use in
pesticide analysis. This highly specific
detector consists of titration cells for
the measurement of chloride-containing
and sulfur-containing compounds. The
sample component emerging from the
column is combusted to produce HC1
or SO , respectively. HC1 is continu-
ously titrated by silver ions present in
the cell; the amount of current required
to regenerate these silver ions is
recorded as a peak. The system for
sulfur containing compounds is analo-
gous except that SO^ produced is con-
tinuously titrated by which is
subsequently regenerated.
Another microeoulometric detector has
recently been applied to the specific
determination of nitrogen. It is based
on the reduction of nitrogen-containing
compounds to NH which is then titrated
by H in the titration cell.
4 Coulson Electrolytic Conductivity
This detector was primarily developed
for the detection of organic halides,
organic nitrogen compounds, and
organic sulfur compounds.
The unit consists of pyrolyzer with a
separately heated inlet block, water
circulating and purification system,
detector cell and dc conductivity
bridge. The sample is oxidized or
reduced and reaction products form
electrolytes when dissolved in the
deionized water. Changes in con-
ductivity between two platinum
electrodes are measured by the dc
bridge. (Figure 2 a)
E Recorder
The recorder system registers the
response of the detector to sample
components. In the case of ionization
detectors, it is often necessary to employ
an electrometer in order to amplify the
small current changes.
Expensive integration and digital read-out
equipment is also available to facilitate
measurement of peak areas.
26-4
-------
Introduction to Gas-Liquid Chromatography
QUA9IZ
TU3E
GAS-L 1Q!; 10
CONTACTOR
CCN2UCF V.T Y
CELL
:PT '
• ELECMC^ES
WifE* *I5£ V/0 ¦*
Figure 2 a. FLOW DIAGRAM OF ELECTROLYTIC CONDUCTIVITY DETECTOR
(Coulson Instruments Company)
1 ! 1
SAMPLE: 5 |xl of Standard Pesticide Mix-
ture (1 ng of each Pesticide
COLUMN: Length -6' XG mn
Stationary Pha.se - 10% OC 200
on Anakrom AB5 (DO / JOG Mush)
Mobile Fhuao - 180 ml /min N<>
Temperature- - 2 0°C
DETECTOR
Mootron Culture
HALF-WIDTH
5 7 9 U
TIME 11N MINUTES)
Figure 3. GAS CHROMATOGRAM OF PESTICIDE MIXTURE
26-5
-------
Introduction to Gas-Liquid Chromatography
IH QUA LITATIVE ANA LYSIS
A Retention Time
The retention time of a sample component
is defined as the time it takes for that
component to travel through the column.
There are a number of variables which
affect the retention time of a compound,
1 Physical parameters of column
operation
a Column length
b Column temperature
c Carrier gas flow rate
2 The nature and amount of stationary
liquid itself
For a given set of column conditions, a
specific compound will have a specific
retention time (See Figure 3 and Table 5).
Various column and detector combinations
can be used to confirm identification.
B Retention Volume
Retention volume is defined as the total
volume of gas required to move a com-
ponent through the column.
C Relative Retention Times and Volumes
It is possible to interpret data more easily
by reporting retention data relative to a
particular compound (e, g., aldrin as in
Table 5).
IV QUANTITATIVE ANALYSIS
A Measurement of Peak Area
The quantity of sample component present
is directly proportional to the area under
its peak. (NOTE: This assumption can
only be made if it has been previously
determined that a linear response is
obtained in the range under study.) The
following are a few of the ways in which
this area can be measured.
1 Planimeter
2 Triangulation
3 Peak height X half-width (see dieldrin
peak in Figure 3)
AREA = peak height X peak half-width
4 Disc integrator
B Measurement of Peak Height
RETENTION _ RETENTION FLOW
VOLUME (Rv) ~ TIME (R,p) RATE
With the electron capture detector it may
be possible to use peak height for quanti-
tative measurements where the following
conditions are met.
Table 5. RETENTION DATA FOR FIGURE 3
Pesticide
Retention Time (R^,)
Relative Retention
Time
Heptachlor
3.3 minutes
0.79
Aldrin
4.2
o
o
T—1
Heptachlor Epoxide
5.3
1. 26
Dieldrin
7.7
CO
H
26-6
-------
Introduction to Gas-Liquid Chromatography
1 A steady busline is obtained,
2 Retention times can be reproduced
from one injection to the next.
V SUMMARY
The basic components of a gas chromatograph
have been described. Elementary aspects of
quantitative and qualitative analysis are
presented.
NEWSLETTERS
1 Aerograph Gas Chromatography Newsletter.
Wilkens Instrument and Research, Inc.,
P.O. Box 313 Walnut Creek, California.
2 F & M Gas Chromatography Ncwsleter,
F & M Scientific Corporation. Starr
Road and Route 41, Avondale, Pa.
3 Gas-Chrom Newsletter. Applied Science
Laboratory, Inc. State College, Pa.
16801.
ROOKS
1 Dal Nogare, S. and Juvet, U.S., Jr.
Gas-Liquid Chromatography. New
York: Intcrscience, 1962.
2 Littlewood, A.B. Gas Chromatography.
New York: Academic Press. 1962.
This outline was prepared by R. A, Punghorst,
Chemist, formerlv with National Training
Center, MDS, WPQ EPA,
Cincinnati, OH 45268.
Descriptors : Adsorbents, Chromatography,
Gas Chromatography, Organic Matter,
Separation Techniques, Water Analysis
26-7
-------
QUANTITATION IN GAS CHROMATOGRAPHY
I INTRODUCTION
B Direct Calibration
One of the factors which has led to gas
chromatography's rapid development in the
field of instrument analysis is the quantitative
precision with which samples can be "analyzed.
There are several equally important factors
involved in quantitative analysis. These are:
Accurate sample introduction, constant
operating parameters, accuracy of peak area
measurement, sensitivity factors of individ-
ual compounds, linearity of detector and
columns which give well resolved peaks.
II CALIBRATION PROCEDURE
A. Peak Height and Peak Area
Blends of components in question are
prepared and chromatographed. The
value of peak heights or areas are then
plotted against the weight of sample
injected. The unknown sample is then
injected and its peak height or area is
compared to that of the standard.
The main disadvantages of direct calibra-
tion are that the precise amount of
sample injected must be known and that
calibration is time consuming. Also,
the sensitivity of the detector must remain
constant from run to run and day to day
in order to compare results with the
calibration graph.
Quantitative work is based on peak height
or area. Peak height measurement is
more rapid than peak area; however, plots
of peak height vs. sample size are more
non-linear than corresponding plots for
peak area. This is because peak heights
and widths are frequently dependent on
sample size and sample feed volume, how-
ever; total area is not. Peak heights are
generally used if samples are less than
10 (jLg for packed columns and 0. 1 (ig for
capillary columns.
or
<
ct
o
X
o
X
3
UJ
C Internal Standardization
This is a common procedure used in
biomedical analyses, such as alcohol in
blood, and steroids in serum and urine.
Known weight ratios of the component in
question and a standard (marker) are
prepared and chromatographed. The
component/standard area ratios obtained
are then plotted vs. the component/
standard weight ratios.
Y/
WEIGHT OF COMPONENT
C.H. MET. 29. 1. 74
27-1
-------
Quantitation in Gas Chromatography
To the unknown, add a known volume con-
centration of the standard. This mixture
is then chromatographed and the
component/standard area ratios are deter-
mined. By determining the corresponding
component/standard weight ratio from the
graph, the unknown amount of the com-
ponent can be determined.
e. g.; To the unknown, add 5 ml of a solu-
tion containing the standard whose
concentration is 100 jig/ml. Upon
chromatographing the mixture, the
area ratio was found to be 8; there-
fore, the weight ratio is 7 {see
graph). Knowing standard concentra-
tion to be 100 p.g/ml, then the
component concentration is 7 X 100
^g/ml. Since we added 5 ml of
standard solution, then the total
amount of the unknown is 5 ml X 700
= 3500 )j.g or 3. 5 mg.
D Internal Normalization
This is used when only approximate
data are required. Assuming all the
components have been eluted, the per-
cent composition of a component within
a mixture is its area divided by the
sum of the areas.
1 Composition A » 10D
A
Standard
Component
WEIGHT
component
standard
A particular advantage of using the
internal standardization method is that
the amount injected need not be accurately
measured. Also, the detector response
need not be known or remain constant
since any change in sensitivity will not
affect the area ratio.
This method also assumes that area
percent equals weight percent. This
may only be true when analyzing close
boiling components of a homologous
series. To obtain the weight percent,
it is necessary to multiply each area
by a correction factor.
The chief disadvantage of this method
is the difficulty in finding a standard
that does not interfere with a component
in the sample. It is also time consuming.
27-2
-------
Quantitation ill Gas Chromatography
III NORMALIZATION OF AREAS
(CORRECTION FACTORS)
A Normalization for Thermal Conductivity
Detectors
This procedure relies on the fact that
each compound has a unique thermal
conductivity cell response. For quanti-
tative analysis, the thermal conductivity
of the sample should be approximately a
linear function of its composition in the
range concerned. From the areas under
each peak and the relative responses that
are characteristic of each peak, it is
possible to determine the quantity of each
component in the sample.
Sample calculation:
The weight percent is calculated and
the known densities are used to convert
percent.
Inject several samples and plot peak
area (or height) vs. volume injected.
COMPOUND
Ethanol
Heptane
Benzene
Ethyl Acetate
AREA
5,0
9.0
4,0
7,0
RELATIVE T. C.
RESPONSE PER MOLE (2^
72
143
100
111
Ethanol
Heptane
Benzene
Ethyl Acetate
TOTAL
5.0
72
9,0
143
4.0
100
7.0
111
0,070
0.063
0.040
NORMALIZE
0. 070
0.236
0.053
0.236
0.040
0.236
0.063
0.236
26.7
17.0
26.7
100.0%
B Normalization for Mass Detectors
A standard solution of compounds a,
b, c, d, and e in a solvent is prepared.
27-3
-------
Quantitation in Gas Chromatography
A -1
M— —I—¦— —¦—
-1 0 1 2 3 4 5 6
m\ injected
4 Note that intercept on "X" axis
corresponds to an injection error of
0. 5 a). Therefore, actual amount
injected = uncorrected +0.5 i±l. For
2. 5 |jl (corr.) injected, the density
values are used to determine the actual
weight W. injected.
5 The corresponding area of each peak is
determined and the response in cm2/(ig
is determined.
6 Using compound "a" as standard
(correction factor = 1), the correspond-
ing correction factors are determined.
See the Table on Page 8-6.
IV STATISTICAL TREATMENT OF DATA
A Accuracy and Precision Data
(Reproducibility)^'
Accuracy is a measurement of the
difference between the true value and the
determined values. In those cases in
which the true value is not known, it is
necessary to express the exactness of a
measurement in another way. This may
be done by obtaining the average of a
number of measurements and finding the
difference (deviation) of each value from
the average value. The magnitude of
deviation is a measure of the precision of
measurement.
It is seen that accuracy expresses the
correctness of a measurement, whereas
precision expresses the reproducibility of
a measurement.
The accuracy and reproducibility achieved
in gas chromatography depend on many
things; among them, the correct choice of
column, temperature, flow rate, sample
size, detector, and injection system.
Highest performance demands considerable
understanding of the chromatographic
process and of the effects of change in a
large number of variables. However, even
with ill-designed apparatus, analyses can
usually be conducted with an accuracy
better than + 10 percent per component.
When precautions are taken, an accuracy
and reproducibility of about + 1 percent
per component is easily attainable.
B Errors
Errors fall into two classes. Determinate
errors are those whose cause and magni-
tude can be determined. (Errors in method,
27-4
-------
Quantitation in Gas Chromatography
apparatus, operator, etc.) Indeterminate 3
errors are random errors which cannot be
eliminated. The distribution of indeter-
minate errors follows the normal probability
law as shown in the error curve. This
curve shows that positive and negative
deviations are equally probable and that
small deviations occur much more
frequently than large ones.
-20 -a o +a +20
0 - Standard Deviation
±0 - 68%
±20 * 95 %
4
V MEASUREMENT OF AREAS
A Methods
1 Cutting and weighing is a fairly accurate
but tedious procedure. Besides the
errors that may arise from thickness
and moisture content of the paper, the
chromatogram is destroyed. It is not
satisfactory for estimating areas of
overlapping peaks.
2 The planimeter is a devise whose
acquisition is rarely justified. In
addition to being very fatiguing, it is
necessary to acquire considerable
practice in its manipulation. The
sensitivity of a normal planimeter is
usually 10 mm^, which is in many
cases insufficient for analytical
purposes. ^
Integrators
a Mechanical integrators are exem-
plified by the Disc Integrator. This
is a device which is attached to most
strip chart recorders. The integra-
tor pen tracing is displayed at the
bottom of the chromatogram and the
pen speed is proportional to the dis-
placement of the recorder pen from
baseline. (See triplicate runs of the
nonanedecane mixture.) This is a
frequently used, accurate device.
b Electronic integrators are devices
which automatically print cumulative
integrals of peak area. In addition
to offering outstanding precision,
electronic integrators automate
chromatographic operations. This
eliminates the time consuming
necessity of the chemist keeping
a watchful eye on the operation.
Its biggest disadvantage is its cost.
Triangulation
a Gaussian peaks
27-5
-------
to
-q
I
O
SAMPLE
WT. %
DENSITY
VOL. ?«
VOL. INJECTED, /ll
W
WT. INJECTED
A_
AREA
cmz/ug_
F
CORRECTION FACTOR
gm/cm®
uncorr.
corr. (actual)
MS
cm2
Solvent
99. 79
.88
99. 781
1. 995S2
2,49453
2195.19
—
—
—
a*
.02
0. 50
. 035
.00070
.00087
. 435
4. 0
9.19
1. 00
b
.03
0.75
. 035
.00070
.00087
. 653
6. 5
9. 95
1. 08
c
.04
0. 90
. 0387
.00077
.00096
. 864
7.6
8. 79
.96
d
.04
0. 90
.0387
.00077
.00096
. 864
8.1
9.38
1.02
e
.08
1.00
.0704
. 00141
.00176
1. 760
15.0
8. 52
. 93
Total
100. 00
100. 00
2.00
2. 50
2199. 706
<0
e
P
3
O
3
o
p
CO
O
o
o
HQ
P
~Correction factors calculated relative to Ha" as standard.
-------
Quantitation in Gas Chromatography
b Tailing or leading peaks, trapezoidal
construction.
Triplicate analysis of nonane-decane mixture
O.lSh
(wfl )S+wa(S)h
B Comparison of Integration Methods
Using several of the above methods on the
triplicate runs of the nonanedecane mix-
ture, the following table was obtained:
J L J L J
v...
COMPARISON OF INTEGRATION METHODS
#1
#2
MODEL 471 DIGITAL INTEGKATOH
u-Nonnne 39.156 39.150
n-Decane 60. S-U 60. 850
DISC INTEGRATOR
n-Nonane 39,33 38.97
n-Decane 60. 67 61.03
TRIANGULA TION
n-Nonane 40.77 40.68
n-Decar.e 59.23 59.32
WEIGHING PAPER
n~Nonane 42.58 41.73
n-Decane 57.42 58.27
#3
39.193
60.807
39.91
61.09
40.07
59.93
42. 83
57.17
Avg.
39.166
60.834
39.07
60.93
40.51
59.49
42.38
57,62
a abs
0.0716
0.0227
0.22
0.23
0. 3S
0.39
0.58
0.58
err el
0.184%
0.037%
0.56%
0.38%
0.94%
0.66%
1,37%
1.01%
Summary: Electronic digital integration is the most precise quantitation
method; weighing paper is the least precise. Based upon this and other
data in this publication, the electronic integrator give 2 to 5 times more
precision than Disc Integration, 4 to 10 times more precision than
triangulation, and 7 to 25 times more precision than cutting and weighing
paper.
27-7
-------
Quantitation in Gas Chromatography
C Measurement of Overlapping Peaks
1 Components present in equal amounts.
Since broken line tan-
gent to peak y intersects
baseline after peak x
has reached its maxi-
mum, then peak heights
may be used. If broken
line tangent to peak y
intersects baseline be-
fore peak x has reached
its maximum, then
various known mixtures
of x and y are prepared,
chromatographed, and
compared to the unknown
x and y.
2 Trace analysis (less than 20 ppm)
a Trace component eluting before
major peak.
Trace component on tail of major
peak.
Assuming one
has tried all
conceivable
columns and
conditions, ex-
tend baseline
as a continuation
of the solvent
peak and deter-
mine area.
Compare this
area with a
synthetic mix-
ture. Also, one
can "spike" the
sample with
known amount
of the trace
component and
compare area
before and after.
In any trace
analysis, one
should choose
a column and
conditions which
will allow the
minor peak to
elute before the
major peak.
This is easier
to quantitate,
and generally,
peak height
methods are
used.
BIBLIOGRAPHY
1 Aerograph Previews and Reviews August
1964 p. 6 .
2 Messner, A. E. , Rosie, D. M,, and
Argabright, P. A. Analytical Chemistry
31, 230 1959 .
3 Aerograph Research Notes, Fall Issue,
1965.
4 Purnell, Howard Gas Chromatography,
Wiley. 1962.
5 Lewin, S. Z. Journal of Chemical Educa-
tion, 41, No. 4, A.235-A259, April 1964.
This outline was prepared by E.J. Bonelli,
Applications Manager, Varian Aerograph
Company, Walnut Creek, California,
Descriptors; Chemical Analysis, Chroma-
tography, Gas Chromatography, Instrumen-
tation, Measurement, Separation Techniques,
Water Analysis
27-8
-------
POLY CHLORINATED BIPHENYLS
I INTRODUCTION
Polychlorinated Biphenyls (PCB's) have been
widely discussed in the literature and news
media in recent years. Although the topic
levels have not been thoroughly evaluated,
limits for concentrations in drinking water
have been set at lOOyg/1. Current studies
have indicated PCB's to have a lower chronic
toxicity than DDT by a factor of at least 2,
Toxic levels for animal life vary considerably
with species. However, due to the pronounced
capacity of aquatic life to accumulate PCB's
in the mg/1 range and hence become toxic or
killed from exposure to PCB's in the low ug/1
range, a maximum allowable concentration
of 25 mg/1 has been recommended.
II OCCURRENCE
The widespread use of PCB's have resulted in
the release into the environment of these
materials. They have been found in rainwater,
human tissue and many species of wildlife.
Originally intended for industrial chemicals
because of their nonflammability, high
dielectric constant and plasticizing abilities,
their use has grown steadily amounting to an
estimated use of 400, 000 tons in 1972 in the
United States alone. Since the findings of PCB
in the environment, the use of these materials
have been reduced to about 20, 000 tons/year.
Sales of PCB's for all general plasticizer
applications were discontinued on August 30,
1970, and are being phased out in other
applications. These compounds are, like the
chlorinated hydrocarbon pesticides such as
DDT, very slow to degrade once they have
entered the environment, and once ingested,
are stored in the body's fatty tissue. The
PCB's produced commercially are mixtures,
incorporating some 50 or more of the 210
different PCB compounds. These mixtures
are produced commercially in the United
States solely by one company under the trade
name of Arochlor.
Ill ANALYTICAL METHODOLOGY
The method of analysis for these compounds
uses gas chromatography and is part of the
National Pollutant Discharge Elimination
System, The method can be found in the Federal
Register, 38, No. 75, Part II, Because of
the similarity in nature between the PCB and
chlorinated pesticides, the same method can
determine both. In fact, if both are present,
they cause interferences in the identification
of each other. Consequently, the analytical
method contains a technique to separate the two.
Basically the method utilizes an extraction
step with 15% methylene chloride in hexane
and subsequent concentration. Then an initial
run is made on a gas chromatograph to
determine the complexity of the sample. If
other interferences, or the degree of complexity,
is too large, an additional clean up procedure
must be carried out. Ultimately the PCB's
are determined on the G. C. This determination
is expressed as the Arochlor number after the
basic chromatogram for the particular mixture
has been identified. The method covers the
determination of certain polychlorinated biphenyl
mixtures including: Arochlors 1221, 1232, 1242,
124B, 1254, 1260, and 1016. The limit of
detection is approximately 1 yg/l for each
Arochlor mixture.
REFERENCES
1 Method for Polychlorinated Biphenyls (PCB's)
in Industrial Effluents. Federal Register,
38, No, 75, Part II. USE PA Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268. 197 3.
2 Lee, F. C. and Veith, G. Position Paper
on Chlorinated Biphenyls. University of
Wisconsin. Madison, Wisconsin. August
1970.
LTI. PES. 23. 12. 75
28-1
-------
Poly chlorinated Biphenyls
3 Method for Organochlorine Pesticides in 10
Industrial Effluents. TJSEPA, National
Environmental Research Center,
Analytical Quality Control Laboratory,
Cincinnati, Ohio 45268. 1973. -
4 Leoni, V. The Separation of Fifty Pesticides
and Related Compounds and Polychlori- 11
nated Biphenyls into Four Groups by
Silica Gel Microcolumn Chromatography.
Journal of Chromatography, 62, 63.
1971. _
Webb, R. G. and McCall, A. C. Quantitative
PCB Standards for Electron Capture Gas
Chromatography. Presented at the 164th
National ACS Meeting. New York.
August 29, 1972. (Submitted to the Journal
of Chromatographic Science for publication.)
Goer lit z, D. F. and Law, L. M. Note on
Removal of Sulfur Interferences from
Sediment Extracts for Pesticide Analysis.
Bulletin of Environmental Contamination
and Toxicology, 6, 9. 1971.
5 McClure, V. E. Precisely Deactivated
Adsorbents Applied to the Separation of
Chlorinated Hydrocarbons. Journal of
Chromatography, 70, 168. 1972.
6 Methods for Organic Pesticides in Water
and Wastewater. USEPA, National
Environmental Research Center,
Analytical Quality Control Laboratory,
Cincinnati, Ohio 45268. 1971.
7 Handbook for Analytical Quality Control in
Water and Wastewater Laboratories.
Chapter 6, Section 6.4, USEPA, National
Environmental Research Center,
Analytical Quality Control Laboratory,
Cincinnati, Ohio 45268. 1972.
12
Mills, P. A. Variation of Florisil Activity:
Sample Method for Measuring Adsorbent
Capacity and Its Use in Standardizing
Florisil Columns. Journal of the Associ-
13
ation of Official Analytical Chemists, 51,
29. 1968.
Steere, N, V., Editor. Handbook of
Laboratory Safety. Chemical Rubber
Company, 18901 Cranwood Parkway,
Cleveland, Ohio 44128. pp. 250-254.
1971.
Pesticide Analytical Manual. U. S. Dept.
of Health, Education, and Welfare, Food
and Drug Administration, Washington,
D. C.
Bellar, T. A. and Lichtenberg, J. J.
Method for the Determination of Poly-
chlorinated Biphenyls in Water and
Sediment. USEPA, National Environ-
mental Research Center, Analytical
Quality Control Laboratory, Cincinnati,
Ohio 45268. 1973.
This outline was prepared by J. D. Pfaff,
Chemist, National Training Center, MOTD,
OWPO, USEPA, Cincinnati, Ohio 45268.
Descriptors; Chemical Analysis, Water Analysis,
Polychlorinated Biphenyls, PCB, Arochlors,
Chlorinated Hydrocarbon Pesticides, Pesticide
Residues.
28-2
-------
METHOD FOR POLYCHLORINATED BIPHENYLS
(PCB'S) IN INDUSTRIAL EFFLUENTS
I SCOPE AND APPLICATION
A This method covers the determination
of certain polychlorinated biphenyl
(PCB) mixtures including: Aroclors
1221, 1232, 1242, 1248, 1254, 1260
and 1016,
B The method is an extension of the
method for organochlorine pesticides
in industrial effluents. ^' It is
designed so that determination of
both the PCB's and the organochlorine
pesticides may be made on the same
sample.
C The limit of detection is approximately
1 Mg/1 f°r each Aroclor mixture,
II SUMMARY
A The PCB's and the organochlorine
pesticides are co-extracted by
liquid-liquid extraction and, insofar
as possible, the two classes of com-
pounds separated from one another
prior to gas chromatographic deter-
mination, A combination of the
standard Florisil column cleanup
procedure and a silica gel micro-
column separation procedure are
employed.12, 3) identification is
made from gas chromatographic
patterns obtained through the use of
two or more unlike columns. De-
tection and measurement is accom-
plished using an electron capture,
microcoulometric, or electrolytic
conductivity detector. Techniques
for confirming qualitative identi-
fication are suggested,
III INTERFERENCES
All of these materials must be
demonstrated to be free from inter-
ferences under the conditions of the
analysis. Specific selection of reagents
and purification of solvents by distil-
lation in all-glaSs systems may be
required. Refer to Part I,
Section 1.4 and 1.5.
B The interferences in industrial effluents
are high and varied and pose great
difficulty in obtaining accurate and
precise measurement of PCB's and
organochlorine pesticides. Separation
and cleanup procedures are generally
required to eliminate these inter-
ferences; however, such techniques may
result in the loss of certain organo-
chlorine compounds. For this reason
great care should be exercised in the
selection and use of methods for
eliminating or minimizing interferences.
It is not possible to describe pro-
cedures for overcoming all of the inter-
ferences that may be encountered in
industrial wastes.
C Phthalate esters, certain organophos-
phorus pesticides, and elemental sul-
fur will interfere when using electron
capture for detection. These materials
do not interfere when the microcoulo-
metric or electrolytic conductivity
detectors are used in the halogen mode.
D Organochlorine pesticides and other
halogenated compounds constitute inter-
ferences in the determination of PCB's.
Most of these are separated by the
method described below. However,
certain compounds, if present in the
sample, will occur with the PCB's.
Included are; Sulfur, Heptachlor, aldrin,
DDE, technical chlordane, mirex, and
to some extent o.p'-DDT and p, p'-DDT.
A Solvents, reagents, glassware, and
other sample processing hardware IV APPARATUS AND MATERIALS
may yield discrete artifacts and/or
elevated baselines causing misinter- A Gas Chromatograph - Equipped with
pretation of gas chromatograms. glass lined injection port.
CH. PES. lab. 18a. 8. 80
29-1
-------
Method for Poly chlorinated Biphenyls In Industrial Effluents
B Detector Options;
1 Electron Capture - Radioactive
(tritium or nickel-63)
2 Microcoulometric Titration
3 Electrolytic Conductivity
C Recorder - Potentiometric strip
chart (10 in.) compatible with
detector system.
D Gas Chromatographic Column
Materials;
1 Tubing - Pyrex (180 cm long x
4 mm ID)
2 Glass Wool - Silanized
3 Solid Support - Gas-Chrom Q
(100-120 mesh)
4 Liquid Phases - Expressed as
weight percent coated on solid
support:
a SE-30 or OV-1, 3%
b OV-17, 1.5% + QF-1, 1.95%
E Kuderna-Danish (K-D) Glassware
(Kontes)
1 Snyder Columns - three ball
(macro)
2 Evaporate Flask - 500 ml
3 Receiver Ampuls - 10 ml,
graduated
4 Ampul stoppers
F Chromatographic Column - Chroma-
flex (400 mm long x 19 mm ID) with
coarse fritted plate on bottom and V
Teflon stopcock; 250 ml reservoir
bulb at top of column with flared out
funnel shape at top of bulb - a special
order (Kontes K-420540-9011).
G Chromatographic Column - Pyrex
(approximately 400 mm long x 20 mm
ID) with a coarse fritted plate on bottom.
H Micro Column Pyrex - constructed
according to Figure 1.
I Capillary pipets disposable (5-3/4 in.)
with rubber bulb. (Scientific Pro-
ducts P5205-1).
J Low pressure regulator - 0 to 5 PSIG -
with low-flow needle valve (See Figure 1,
Matheson Model 70).
K Beaker - 100 ml
L Micro syringes - 10, 25, 50 and
100 pi.
M Separatory Funnels - 125 ml, 100 ml,
and 2000 ml with Teflon stopcocks.
N Graduated Cylinders - 100 ml, 250 ml
and 1000 ml.
O Blender - High Speed, glass or stain-
less cup.
P Florisil - PR Grade (60-100 mesh);
purchase activated at 1250 F and
store in the dark in glass containers
with glass stoppers or foil-lined screw
caps. Before use, activate each batch
overnight at 130 in foil-covered glass
container. Determine lauric-acid value
(See Appendix I).
Q Silica gel - Davison code 950-08-08-
226 (60/80 mesh).
R Glass Wool - Hexane extracted,
S Centrifuge Tubes - Pyrex calibrated
(15 ml).
REAGENTS, SOLVENTS AND STANDARDS
A Ferrous Sulfate - (ACS) 30% solution
in distilled water.
B Potassium Iodide - (ACS) 10% solution
in distilled water.
29-2
-------
Method for Folychlormated Biphenyls in Industrial Effluents
Sodium Chloride - (ACS) Saturated
solution (pre-rinse NaCl with
hexane) in distilled water.
VI CALIBRATION
D Sodium Hydroxide
distilled water.
(ACS) 10 N in
E Sodium Sulfate - (ACS) Granular,
anhydrous, conditioned for 4 hours
@ 400 C.
F Sulfuric Acid - (ACS) Mix equal
volumes of concentrated H2SO4
with distilled water,
G Diethyl Ether - Nanograde, redis-
tilled in glass, if necessary,
1 Must contain 2% alcohol and be
free of peroxides by following
test: to 10 ml of ether in glass-
stoppered cylinder previously
rinsed with ether, add one ml of
freshly prepared 10% KI solution.
Shake and let stand one minute. '
No yellow color should be ob-
served in either layer,
2 Decompose ether peroxides by
adding 40 g of 30% ferrous sul-
fate solution to each liter of sol-
vent, CAUTION: Reaction may be
vigorous if the solvent contains a
high concentration of peroxides.
3 Distill deperoxidized ether in glass
and add 2% ethanol.
B
Gas chromatographic operating
conditions are considered acceptable
when the response to dicapthon is at
least 50% of full scale when < . 06 ng
is injected for electron capture de-
tection and < 100 ng is injected for
microcoulometric or electrolytic con-
ductivity detection. For all quantitative
measurements, the detector must be
operated within its linear response
range and the detector noise level
should be less than 2% of full scale.
Standards are injected frequently as a
check on the stability of operating con-
ditions, detector and column. Example
chromatograms are shown in Figures 3
through 8 and provide reference oper-
ating conditions.
VII QUALITY CONTROL
A Duplicate and spiked sample analyses
are recommended as a quality control
check. When the routine occurrence
of a pollution parameter is observed,
quality control charts are also recom-
mended, ^
B Each time a set of samples is ex-
tracted, a method blank is determined
on a volume of distilled water equal
to that used to dilute the sample.
II n-Hexane - Pesticide quality (NOT
MIXED HEXANES).
I Aeetonitri'lc, Hexane, Methanol,
Methylene Chloride, Petroleum Ether
(Boiling range 30- 6C°C) - pesticide
quality, redistill in glass if necessary.
J Standards - Aroclors 1221, 1232,
1242, 1248, 1254, 1260, and 1016*
K Anti- static Solution - STATNUL,
Daystrom, Inc., Weston Instrument
Division, Newark, N.J. 95212.
*May be ordered from US EPA, EMSL,
Quality Assurance Branch, Cincinnati,
Ohio 45268.
SAMPLE PREPARATION
A Blend the sample if suspended matter
is present and adjust pH to near
neutral (pH 6.5-7,5) with 50% sulfuric
acid or 10 N sodium hydroxide.
B For a sensitivity requirement of 1 /ig/1,
when using microoulometric or electro-
lytic conductivity methods for detection
take 1000 ml of sample for analysis.
If interferences pose no problem the
Sensitivity of the electron capture
detector should permit as little as
100 ml of sample to be used. Back-
ground information on the extent and
nature of interferences will assist the
-------
Method for Polychloriiiated Biphenyls in Industrial Effluents
analyst in choosing the required
sample size and preferred detector,
C Quantitatively transfer the proper
aliquot into a two-liter separatory
funnel and dilute to one liter.
IX EXTRACTION
A Add 60 ml of 15% methylene chloride
in hexane (vw ) to the sample in the
separatory funnel and shake vigor-
ously for two minutes.
B Allow the mixed solvent to separate
from the sample, then draw the
water into a one-liter Erlenmeyer
flask. Pour the organic layer into
a 100 ml beaker and then pass it
through a column containing 3-4
inches of anhydrous sodium sulfate,
and collect it in a 500 ml K-D
flask equipped with a 10 ml ampul.
Return the water phase to the separ-
atory funnel. Rinse the Erlenmeyer
flask with a second 60 ml volume
of solvent; add the solvent to the
separatory funnel and complete the
extraction procedure a second time.
Perform a third extraction in the
sanje manner,
C Concentrate the extract to 6-10 ml
in the K-D evaporator on a hot
water bath.
D Qualitatively analyze the sample
by gas chromatography with an
electron capture detector. From the
response obtained decide:
1 If there are any organochlorine
pesticides present,
2 If there are any PCB's present,
3 If there is a combination of
1 and 2,
4 If elemental sulfur is present,
5 If the response is too complex to
determine 1, 2, or 3.
a9-
-------
Method for Polychlorinated Biphcnyls In Industrial Effluents
measurement of the pesticides, or
affect column life or detector sensi-
tivity, proceed as directed below.
B Acetonitrile Partition - This pro-
cedure is used to remove fats and
oils from the sample extracts. It
should be noted that not all pesti-
cides are quantitatively recovered by
this procedure. The analyst must be
aware of this and demonstrate the
efficiency of the partitioning for the
compounds of interest.
1 Quantitatively transfer the pre-
viously concentrated extract to a
125 ml .separatory funnel with
enough hexane to bring the final
volume to 15 ml. Extract the
sample four times by shaking
vigorously for one minute with
30 ml portions of hexane-saturated
acetonitrile.
2 Combine and transfer the acetoni-
trile phases to a one-liter separa-
tory funnel and add 65 0 ml of
distilled water and 40 ml of sat-
urated sodium chloride solution.
Mix thoroughly for 30-35 seconds.
Extract with two 100 ml portions
of hexane by vigorously shaking
about 15 seconds.
3 Combine the hexane extracts in a
one-liter separatory funnel and
wash with two 100 ml portions of
distilled water. Discard the water
layer and pour the hexane layer
through a 3-4 inch anhydrous
sodium sulfate column into a 500
ml K -D flask equipped with a
10 ml ampul. Rinse the separa-
tory funnel and column with three
10 ml portions of hexane,
4 Concentrate the extracts to 6-10 ml
in the K-D evaporator in a hot
water bath.
5 Analyze by gas chromatography
unless a need for further cleanup
is indicated.
C Florisil Column Adsorption Chroma-
tography
1 Adjust the sample extract volume
to 10 ml.
2 Place a charge of activated Florisil
(weight determined by lauric acid
value, see Appendix I) in a Chroma-
flex column. After settling the
Florisil by tapping the column, add
about one-half inch layer of anhy-
drous granular sodium sulfate to
the top.
3 Pre-elute the column, after cooling,
with 50-GO ml of petroleum ether.
Discard the eluate and just, prior to
exposure of the sulfate layer to air
quantitatively transfer the sample
extract into the column by decan-
tation and subsequent petroleum
ether washings. Adjust the elution
rate to about 5 ml per minute and,
separately, collect up to three
eluates in 500 ml K-D flasks
equipped with 10 ml ampuls. (See
Eluate Composition below). Perform
the first elution with 2 00 ml of 6%
ethyl ether in petroleum ether, and
the second elution with 2 00 ml of
15% ethyl ether in petroleum ether.
Perform the third elution with 200 ml
of 5 0% ethyl ether - petroleum ether
and the fourth elution with 200 ml
of 100% ethyl ether.
Eluate Composition - By using an
equivalent quantity of any batch of
Florisil as determined by its lauric
acid value, the pesticides will be
separated into the eluates indicated
on the following page:
29-5
-------
Method for Polychlor inated Biphenyls in Industrial Effluents
6% Eluate
this manner for several days.
Aldrin
BHC
Chlordane
DDD
DDE
DDT
Heptachlor
Heptachlor Epoxide
Lindane
Methoxychlor
Mirex
Pentachloro-
nitrobenzene
Strobane
Toxaphene
Trifluralin
PCB's
50% Eluate
Endosulfan II
C apt an
15% Eluate
Endosulfan I
Endrin
Dieldrin
Dichloran
Phthalate esters
Certain thiophosphate pesticides
will occur in each of the above
fractions as well as the 100%
fraction. For additional infor-
mation regarding eluate composi-
tion, refer to the FDA Pesticide
Analytical Manual.(6)
4 Concentrate the eluates to 6- 10 ml
in the K-D evaporator in a hot
water bath.
5 Analyze by gas chromatography,
D Silica Gel Micro-Column Separa-
tion Procedure (7)
1 Activation for Silica Gel
a Place about 20 gm of silica
gel in a 100 ml beaker. Act-
ivate at 180 C for approxi-
mately 16 hours. Transfer
the silica gel to a 100 ml glass
stoppered bottle. When cool,
cover with about 35 ml of
0.50% diethyl ether in benzene
(volume:volume). Keep bottle
well sealed. If silica gel
collects on the ground glass
surfaces, wash off with the
above solvent before re-
sealing. Always maintain an
excess of the mixed solvent in
bottle (approximately 1/2 in.
above silica gel). Silica gel
can be effectively stored in
2 Preparation of the Chromatographic
Column
a Pack the lower 2 mm ID Section
of the microcolumn with glass
wool. Permanently mark the
column 120 mm above the glass
wool. Using a clean rubber bulb
from a disposable pipet seal the
lower end of the microcolumn.
Fill the microcolumn with 0. 50%
ether in benzene (v.v) to the
bottom of the 10/30 joint (Figure
1). Using a disposable capillary
pipet, transfer several aliquots
of the silica get slurry into the
microcolumn. After approxi-
mately 1 cm of silica gel collects
in the bottom of the microcolumn,
remove the rubber bulb seal,
tap the column to insure that the
silica gel settles uniformly. Care-
fully pack column until the silica
gel reaches the 120 + 2 mm mark.
Be sure that there are no air
bubbles in the column. Add about
10 mm of sodium sulfate to the
top of the silica gel. Under low
humidity conditions, the silica
gel may coat the sides of the
column and not settle properly.
This can be minimized by wiping
the outside of the column with an
anti-static solution.
b Deactivation of the Silica Gel
1) Fill the microcolumn to the
base of the 10/30 joint with
the 0. 50% ether-benzene mix-
ture, assemble reservoir
(using spring clamps) and fill
with approximately 15 ml of
the 0.50% ether-benzene mix-
ture. Attach the air pressure
device (using spring clamps)
and adjust the elution rate to
approximately 1 ml/min. with
the air pressure control. Re-
lease the air pressure and
detach reservoir just as the
last of the solvent enters the
29-6
i
-------
Method for Polychlorinated Biphenyls in Industrial Effluents
sodium sulfate. Fill the
column with n-hexane (not
mixed hexanes) to the base
of the 10/30 fitting. Evap-
orate all residual benzene
from the reservoir, assem-
ble the reservoir - section
and fill with 5 ml of n-hexane.
Apply air pressure and ad-
just the flow to 1 ml/min.
{The n-hexane flows slightly
faster than the benzene).
Release the air pressure and
remove the reservoir just
as the n-hexane enters the
sodium sulfate. The column
is now ready for use.
2) Pipet a 1.0 ml aliquot of
the concentrated sample
extract (previously reduced
to a total volume of 2,0 ml)
on to the column. As the
last of the sample passes into
the sodium sulfate layer,
rinse down the internal wall
of the column twice with
0. 25 ml of n-hexane. Then
assemble the upper section
of the column. As the last
of the n-hexane rinse reaches
the surface of the sodium
sulfate, add enough n-hexane
(volume predetermined, see
X D 3 below) to just elute
all of the PCB's present in
the sample. Apply air
pressure and adjust until the
flow is 1 ml/min. Collect
the desired volume of eluate
(predetermined, see X D 3
below) in an accurately cali-
brated ampul. As the last
of the n-hexane reaches the
surface of the sodium sul-
fate, release the air pres-
sure and change the collec-
tion ampul.
3) Fill the column with 0.50%
diethyl ether in benzene,
again apply air pressure and
adjust flow to 1 ml/min.
Collect the eluate until all
of the organochlorine pesti-
cides of interest have been
eluted (volume predetermined,
see X D3 below).
4) Analyze the eluates by gas
chromatography.
3 Determination of Elution Volumes
a The elution volumes for the.
PCB's and the pesticides depend
upon a number of factors which
are difficult to control. These
include variation in;
1) Mesh size of the silica gel
2) Adsorption properties of the
silica gel
3) Polar contaminants present
in the eluting solvent
4) Polar materials present in
the sample and sample
solvent
5) The dimensions of the micro-
columns
Therefore, the optimum elution
volume must be experimentally
determined each time a factor
is changed. To determine the
elution volumes, add standard
mixtures of Aroclors and pesti-
cides to the column and serially
collect 1 ml elution volumes.
Analyze the individual eluates by
gas chromatography and determine
the cut-off volume for n-hexane
and for ether-benzene. Figure 2
shows the retention order of the
various PCB components and of
the pesticides. Using this infor-
mation, prepare the mixtures
required for calibration of the
microcolumn.
b In determining the volume of
hexane required to elute the PCB's
the sample volume (1 ml) and
the volume of n-hexane used to
29-7
-------
Method for Polychlorinated Biphenyls in Industrial Effluents
rinse the column wall must
be considered. Thus, if it
is determined that a 10, 0 ml
elution volume is required to
elute the PCB's, the volume
of hexane to be added in addi-
tion to the sample volume
but including the rinse volume
should be 9.5 ml.
c Figure 2 shows that as the
average chlorine content of
a PCB mixture decreases
the solvent volume for com-
plete elution increases. Quali-
tative determination (IX D) indi-
cates which Aroclors are
present and provides the basis
for selection of the ideal elu-
tion volume. This helps to
minimize the quantity of organo-
chlorinc pesticides which will
elute along with the low percent
chlorine PCB's and insures the
most efficient separations possible
for accurate analysis.
d Fcr critical analysis where the
PCB's and pesticides are not
separated completely, the column
should be accurately calibrated
according to (X D 3 a) to de-
termine the percent of material
of interest that elutes in each
fraction. Then flush the column
with an additional 15 ml of 0. 50%
ether in benzene followed by 5 ml
of n-hexane and use this recon-
ditioned column for the sample
separation. Using this technique
one can accurately predict the
amount (%) of materials in each
micro column fraction.
E Micro Column Separation of Sulfur,
PCB's, and Pesticides
1 See procedure for preparation and
packing micro column in PCB
analysis section (X D 1 and X D 2).
2 Microcolumn Calibration
a Calibrate the microcolumn for
OLH-%
sulfur and PCB separation by
collecting 1,0 ml fractions and
analyzing them by gas chroma-
tography to determine the
following:
1) The fraction with the first
eluting PCB's (those present
in 1260),
2) The fraction with the last
eluting PCB's (those present
in 1221),
3) The elution volume for
sulfur,
4) The elution volume for the
pesticides of interest in
the 0.50% ether-benzene
fraction.
From these data determine the
following:
1) The eluting volume containing
only sulfur (Fraction I),
2) The eluting volume containing
the last of the sulfur and the
early eluting PCB's (Frac-
tion II),
3) The eluting volume containing
the remaining PCB's (Frac-
tion ni),
4) The ether-benzene eluting
volume containing the pesti-
cides of interest (Fraction IV).
3 Separation Procedure
a Carefully concentrate the 6%
eluate from the florisil column
to 2.0 ml in the graduated ampul
on a warm water bath.
b Place 1.0 ml (50%) of the con-
centrate into the microcolumn
with a 1 ml pipet. Be careful
not to get any sulfur crystals
into the pipet.
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
c Collect Fractions I and II in
calibrated centrifuge tubes.
Collect Fractions III and IV in
calibrated ground glass stoppered
ampules.
d Sulfur Removal'^ - Add 1 to 2
drops of mercury to Fraction II
stopper and place on a wrist-
action shaker. A black precip-
itate indicates the presence of
sulfur. After approximately
20 minutes the mercury may-
become entirely reacted or de-
activated by the precipitate. The
sample should be quantitatively
transferred to a clean centrifuge
tube and additional mercury added.
When crystals are present in the
sample, three treatments may be
necessary to remove all the
sulfur. After all the sulfur has
been removed from Fraction II
(check using gas chromatography)
combine Fractions II and III. Adjust
the volume to 10 ml and analyze
gas chromatographically. Be
Sure no mercury is transferred
to the combined Fractions II and
III, since it can react with certain
pesticides.
By combining Fractions II and III,
.if PCB's are present, it is
possible to identify the Aroclor(s)
present and a quantitative analysis
can be performed accordingly.
Fraction I can be discarded since
it only contains the bulk of the
sulfur. Analyze Fractions III
and IV for the PCB's and pesti -
cides. If DDT and its homologs,
aldrin, heptachlor, or technical
chlordane are present along with
the PCB's, an additional micro-
column separation can be per-
formed which may help to further
separate the PCB's from the
pesticides (See X D).
XI QUANTITATIVE DETERMINATION
A Measure the volume of n-hexane
eluate, containing the PCB's and inject
1 to 5 p. 1 into the gas chromatograph.
If necessary, adjust the volume of the
eluate to give linear response to the
electron capture detector. The
microcoulometric or the electrolytic
detector may be employed to improve
specificity for samples having higher
concentrations of PCB's.
B Calculations
1 When a single A roc lor is present,
compare quantitative Aroclor
reference standard (e.g., 1242,
1260) to the unknown. Measure and
sum the areas of the unknown and
the reference Aroclor and calculate
the result as follows:
[A] fB] [Vt]
Microgram /liter = x [N]
((Vi> (Vs)]
a _ ng of Standard Injected _ ng
~~ of Standard Peak Areas ~ 2
mm
B - £ of Sample Peak Areas = (mm2)
V.= Volume of sample injected (^1)
V^= Volume of Extract (|jl1) from which
sample is injected into gas chroma-
tograph
V = Volume of water sample extracted
S (ml)
\r = 2 when micro column used
1 when micro column not used
Peak Area = Peak height (mm x Peak
Width at 1/2 height
2 For complex situations, use the
calibration method described below.
Small variations in components
between different Aroclor batches
make it necessary to obtain samples
of several specific Aroclors. These
reference Aroclors can be obtained
from Dr. Ronald Webb, Southeast
Environmental Research Laboratory,
EPA, Athens, Georgia 30601. The
procedure is as follows;
a Using the OV-1 column, chroma-
tograph a known quantity of each
Aroclor reference standard. Also
chromatograph a sample of p, p' -
DDE. Suggested concentration of
each standard is 0. 1 ng/ul for the
Aroclors and 0,02 ng/fil for the
p, p' - DDE.
29-9
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
Determine the relative retention
time (RRT) of each PCB peak in
the resulting chromatograms
using p, p' - DDE as 100. See
Figures 3 through 5.
RRT
RT x 100
RT.
RRT
RT
RT
DDE
Relative Retention
Time
Retention time of
peak of interest
DDE = Retention time of
p, p' -DDE
Retention time is measured as
that distance in mm between the
first appearance of the solvent
peak and the maximum for the
compound.
To calibrate the instrument for
each PCB measure the area of
each peak.
Area = Peak height (mm) x Peak
width at 1/2 height. Using Tables
1 through 6 obtain the proper
mean weight factor, then deter- g
mine the response factor ng/mm .
ng/mm
ng;
(ng.) (mean weight percent)
2 1 TTmT
fffreal
ng of Aroclor Standard Injected
Mean weight percent = obtained from
Tables 1 through
Where Area = Areajfmm ) of
sample peak ng/mm = Response
factor for that peak measured.
Then add the nanograms of PCB's
present in the injection to get the
total number of nanograms of
PCB's present. Use the following
formula to calculate the concen-
tration of PCB's in the sample:
Micrograms / Liter
[ng] [Vt]
[Vs] [V.]
x [N]
t
volume of water extracted (ml)
volume of extract (jxl)
Y| = volume of sample injected (ul)
Eng = sum of all the PCB's in nano-
grams for that Aroclor identified
N = 2 when microcolumn used
N = 1 when microcolumn not used
The value can then be reported
as Micrograms/ Liter PCB's
reported as the Aroclor. For
samples containing more than one
Aroclor, use Figure 9 chromato-
gram divisional flow chart to
assign a proper response factor
to each peak and also identify the
"most likely" Aroclors present.
Calculate the ng of each PCB
isomer present and sum them
according to the divisional flow
chart. Using the formula above,
calculate the concentration of the
various Aroclors present in the
sample.
Calculate the RRT value and the
area for each PCB peak in the
sample chromatogram. Compare
the sample chromatogram to those
obtained for each reference
Aroclor standard. If it is appar-
ent that the PCB peaks present
are due to only one Aroclor then
calculate the concentration of
each PCB using the following
formula;
XII REPORTING RESULTS
A Report results in micrograms per liter
without correction for recovery data.
When duplicate and spiked samples are
analyzed, all data obtained should be
reported.
ng PCB = ng/mm x Area
29-10
-------
Method for Polychlorinated Biphenyls in Industrial Effluents
Table 1
Composition of Aroclor 1221^
RRTa
Mean
Weight
Percent
Relative
Std. Dev.b
Number of
Chlorines0
11
31.8
15. 8
1
14
19. 3
9. 1
1
16
10. 1
9. 7
2
19
2.8
9. 7
2
21
20. 8
9. 3
2
28
5.4
13. 9
2
85%
3
15%
32
1.4
30, 1
2"
10%
3
90%
37
1.7
48. 8
3
40
3
Total
93. 3
^Retention time relative to p, p' DDE- 100. Measured from first
appearance of solvent. Overlapping peaks that are quantitated
as one peak are bracketed.
^Standard deviation of seventeen results as a percentage of the
mean of the results.
£
From GC-MS data. Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.
29-11
-------
Method for Polychlorinated Biphenyls in Industrial Effluents
Table 2
Composition of Aroclor 1232 W
RRT2
Mean
Weight
Percent
Relative,
Std. Dev.
Number of
Chlorines0
11
16.2
3.4
1
14
,9.9
2.5
1
16
7.1
6.8
2
f20
17.8
2.4
2
[21
2
28
9.6
3.4
2l
40%
3.
. 60%
32
3.9
4. 7
3
37
6. 8
2.5
3
40
6.4
2.7
3
47
4.2
4. 1
4
54
3.4
3.4
3
33%
4
67%
58
2.6
3.7
4
70
4. 6
3. 1
4 "
90%
5
10%
78
1.7
7.5
4
Total
94.2
aRetention time relative to p, p1 - DDE- 100. Measured from first
appearance of solvent. Overlapping peaks that are quantitated
as one peak are bracketed.
^Standard deviation of four results as a mean of the results.
CFrom GC-MS data. Peaks containing mixtures of isomers of
different chlorine numbers are bracketed.
29-12
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
Table 3
Composition of Aroclor 1242 ^
RRTa
Mean
Weight
Percent
Relative ,
Std. Dev.
Number of
Chlorines
11
1. 1
35.7
1
16
2. 9
4.2
2
21
11. 3
3.0
2
28
11.0
5. 0
21
25%
3J
75%
32
6.1
4.7
3
37
11.5
5. 7
3
40
11. 1
6.2
3
47
8. 8
4.3
4
54
6. 8
2.9
31
33%
4.
67%
58
5.6
3. 3
4
70
10. 3
2. 8
41
90%
5
10%
78
3.6
4.2
4
84
2.7
9.7
5
98
1.5
9.4
5
104
2.3
16.4
5
125
1.8
20.4
5"
85%
6.
15%
146
1.0
19.9
5"
75%
6
25%
Total
98.5
a Retention time relative to p, p'-DDE= 100. Measured from first
appearance of solvent.
'
Standard deviation of six results as a percentage of the mean
of the results.
cFrom GC-MS data. Peaks containing mixtures of isomers of
different chlorine number are bracketed.
29-13
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
Table 4
Composition of Aroclor 124 B ^
RRTa
Mean
W eight
Percent
Relative,
Std. Dev.
Number of
c
Chlorines
21
1.2
23.9
2
28
5.2
3.3
3
32
3.2
3. 8
3
37
8.3
3. 6
3
40
8. 3
3.9
3]
85%
4.
15%
47
15.6
1. 1
4
54
9.7
6. 0
31
10%
4.
90%
58
9.3
5. 8
4
70
19. 0
1.4
4]
80%
5.
20%
78
6.6
2.7
4
84
4.9
2. 6
5
98
3.2
3. 2
5
104
3.3
3.6
41
10%
5
90%
112
1.2
6.6
5
125
2.6
5.9
5 "
90%
6.
10%
146
1.5
o
o
5 '
85%
6
15%
Total
103. 1
&
Retention time relative to p,p'-DDE= 100. Measured from
first appearance of solvent.
Standard deviation of six results as a percentage of the mean
of the results.
c
From GC-MS data. Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.
29-14
-------
Method for Polychlorinatcd Biphenyls in Industrial Effluents
Table 5
Composition of Aroclor 1254 ^
RRTa
Mean
W eight
Percent
Relative,
Std. Dev.
Number of
Chlorines0
47
6.2
3.7
4
54
2.9
2.6
4
58
1.4
2. 8
4
70
13.2
2.7
4"
25%
5.
75%
84
17. 3
1.9
5
98
7.5
5.3
5
104
13.6
3. 8
5
125
15. 0
2.4
5"
70%
6
30%
146
10.4
2.7
5"
30%
6
70%
160
1.3
8.4
6
174
3.4
5.5
6
203
1.8
18.6
6
232
1.0
26. 1
7
Total
100.0
^Retention time relative to p, p'-DDE- 100. Measured from first
appearance of solvent.
^Standard deviation of six results as a percentage of the mean
of the results.
cFrom GC-MS data. Peaks containing mixtures of isomers are
bracketed.
29-15
-------
Method for Polychlorinated Biphenyls in Industrial Effluents
Table 6
Composition of Aroclor 1260^
HRTa
Mean
W eight
Percent
Relative ,
Std. Dev.
Number of
Chlorines0
70
2.7
6.3
5
84
4. 7
1.6
5
r 98
3.8
3.5
1
d
L104
5
60%
6.
40%
117
3.3
6.7
6
125
12. 3
3.3
5
15%
6.
85%
146
14. 1
3.6
6
160
4.9
2.2
61
50%
7.
50%
174
12.4
2. 7
6
2 03
9.3
4.0
6
10%
7.
90%
f232
"
e
1.2 44
9. 8
3.4
6
10%
8_
90%
280
11.0
2.4
7
332
4.2
5.0
7
372
4. 0
8.6
8
448
. 6
25.3
8
528
1.5
10.2
8
Total
98.6
Retention time relative to p, p'-DDE- 100. Measured from first
appearance of solvent. Overlapping peaks that are quantitated
as one peak are bracketed.
¦L
Standard deviation of six results as a mean of the results.
Q
From GC-MS data. Peaks containing mixtures of isomers of
different chlorine numbers are bracketed.
Composition determined at the center of peak 104.
Composition determined at the center of peak 232.
29-16
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
©PRESSURE
GAUGE
—
REGULATOR
COMPRESSED
AIR =
SUPPLY
SHUT-OFF
VALVE
NEEDLE
VALVE
FLEXIBLE
TUBING
I cm
SILICA GEL
5 cm
GLASS
WOOL
I cm
15 ml
RESERVOIR
10 / 30
23 cm x 4.2 mm 1.0.
J | 2 cm
FIGURE I. MICROCOLUMN SYSTEM
29-17
-------
to
CD
1
1 SULFUR I
HEPTACHLOR
<
i-
b
6-
li-
O
z
u
o
¦X.
-•J
DDE
1 MIREX
ALDRiN
OP* a PP* DDT
I TECHNICAL CH LOR DANE
1 y-CHLOROANE
[254 /
S'xxlM \
/
.£/ £$
4 6 8 10 12
VOLUME n-HEXANE ml
Figure 2. Aroclor Eiution Patterns
o
&
Mi
o
hd
o
n
tr
i—i
o
3
P
o
a
w
rr
n>
3
*
-------
47 54
104
125
146
Figure 3. Column: 3% 9V-1, Carrier Sas: Nitrogsn at 80 ml/rnin,
Column Temperature: 170 0, Detector: Electron Capture
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
29-20
-------
146
174
125
280
160
117
203
232
372
520
Figure 5. Column: 3% OV-1, Carrier Gas: Nitrogen at SG snl/min,
Column Temperature: 170 C, Detector: Electron Capture.
-------
Method for Poly chlorinated Biphenyls In Industrial Effluents
RETENTION TIME IN MINUTES
Figure 6. Column: 1.5% OV-17 + 1.05% QF-1, Carrier Gas: Nitrogen
at 60 ml/niin, Column Temperature: 200 C, Detector: Electron Capture.
29-22
-------
AROCLOR 1254
Figure 7. Column: 1.5% QV-17 + 1.35%
Detector: Electron Capture.
RETENTION TIME IN HINUTES
QF-1, Carrisr Gas: Nitrogen at 60 ml/rain,
Column Temperature: 2DO C,
ts?
CD
I
JS3
W
sr
o
a
Ma
o
4
t)
O
H-J
t
p-
W
i—'•
=r
3
a
£
CO
i—
P
2
c
(T>
3
-------
51
39
45
48
27
33
35
42
9
12
6
3
15
18
21
RETENTION TIKE ill UlSliTES
Figure 8. Column; 1.5% OV-17 + 1.95% QF-1. Carrier Ess; Nitrogen at EC ml/rain, Cote Tasajisrstcre: 200C, Detector: Electron Capture.
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
RRT of first peak < 4 7?
Is there a slistsnc
peak with ROT 78
RRT 47-5 0?
\
Use 1242 far | | Use 1242 far
peahs - RRT 04 | j pcaks - HfiT 7 Of 8 'or pcaxs
Is there a distinct
Use 12G0 fcr
all p eahs
peak with RRT 117 ? j
Use 1254 for a
peaks- RRT 174
y
Use 1280 far
fill ether p e ;¦s
Figure 9. ChroaaiogrsRi Division Flowchart (3).
29-25
-------
Method for Folychlorlnated Biphenyls in Industrial Effluents
REFERENCES
1 "Method of Organochlorine Pesticides in
Industrial Effluents, " U.S. Envir-
onmental Protection Agency, National
Environmental Research Center,
Analytical Quality Control Laboratory,
Cincinnati, Ohio 45268, 1973.
2 Leoni, V. "The Separation of Fifty
Pesticides and Related Compounds
and Polychlorinated Biphenyls into
Four Groups by Silica Gel Micro-
column Chromatography, " Journal of
Chromatography, 62, 63 (1971).
3 McClure, V.E. "Precisely Deactivated
Adsorbents Applied to the Separation
of Chlorinated Hydrocarbons, "
Journal of Chromatography, 70, 168
(1972). '
4 "Methods for Organic Pesticides in Water
and Wastewater," U.S. Environmental
Protection Agency, National Environ-
mental Research Center, Cincinnati,
Ohio, 45268, 1971.
5 "Handbook for Analytical Quality Control
in Water and Wastewater Laboratories, '*
Chapter 6, Section 6.4, U.S. Environ-
mental Protection Agency, National
Environmental Research Center,
Analytical Quality Control Laboratory,
Cincinnati, Ohio 45268, 1972.
6 "Pesticide Analytical Manual, " U.S.
Dept. of Health, Education, and
Welfare, Food and Drug Administration,
Washington, D.C,
7 Bellar, T.A. and Lichtenberg, J. J.
"Method for the Determination of
Polychlorinated Biphenyls in Water
and Sediment, " U.S. Environmental
Protection Agency, National Environ-
mental Research Center, Analytical
Quality Control Laboratory, Cincinnati,
Ohio 45268, 1973.
8 Webb, R.G. and McCall, A.C. "Quanti-
tative PCB Standards for Electron Cap-
ture Gas Chromatography. " Presented
at the 164th National ACS Meeting, New
York, August 29, 1972. (Submitted to
the Journal of Chromatographic Science
for publication).
9 Goerlitz, D.F. and Law, L.M. "Note on
Removal of Sulfur Interferences from
Sediment Extracts for Pesticide Analysis, "
Bulletin of Environmental Contamination
and Toxicology, 6, 9 (1971).
10 Mills, P. A. "Variation of Florisil Acti-
vity: Sample Method for Measuring
Adsorbent Capacity and its Use in
Standardizing Florisil Columns, "
Journal of the Association of Official
Analytical Chemists, 51, 29 (1968).
11 Steere, N.V., editor, "Handbook of Lab-
oratory Safety, " Chemical Rubber
Company, 18901 Cranwood Parkway,
Cleveland, Ohio 44128, 1971, pp. 250-
254.
Descriptors: Chemical Analysis, Poly-
chlorinated Biphenyls, Chromatography,
Gas Chromatography
29-26
-------
Method for Poly chlorinated Biphenyls in Industrial Effluents
XIII STANDARDIZATION OF FLORISIL
COLUMN BY WEIGHT ADJUSTMENT
BASED ON ADSORPTION OF LAURIC
ACID
A A rapid method for determining
adsorptive capacity of Florisil is based
on adsorption of lauric acid from hexane
solution (6), (8). An excess of lauric
acid is used and amount not adsorbed
is measured by alkali titration. Weight
of lauric acid adsorbed is used to cal-
culate, by simple proportion, equiv-
alent quantities of Florisil for batches
having different adsorptive capacities.
B Apparatus
1 Buret. — 25 ml with 1/10 ml
graduations.
2 Erlenmeyer flasks. — 125 ml narrow
mouth and 25 ml, glass stoppered,
3 Pipet. — 10 and 20 ml transfer,
4 Volumetric flasks. — 500 ml.
C Reagents and Solvents
1 Alcohol, ethyl. — USP or absolute,
neutralized to phenolphthalein.
2 Hexane. — Distilled from all glass
apparatus.
3 Lauric acid. — Purified, CP.
4 Laurie acid solution. — Transfer
10.000 g lauric acid to 500 ml
volumetric flask, dissolve in hexane,
and dilute to 500 ml (1 ml =20 mg).
5 Phenolphthalein Indicator. — Dissolve
1 g in alcohol and dilute to 100 ml.
6 Sodium hydroxide. — Dissolve 20 g
NaOH (pellets, reagent grade) in
water and dilute to 500 ml (IN).
Dilute 25 ml IN NaOH to 500~ml
with water (0.05N). Standardize as
follows: Weigh 11)0-200 mg lauric
acid into 125 ml Erlenmeyer flask.
Add 50 ml neutralized ethyl alcohol
and 3 drops phenolphthalein indi-
cator; titrate to permanent end point.
Calculate mg lauric acid/ml
0. 05 N NaOH (about 10 m g/ml).
ENDIX I
D Procedure
1 Transfer 2. 000 g Florisil to 25 ml
glass stoppered Erlenmeyer flasks.
Cover loosely with aluminum foil
and heat overnight at 130°C.
Stopper, cool to room temperature,
add 20.0 ml lauric acid solution
(400 mg), stopper, and shake
occasionally for 15 min. Let
adsorbent settle and pipet 10.0 ml
of supernatant into 125 ml Erlen-
meyer flask. Avoid inclusion of
any Florisil.
2 Add 50 ml neutral alcohol and
3 drops indicator solution; titrate
with 0. 05N to a permanent end
point. —
E Calculation of Lauric Acid Value and
Adjustment of Column Weight
1 Calculate amount of lauric acid
adsorbed on Florisil as follows:
Lauric Acid value - mg lauric
acid/g Florisil = 200 - (ml
required for titration X mg lauric
acid/ml 0. 05N NaOH).
2 To obtain an equivalent quantity
of any batch of Florisil, divide
110 by lauric acid value for that
batch and multiply by 20 g.
Verify proper elution of pesti-
cides by 13.6.
F Test for Proper Elution Pattern and
Recovery of Pesticides: Prepare
a test mixture containing a Id r in,
heptachlor epoxide, p, p'-DDE,
dieldrin, Parathion and malathion.
Dieldrin and Parathion should elute
in the 15% eluate; all but a trace of
malathion in the 50% eluate and the
others in the 6% eluate.
29-27
-------
METHOD FOR ORGANOCHLORINE PESTICIDES
IN INDUSTRIAL EFFLUENTS
I SCOPE AND APPLICATION
A This method covers the determination
of various organochlorine pesticides,
including some pesticidal degradation
products and related compounds in
industrial effluents. Such compounds
are composed of carbon, hydrogen,
and chlorine, but may also contain
oxygen, sulfur, phosphorus, nitrogen
or other halogens.
B The following compounds may be
determined individually by this method
with a sensitivity of 1 jug/liter: BHC,
lindane, heptachlor, aldrin, heptachlor
epoxide, dieldrin, endrin, C apt an,
DDE, DDD, DDT, methoxychlor, endo-
sulfan, dichloran, mirex, pentachloro-
nitrobenzene and trifluralin. Under
favorable circumstances, Strobane, III
toxaphene, chlordane (tech,) and
others may also be determined. The
usefulness of the method for other
specific pesticides must be demon-
strated by the analyst before any
attempt is made to apply it to sample
analysis.
C When organochlorine pesticides exist
as complex mixtures, the individual
compounds may be difficult to dis-
tinguish. High, low, or otherwise
unreliable results may be obtained
through misidentification and/or one
compound obscuring another of lesser
concentration. Provisions incorporated
in this method are intended to minimize
the occurrence of such interferences.
II SUMMARY
A The method offers several analytical
alternatives, dependent on the analyst's
assessment of the nature and extent
of interferences and/or the complexity
of the pesticide mixtures found.
Specifically, the procedure describes
the use of an effective co-solvent for
efficient sample extraction; provides.
through use of column chromatography
and liquid-liquid partition, methods
for elimination of non-pesticide inter-
ferences and the pre-separation of
pesticide mixtures. Identification is
made by selective gas chromatographic
Separations and may be corroborated
through the use of two or more unlike
columns. Detection and measurement is
accomplished by electron capture, micro-
coulometric or electrolytic conductivity
gas chromatography. Results are re-
ported in micrograms per liter.
B This method is recommended for use
only by experienced pesticide analysts
or under the close supervision of such
qualified persons.
INTERFERENCES
A Solvents, reagents, glassware, and
other sample processing hardware may
yield discrete artifacts and/or elevated
baselines causing misinterpretation of
gas chromatograms. All of these ma-
terials must be demonstrated to be free
from interferences under the conditions
of the anlaysis. Specific selection of
reagents and purification solvents by
distillation in all-glass systems may be
required. Refer to Part I, Sections 1.4
and 1.5.
B The interferences in industrial effluents
are high and varied and often pose great
difficulty in obtaining accurate and pre-
cise measurement of organochlorine
pesticides. Sample clean-up procedures
are generally required and may result
in the loss of certain organochlorine
pesticides. Therefore, great care
should be exercised in the selection
and use of methods for eliminating or
minimizing interferences. It is not
possible to describe procedures for
overcoming all of the interferences that
may be encountered in industrial
effluents.
CH. PES. lab. 16. 11 . 77
30-1
-------
Method for Organochlorine Pesticides
C Polychlorinated Biphenyls (PCB's) - C
Special attention is called to indust-
rial plasticizers and hydraulic fluids
such as the PCB's which are a
potential source of interference in D
pesticide analysis. The presence of
PCB's is indicated by a large number
of partially resolved or unresolved
peaks which may occur throughout
the entire chromatogram. Particular-
ly severe PCB interference will require
special separation procedures.^' ^
D Phthalate Esters - These compounds,
widely used as plasticizers, respond
to the electron capture detector and
are a source of interference in the
determination of organochlorine pest-
icides using this detector. Water
leaches these materials from plastics,
such as polyethylene bottles and tygon
tubing. The presence of phthalate
esters is implicated in samples that
respond to electron capture but not
to the microcoulometric or electro-
lytic conductivity halogen detectors
or to the flame photometric detector, E
E Organophosphorus Pesticides - A
number of organophosphorus pesticides,
such as those containing a nitro group,
e.g., parathion, also respond to the
electron capture detector and may
interfere with the determination of
the organochlorine pesticides. Such
compounds can be identified by their
response to the flame photometric
detector, (4) jr
IV APPARATUS AND MATERIALS
A Gas Chromatograph - Equipped with
glass lined injection port,
B Detector Options: G
1 Electron Capture - Radioactive
(tritium or nickel 63)
H
2 Microcoulometric Titration
I
3 Electrolytic Conductivity
Recorder - Potentiometric strip
chart (10 in.) compatible with the
detector.
Gas Chromatographic Column Materials:
1 Tubing - Pyrex (180 cm long x
4 mm ID)
2 Glass Wool - Silanized
3 Solid Support - Gas - Chrom Q
(100-120 mesh)
4 Liquid Phases - Expressed as
weight percent coated on solid
support,
a OV-1, 3%
b OV- 210, 5%
c OV-17, 1.5% plus QF-1, 1.95%
d QF-1, 6% plus SE-30, 4%
Kuderna-Danish (K-D) Glassware (Kontes)
1 Snyder Column - three ball (macro)
and two ball (micro)
2 Evaporative Flasks - 500 ml
3 Receiver Ampuls - 10 ml, graduated
4 Ampul Stoppers
Chromatographic Column - Chromaflex
(400 mm long x 19 mm ID) with coarse
fritted plate on bottom and Teflon
stopcock; 250 ml reservoir bulb at top
of column with flared out funnel shape
at top of bulb - a special order
(Kontes K-420540-9011),
Chromatographic Column - pyrex
(approximately 400 mm long x 20 mm ID)
with coarse fritted plate on bottom.
Micro Syringes - 10, 25, 50 and 100
Separatory Funnels - 125 ml, 1000 ml
and 2000 ml with Teflon stopcock.
30-2
-------
Method for Organochlorine Pesticides
J Blender - High speed, glass or
stainless steel cup.
K Graduated cylinders - 100, 250
and 1000 ml.
L Florisil - PR Grade (60-100 mesh);
purchase activated at 1250 F and'
store in the dark in glass con-
tainers with glass stoppers or foil-
lined screw caps. Before use,
activate each batch overnight at
130 C in foil-covered glass con-
tainer, Determine lauric-acid
value (See Appendix I).
V REAGENTS, SOLVENTS, AND STANDARDS
VI
A Ferrous Sulfate - (ACS) 30% solution
in distilled water.
B Potassium Iodide - (ACS) 10% solu-
tion in distilled water.
C Sodium Chloride - (ACS) Saturated
solution in distilled water (pre-rinse
NaCl with hexane),
D Sodium Hydroxide - (ACS) 10 N in
distilled water.
E Sodium Sulfate - (ASC) Granular,
anhydrous (conditioned @ 400 C for
4 hours).
F Sulfuric Acid - (ASC) Mix equal
volumes of concentration 1 IgSO^
with distilled water.
G Diethyl Ether - Nanograde, re-
distilled in glass, if necessary,
1 Must contain 2% alcohol and be
free of peroxides by following
test: To 10 ml of ether in glass-
stoppered cylinder previously VII
rinsed with ether, add one ml of
freshly prepared 10% KI solution.
Shake and let stand one minute.
No yellow color should be ob-
served in either layer.
*May be ordered from US EPA, EMSL,
Quality Assurance Branch, Cincinnati,
Ohio 45268.
2 Decompose ether peroxides by
adding 40 g of 30% ferrous sulfate
solution to each liter of solvent.
CAUTION : Reaction may be
vigorous if the solvent contains a
high concentration of peroxides.
3 Distill deperoxidized ether in
glass and add 2% ethanol.
H Acetonitrile, Hexane, Methanol, Methy-
lene Chloride, Petroleum Ether
(boiling range 30-60 C) - nanograde,
redistill in glass if necessary.
I Pesticide Standards - Reference grade?
CA LIBRA TION
A Gas chromatographic operating con-
ditions are considered acceptable if
the response to dicapthon is at least
50% of full scale when < 0. 06 ng is
injected for electron capture detection
and 100 ng is injected for micro
coulonietric or electrolytic conductivity
detection. For all quantitative
measurements, the detector must be
operated within its linear response
range and the detector noise level
should be less than 2% of full scale,
B Standards are injected frequently as a
check on the stability of operating
conditions. Gas chromatograms of
several standard pesticides are shown
in Figures 1, 2, 3 and 4 and provide
reference operating conditions for the
four recommended columns.
C The elution order and retention ratios
of various organochlorine pesticides
are provided in Table 1, as a guide.
QUALITY CONTROL
A Duplicate and spiked sample analyses
are recommended as quality control
checks. When the routine occurrence
of a pesticide is being observed, the
use of quality control charts is
recommended. (5)
30-3
-------
Method for Organochlorine Pesticides
B Each time a set of samples is
extracted, a method blank is deter-
mined on a volume of distilled
water equivalent to that used to
dilute the sample.
VIII SAMPLE PREPARATION
A Blend the sample if suspended
matter is present and adjust pH
to near neutral (pH 6.5-7.5) with
50% sulfuric acid or 10 N sodium
hydroxide.
B For a sensitivity requirement of
Id g/'l, when using microcoulo-
metric or electrolytic conductivity
methods for detection, 100 ml or
more of sample will be required
for analysis. If interferences pose
no problem, the sensitivity of the
electron capture detector should
permit as little as 50 ml of sample
to be used. Background information
on the extent and nature of inter-
ferences will assist the analyst in
choosing the required sample size
and preferred detector.
C Quantitatively transfer the proper
aliquot into a two-liter separatory
funnel and dilute to one liter.
IX EXTRACTION
A Add 60 ml of 15% methylene chloride
in hexane (v:v) to the sample in the
separatory funnel and shake vigor-
ously for two minutes.
B Allow the mixed solvent to separate
from the sample, then draw the
water into a one-liter Erlenmeyer
flask. After prewctting 3-4 inches of
anhydrous sodium sulfate in a column,
pour the organic layer into a 100 ml
beaker, pass it through the column,
and collect it in a 500 ml K~D flask
equipped with a 10 ml ampul. Re-
turn the water phase to the separatory
funnel. Rinse the Erlenmeyer flask
with a second 60 ml volume of
solvent; add the solvent to the
separatory funnel and complete the
extraction procedure a second time.
Perform a third extraction in the
same manner.
C Concentrate the extract in the K-D
evaporator on a hot water bath.
D Analyze by gas chromatography unless
a need for cleanup is indicated (See
Section X).
CLEAN-UP AND SEPARATION PROCEDURES
A Interferences in the form of distinct
peaks and/or high background in the
initial gas chromatographic analysis,
as well as the physical characteristics
of the extract (color, cloudiness,
viscosity) and background knowledge of
the sample will indicate whether clean-
up is required. When these interfere
with measurement of the pesticides,
or affect column life or detector
sensitivity, proceed as directed below.
B Acetonitrile Partition - This procedure
is used to isolate fats and oils from
the sample extracts. It should be
noted that not all pesticides are quanti-
tatively recovered by this procedure.
The analyst must be aware of this and
demonstrate the efficiency of the par-
titioning for specific pesticides. Of the
pesticides listed in Scope I B only
mirex is not efficiently recovered.
1 Quantitatively transfer the previously
concentrated extract to a 125 ml
separatory funnel with enough hexane
to bring the final volume to 15 ml.
Extract the sample four times by
shaking vigorously for one minute
with 30 ml portions of hexane-
saturated acetonitrile.
2 Combine and transfer the acetonitrile
phases to a one-liter separatory
funnel and add 650 ml of distilled
water and 40 ml of saturated sodium
chloride solution. Mix thoroughly
for 30-45 seconds. Extract with
30-4
-------
Method for Organochlorine Pesticides
two 100 ml portions of hexane
by vigorously shaking about
15 seconds,
3 Combine the hexane extract in
a one-liter separatory funnel and
wash with two 100 ml portions
of distilled water. Discard the
water layer and pour the hexane
through a 3-4 inch anhydrous
sodium sulfate column into a
500 ml K-D flask equipped with
a 10 ml ampul. Rinse the
separatory funnel and column
with three 10 ml portions of
hexane.
4 Concentrate the extracts to 6-10
ml in the K-D evaporator in a
hot water bath.
5 Analyze by gas chromatography
unless a need for further cleanup
is indicated.
C Florisil Column Adsoprtion Chroma-
tography
1 Adjust the sample extract volume
to 10 ml,
2 Place a charge of activated
Florisil (weight determined by
lauric-acid value, see Appendix I)
in a Chromaflex column. After
settling the Florisil by tapping
the column, add about one-half
inch layer of anhydrous granular
sodium sulfate to the top,
3 Pre-elute the column, after cooling,
with 50-60 ml of petroleum ether.
Discard the eluate and just prior
to exposure of the sulfate layer
to air, quantitatively transfer the
sample extract into the column by
decantation and subsequent petro-
leum ether washings. Adjust the
elution rate to about 5 ml per
minute and, separately, collect
up to three eluates in 500 ml K-D
flasks equipped with 10 ml ampuls.
(See Eluate Composition d).
D
Perform the first elution with
200 ml of 6% ethyl ether in
petroleum ether, and the second
elution with 200 ml of 15% ethyl
ether in petroleum ether. Perform
the third elution with 200 ml of
50% ethyl ether - petroleum ether
and the fourth elution with 200 ml
of 100% ethyl ether.
4 Concentrate the eluates to 6-10 ml
in the K-D evaporator in a hot
water bath.
5 Analyze by gas chromatography.
Eluate Composition - By using an
equivalent quantity of any batch of
Florisil as determined by its lauric
acid value, the pesticides will be
separated into the eluates indicated
below;
Aldrin
BHC
Chlordane
DDD
DDE
6% Eluate
DDT
Heptachlor
Pentachloro-
nitrobenzene
Heptachlor Expoxide Strobane
Lindane
Methoxychlor
Mi rex
Toxaphene
Trifluralin
PCB's
5 0% Eluate
Endosulfan II
Captan
15% Eluate
Endosulfan I
Endrin
Dieldrin
Dichloran
Phthalate esters
Certain thiophosphate pesticides will
occur in each of the above fractions
as well as the 100% fraction. For
additional information regarding eluate
composition, refer to the FDA Pesti-
cide Analytical Manual. ^
30-5
-------
Method for Organochlorine Pesticides
XI CALCULATION OF RESULTS
A Determine the pesticide concentra-
tion by using the absolute calibra-
tion procedure described below or
the relative calibration procedure
described in Part I, Section
3.4. 2. (1)
(1) Micrograms/liter ~ (A) (B) (Vt)
(Vj)
-------
Method for Organochlorine Pesticides
Table 1
RETENTION RATIOS
OF VARIOUS
ORGANOCHLORINE
PESTICIDES
RELATIVE TO ALDRIN
Liquid Phase ^
1.5% OV-17 ¦
+
1.95% QF-1
5%
OV-210
3%
OV-1
6% QF-1
+
4% SHI-30
Column Temp.
200 C
180 C
180 C
2 00 C
A rgon/ Methane
Carrier Flow
60 ml/min
70 ml/min
70 ml/min
60 ml/min
Pesticide
RR
RR
RR
RR
Trifluralin
0.39
1. 11
0.33
0. 57
a -BHC
0. 54
0. 64
0.35
0.49
PCNB
0.68
0. 85
0. 49
0, 63
Lindane
0,69
0, 81
0. 44
0. 60
Dichloran
0.77
1. 29
0. 49
0.70
Heptachlor
0. 82
0.87
0. 78
0. 83
Aldrin
1. 00
1. 00
1. 00
1. 00
Heptachlor Epoxide
1.54
1. 93
1. 28
1.43
Endosulfan I
1.95
2.48
1.62
1. 79
p, p'-DDE -
2.23
2. 10
2. 00
1.82
Dieldrin
2.40
3. 00
1. 93
2. 12
Captain
2.59
4.09
1. 22
1.94
Endrin
2.93
3. 56
2. 18
2.42
o, p!- DDT
3. 16
2.70
2. 69
2. 39
p.p'-DDD
3.48
3. 75
2.61
2. 55
Endosulfan II
3. 59
4. 59
2. 25
2. 72
p, p'-DDT
4. 18
4. 07
3. 50
3. 12
Mirex
6. 1
3. 78
6. 6
4. 79
Methoxychlor
7. 6
6. 5
5. 7
4.60
Aldrin
(Min absolute)
3.5
2.6
4. 0
5.6
*A11 columns glass,
180 cm x 4 mm ID, solid support Gas-Chrom Q (100/120 mesh)
30-7
-------
to
0
1
CO
15 10
RETENTION TIME IN MINUTES
Figure 1
Column Packing: 1.5% OV-17 + 1.95% QF-1, Carrier Gas: Argon/Methane at BO ml/min,
Column Temperature: 200 C, Detector: Electron Capture.
-------
Method for Organochlorine Pesticides
RETENTION TIKI III MIMIITES
Figure 2. Column Packing.; 5% 0V-210, Carrier Gas: Areon/Melhane
at 70 ml/rnin, Column Temperature: 180 C, Detector:
Election Capture.
30-9
-------
to
0
1
E3-
O
a
20
15 10
RETENTION TIME IN MINUTES
figure 3. Column Packing: 6% QF 1 + 4% SE 30, Carrier Gas: Argon/Methane at 60 ml/min.
Column Temperature: 200 C, Detector: Electron Capture.
o
O
cm
SJ
o
o
IT
M
O
n
a>
w
a
CD
OT
-------
SETEKTIOK TIME IN KIHUTES
Column Packing: 3% OV 1, Carrier Gas: Argon/MBthare at 70 ml/min.
Column Temperature: 1BQC, Detector: Electron Capture.
-------
Method for Organoc hi ori ne Pesticides
appendix I
XIII STANDARDIZATION OF FLORISIL
COLUMN BY WEIGHT ADJUSTMENT
BASED ON ADSORPTION OF LA URIC
ACID
A A rapid method for determining adsorp-
tive capacity of Florisil is based on
adsorption of lauric acid from hexane
solution. An excess of lauric
acid is used and amount not adsorbed
is measured by alkali titration. Weight
of lauric acid adsorbed is used to cal-
culate, by simple proportion, equiva-
lent quantities of Florisil for batches
having different adsorptive capacities.
B Apparatus
1 Buret. — 25 ml with 1/10 ml
graduations.
2 Erlenmeyer flasks. — 125 ml
narrow mouth and 25 ml, glass
stoppered.
3 Pipet. — 10 and 20 ml transfer.
Add 50 ml neutralized ethyl alcohol
and 3 drops phenolphthalein indi-
cator; titrate to permanent end point.
Calculate mg lauric acid/ml 0.05 N
NaOH {about 10 mg/ml).
D Procedure
1 Transfer 2.000 g Florisil to 25 ml
glass stoppered Erlenmeryer flasks.
Cover loosely with aluminum foil
and heat overnight at 130°C. Stopper,
cool to room temperature, add 20.0
ml lauric acid solution (400 mg),
stopper, and shake occasionally for
15 min. Let adsorbent settle and
pipet 10. 0 ml of supernatant into
125 ml Erlenmeyer flask. Avoid
inclusion of any Florisil.
2 Add 50 ml neutral alcohol and 3 drops
indicator solution; titrate with 0.05N
to a permanent end point,
E Calculation of Lauric Acid Value and
Adjustment of Column Weight
4 Volumetric flasks. — 500 ml.
C Reagents and Solvents
1 Alcohol, ethyl. — USP or absolute,
neutralized to phenolphthalein.
2 Hexane. — Distilled from all glass
apparatus.
3 Lauric acid. — Purified, CP.
4 Lauric acid solution. — Transfer
10, 000 g lauric acid to 500 ml
volumetric flask, dissolve in hexane,
and dilute to 500 ml (1 ml = 20 mg).
5 Phenolphthalein Indicator, — Dissolve
1 g in alcohol and dilute to 100 ml.
6 Sodium hydroxide. — Dissolve 20 g
NaOH (pellets, reagent grade) in
water and dilute to 500 ml (IN).
Dilute 25 ml IN NaOH to 500~ml
with water (0, 05N). Standardize as
follows: Weigh 100-200 mg lauric
acid into 125 ml Erlenmeyer flask.
1 Calculate amount of lauric acid
adsorbed on Florisil as follows:
Lauric Acid value = mg lauric acid/g
Florisil = 200 - (ml required for
titration X mg lauric acid/ml
0. 05 N_ NaOH).
2 To obtain an equivalent quantity of
any batch of Florisil, divide 110
by lauric acid value for that batch
and multiply by 20 g. Verify proper
elution of pesticides by F.
F Test for Proper Elution Pattern and
Recovery of Pesticides: Prepare a test
mixture containing aldrin, heptachlor
epoxide, p. p'-DDE, dieldrin, Parathion
and malathion. Dieldrin and Fkrathion
should elute in the 15% eluate; all but
a trace of malathion in the 50% eluate
and the others in the 6% eluate.
30-12
-------
Method for Qrgariochl or i ne Pesticides
7 "Analysis of Pesticide Residues in Human
and Environmental Samples," U.S.
Environmental Protection Agency, Perrine
Primate Research Laboratories, Perrine,
Florida 33157, 1971,
8 Mills, P. A., "Variation of Florisil
REFERENCES
1 "Method for Organic Pesticides in Water
and Wastewater, " Environmental Pro-
tection Agency, National Environmental
Research Center, Cincinnati, Ohio
45268, 1971.
2 Monsanto Methodology for Aroclors -
Analysis of Environmental Materials for
Biphenyls, Analytical Chemistry Method
71-35, Monsanto Company, St. Louis,
Missouri 63166, 1970.
9
3 "Method for Poly chlorinated Biphenyls
in Industrial Effluents, " Environmental
Protection Agency, National Environmental
Research Center, Cincinnati, Ohio 45268,
1973.
4 "Method for Organophosphorus Pesticides
in Industrial Effluents, " Environmental io
Protection Agency, National Environmental
Research Center, Cincinnati, Ohio 45268,
1973.
5 "Handbook for Analytical Quality Control
in Water and Wastewater Laboratories, " ]
Chapter 6, Section 6.4, U.S. Environmental ,
Protection Agency, National Environmental ,
Research Center, Analytical Quality Con- ,
trol Laboratory, Cincinnati, Ohio 45268,
1973,
6 "Pesticide Analytical Manual, " U. S.
Department of Health, Education and
Welfare, Food and Drug Administration,
Washington, D. C,
Activity: Simple Method for Measuring
Adsorbent Capacity and its Use in Standard-
izing Florisil Columns, " Journal of the
Association of Official Analytical Chemists,
J51, 29 (1968).
Goerlitz, D.F, and Brown, E., "Methods
for Analysis of Organic Substances in
Water, " Techniques of Water Resources
Investigations of the United States Geological
Survey, Book 5, Chapter A3, U. S. Depart-
ment of the Interior, Geological Survey,
Washington, D, C, 20402, 1972, pp. 24-40.
i Steere, N.V., editor, "Handbook of Labor-
atory Safety, " Chemical Rubber Company,
18901 Cranwood Parkway, Cleveland, Ohio
44128, 1971, pp. 250-254.
Descriptors: Organic Compounds Pesticides,
Chlorinated Hydrocarbon Pesticides, Tech-
niqes Analytical, Chromatography, Gas
Chromatography
30-13
-------
Method for Organochlorlne Pesticides in Industrial Effluents
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30-14
------- |