EPA/6QQ/R-93/100
August 1993
METHODS FOR THE DETERMINATION
OF INORGANIC SUBSTANCES IN
ENVIRONMENTAL SAMPLES
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U\S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
Printed on Recycled Paper
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DISCLAIMER
This manual has been reviewed by the Environmental Monitoring Systems
Laboratory - Cincinnati, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
n
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts research
to:
o Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in marine and estuarine waters,
drinking waters, surface waters, groundwaters, wastewaters,
sediments, sludges, and solid wastes.
o Investigate methods for the identification and measurement of
viruses, bacteria and other microbiological organisms in aqueous
samples and to determine the responses of aquatic organisms to water
quality.
o Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants
in marine and estuarine waters, drinking water, surface water,
groundwater, wastewater, sediment and solid waste.
o Develop methods and models to detect and quantify responses in
aquatic and terrestrial organisms exposed to environmental stressors
and to correlate the exposure with effects on chemical and
biological indicators.
This EMSL-Cincinnati publication, "Methods for the Determination of
Inorganic Substances in Environmental Samples," was prepared as the
continuation of an initiative to gather together a compendium of standardized
laboratory analytical methods for the determination of inorganic substances in
water and wastewater. We are pleased to provide this manual and believe that
it will be of considerable value to many public and private laboratories
involved in inorganic analyses for regulatory or other reasons.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
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ABSTRACT
This manual contains ten updated and revised automated, semi-automated or
methods amenable to automation for the determination of a variety of inorganic
substances in water and wastewater.
These methods include and address, in an expanded form, information
concerning safety, quality control, pollution prevention, and waste
management. Methods were selected which minimize the amount of hazardous
reagents required and maximize sample throughput to allow expanded quality
control.
Automated methods are included for nitrate-nitrite, phosphorus, and
sulfate. Semi-automated methods cover cyanide, ammonia, total kjeldahl
nitrogen (TKN), chemical oxygen demand (COD) and generic phenolics. Methods
amenable to automation include turbidity and inorganic anions by ion
chromatography.
IV
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TABLE OF CONTENTS
Method
Number Title Revision Date Page
Disclaimer ii
Foreword lit
Abstract . iv
Acknowledgment. ........ vi
Introduction. ....... .... 1
180.1 Determination of Turbidity 2.0 8/93
by Nephelometry
300.0 Determination of Inorganic Anions 2.1 8/93
by Ion Chromatography
335.4 Determination of Total Cyanide 1.0 8/93
by Semi-Automated Colorimetry
350.1 Determination of Ammonia Nitrogen 2.0 8/93
by Semi-Automated Coloriimetry
351.2 Determination of Total Kjeldahl 2.0 8/93
Nitrogen by Semi-Automated
Colorimetry
353.2 Determination of Nitrate-Nitrite 2.0 8/93
by Automated Colorimetry
365.1 Determination of Phosphorus 2.0 8/93
by Automated Colorimetry
375.2 Determination of Sulfate 2.0 8/93
by Automated Colorimetry
410.4 Determination of Chemical Oxygen 2.0 8/93
Demand by Semi-Automated Colorimetry
420.4 Determination of Total Recoverable 1.0 8/93
Phenolics by Semi-Automated
Colorimetry
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ACKNOWLEDGMENTS
This methods manual was prepared and edited by the Inorganic Chemistry
Branch (ICB) of the Chemistry Research Division, Environmental Monitoring
Systems Laboratory - Cincinnati (EMSL-Cincinnati).
Major contributors from the ICB include John D. Pfaff for anions by ion
chromatography, Billy Potter for cyanide methodology, Theodore Martin and John
Creed for quality control, and Diane Schirmann for manuscript production.
James O'Dell selected the methods that are included from previous versions
published in "Methods for the Chemical Analysis of Water and Wastes," EPA
600/4-79-020, Revised March 1983. He reorganized the previous versions into a
format approved by the Environmental Monitoring Management Council and added
new sections to address safety, quality control, pollution prevention, and
waste management. Last but not least, a very special acknowledgement goes to
former ICB member Morris E. Gales who started this project before his
retirement and was responsible for most of the original versions of these
methods. Thanks again Mo for your years of dedicated service to our
environment, the USEPA, and your fellow employees.
VI
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INTRODUCTION
The original version of this manual was issued in November 1969 by the
Federal Water Pollution Control Administration as "FWPCA Methods for Chemical
Analysis of Water and Wastes." With the creation of the United States
Environmental Protection Agency (USEPA) came "Methods for Chemical Analysis of
Water and Wastes 1971" Publication No. 1602007/71. The second edition was
issued in 1974 as EPA 625/6-74-003, and the third edition in 1979 as EPA
600/4/79-020. The current version, an updated second printing of the third
edition, was revised and issued in March 1983. The methods contained in the
1983 manual form the basis for most of the methodology approved for compliance
monitoring of inorganic parameters specified under the Clean Water Act (NPDES)
and contaminants regulated under the Safe Drinking Water Act.
I
In 1991, a number of new and revised metals methods were incorporated
into a new publication entitled, "Methods for the Determination of Metals in
Environmental Samples." Concurrently, the decision was made to revise and
update selected non-metal methods to be issued under the name "Methods for the
Determination of Inorganic Substances in Environmental Samples."
For both the metals and non-metals manuals, several important features
were adopted:
Consistent use of terminology, a feature especially helpful in the
quality control sections where standardized terminology is not yet
available. The terms were carefully selected to be meaningful without
extensive definition, and therefore should be easy to understand and use.
New sections are included with expanded useful coverage of safety,
quality control, pollution prevention and waste management.
All methods are presented in the new EPA standard Environmental
Monitoring Management Council (EMMC) format.
Although a number of other methods included the 1983 edition of USEPA
"Methods for Chemical Analysis of Water and Wastes", Standard Methods for the
Examination of Water and Wastewater, and American Society for Testing and
Materials Annual Book of Standards (ASTM) are acceptable for compliance
monitoring, the revised methods contained in this publication are considered
to be the most useful in terms of future regulatory requirements. They
represent a selection of air segmented automated, semi-automated, or amenable
to automation methodology that provides the following advantages over their
manual counterparts.
Higher sample throughput for faster analysis and improved precision.
Faster analysis allows more time to perform the updated quality control
required to insure valid results.
Lower per analysis reagent consumption to reduce waste production and
minimize disposal costs.
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The inclusion of multi-laboratory data generated from USEPA performance
evaluation studies.
The following methods are included with specific features and improvements:
A revised version of EPA turbidity Method 180.1 that minimizes the direct
use of hydrazine sulfate.
An updated version of EPA Method 300.0 for anions by ion chromatography.
A new stand alone semi-automated revision of EPA cyanide Method 335.2
that specifies the use of the downsized midi-distillation procedure.
A new semi-automated version of the EPA phenolics Method 420.2.
The optional use of a non-mercury catalyst in EPA TKN Method 351.2.
All methods allow the optional use of reduced reagent and distillation-
digestion volumes.
Most of the methods include the option of limited performance-based
modifications or improvements.
James W. O'Dell, John D. Pfaff, William L. Budde
Chemistry Research Division
August 1993
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METHOD 180.1
DETERMINATION OF TURBIDITY BY NEPHELOMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
180.1-1
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METHOD 180.1
H
DETERMINATION OF TURBIDITY BY NEPHELOMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of turbidity in drinking,
ground, surface, and saline waters, domestic and industrial wastes.
1.2 The applicable range is 0 to 40 nephelometric turbidity units (NTU).
Higher values may be obtained with dilution of the sample.
2.0 SUMMARY OF METHOD
2.1 The method is based upon a comparison of the intensity of light
scattered by the sample under defined conditions with the intensity
of light scattered by a standard reference suspension. The higher
the intensity of scattered light, the higher the turbidity.
Readings, in NTU's, are made in a nephelometer designed according to
specifications given in sections 6.1 and 6.2. A primary standard
suspension is used to calibrate the instrument. A secondary
standard suspension is used as a daily calibration check and is
monitored periodically for deterioration using one of the primary
standards.
2.1.1 Formazin polymer is used as a primary turbidity suspension
for water because it is more reproducible than other types of
standards previously used for turbidity analysis.
2.1.2 A commercially available polymer primary standard is also
approved for use for the National Interim Primary Drinking
Water Regulations. This standard is identified as AMCO-AEPA-
1, available from Advanced Polymer Systems.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogates analytes.
3.2 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.3 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
180.1-2
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present in the laboratory environment, the reagents, or the
apparatus.
3.4 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.5 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.6 PRIMARY CALIBRATION STANDARD (PCAL) A suspension prepared from
the primary dilution stock standard suspension. The PCAL
suspensions are used to calibrate the instrument response with
respect to analyte concentration.
3.7 QUALITY CONTROL SAMPLE (QCS) A solution of the method analyte of
known concentrations that is used to fortify an aliquot of LRB
matrix. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance.
3.8 SECONDARY CALIBRATION STANDARDS (SCAL) « Commercially prepared,
stabilized sealed liquid or gel turbidity standards calibrated
against properly prepared and diluted formazin or styrene
divinyl benzene polymers.
3.9 STOCK STANDARD SUSPENSION (SSS) A concentrated suspension
containing the analyte prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
Stock standard suspension is used to prepare calibration suspensions
and other needed suspensions.
4.0 INTERFERENCES
4.1 The presence of floating debris and coarse sediments which settle
out rapidly will give low readings. Finely divided air bubbles can
cause high readings.
4.2 The presence of true color, that is the color of water which is due
to dissolved substances that absorb light, will cause turbidities to
be low, although this effect is generally not significant with
drinking waters.
4.3 Light absorbing materials such as activated carbon in significant
concentrations can cause low readings.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been fully established. Each chemical should be regarded as
a potential health hazard and exposure should be as low as
reasonably achievable.
180.1-3
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5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 Hydrazine Sulfate (7.2.1) is a carcinogen. It is highly toxic and
may be fatal if inhaled, swallowed, or absorbed through the skin.
Formazin can contain residual hydrazine sulfate. Proper protection
should be employed.
6.0 EQUIPMENT AND SUPPLIES
6.1 The turbidimeter shall consist of a nephelometer, with light source
for illuminating the sample, and one or more photo-electric
detectors with a readout device to indicate the intensity of light
scattered at right angles to the path of the incident light. The
turbidimeter should be designed so that little stray light reaches
the detector in the absence of turbidity and should be free from
significant drift after a short warm-up period.
6.2 Differences in physical design of turbidimeters will cause
differences in measured values for turbidity, even though the same
suspension is used for calibration. To minimize such differences,
the following design criteria should be observed:
6.2.1 Light source: Tungsten lamp operated at a color temperature
between 2200-3000°K.
6.2.2 Distance traversed by incident light and scattered light
within the sample tube: Total not to exceed 10 cm.
6.2.3 Detector: Centered at 90° to the incident light path and not
to exceed ± 30° from 90°. The detector, and filter system if
used, shall have a spectral peak response between 400 and 600
nm.
6.3 The sensitivity of the instrument should permit detection of a
turbidity difference of 0.02 NTU or less in waters having
turbidities less than 1 unit. The instrument should measure from 0
to 40 units turbidity. Several ranges may be necessary to obtain
both adequate coverage and sufficient sensitivity for low
turbidities.
6.4 The sample tubes to be used with the available instrument must be of
clear, colorless glass or plastic. They should be kept scrupulously
clean, both inside and out, and discarded when they become scratched
or etched. A light coating of silicon oil may be used to mask minor
imperfections in glass tubes. They must not be handled at all where
the light strikes them, but should be provided with sufficient extra
length, or with a protective case, so that they may be handled.
180.1-4
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, Tubes should be checked, indexed and read at the orientation that
produces the lowest background blank value.
6.5 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.6 Glassware Class A volumetric flasks and pi pets as required.
7.0 REAGENTS AND STANDARDS
7.1 Reagent water, turbidity-free: Pass deionized distilled water
through a 0.45/a pore size membrane filter, if such filtered water
shows a lower turbidity than unfiltered distilled water.
7.2 Stock standard suspension (Formazin):
7.2.1 Dissolve 1.00 g hydrazine sulfate, (WU2.H2S04, (CASRN 10034-
93-2) in reagent water and dilute to 100 ml in a volumetric
flask. CAUTIONCARCINOGEN
7.2.2 Dissolve 10.00 g hexamethylenetetramine (CASRN 100-97-0) in
reagent water and dilute to 100 ml in a volumetric flask. In
a 100 ml volumetric flask, mix 5.0 ml of each solution (7.2.1
+ 7.2.2). Allow to stand 24 hours at 25 ± 3°C, then dilute
to the mark with reagent water.
7.3 Primary calibration standards: Mix and dilute 10.00 ml of stock
standard suspension (7.2) to 100 ml with reagent water. The
turbidity of this suspension is defined as 40 NTU. For other
values, mix and dilute portions of this suspension as required.
7.3.1 A new stock standard suspension (7.2) should be prepared each
month. Primary calibration standards (7.3) should be
prepared daily by dilution of the stock standard suspension.
7.4 Formazin in commercially prepared primary concentrated stock
standard suspension (SSS) may be diluted and used as required.
Dilute turbidity standards should be prepared daily.
7.5 AMCO-AEPA-1 Styrene Divinyl benzene polymer primary standards are
available for specific instruments and require no preparation or
dilution prior to use.
7.6 Secondary standards may be acceptable as a daily calibration check,
but must be monitored on a routine basis for deterioration and
replaced as required.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with turbidity free
water. Volume collected should be sufficient to insure a
180.1-5
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representative sample, allow for replicate analysis (if required),
and minimize waste disposal.
8.2 No chemical preservation is required. Cool sample to 4°C.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, samples maintained at 4°C may be held for up to
48 h.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory capability
and analysis of laboratory reagent blanks and other solutions as a
continuing check on performance. The laboratory is required to
maintain performance records that define the quality of data
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE.
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS).
9.2.2 Linear Calibration Range (LCR) -- The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analysis of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The source
of the problem must be identified and corrected before
continuing with on-going analyses.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must analyze
at least one LRB with each batch of samples. Data produced
180.1-6
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are used to assess contamination from the laboratory
environment.
9.3.2 Instrument Performance Check Solution (IPC) For all
determinations, the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required) and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following the
last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data. NOTE:
Secondary calibration standards (SS) may also be used as the
1 i U
9.3.3 Where additional reference materials such as Performance
Evaluation samples are available, they should be analyzed to
provide additional performance data. The analysis of
reference samples is a valuable tool for demonstrating the
ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10.1 Turbidimeter calibration: The manufacturer's operating instructions
should be followed. Measure standards on the turbidimeter covering
the range of interest. If the instrument is already calibrated in
standard turbidity units, this procedure will check the accuracy of
the calibration scales. At least one standard should be run in each
instrument range to be used. Some instruments permit adjustments of
sensitivity so that scale values will correspond to turbidities.
Solid standards, such as those made of lucite blocks, should never
be used due to potential calibration changes caused by surface
scratches. If a pre-calibrated scale is not supplied, calibration
curves should be prepared for each range of the instrument.
11.0 PROCEDURE
11.1 Turbidities less than 40 units: If possible, allow samples to come
to room temperature before analysis. Mix the sample to thoroughly
disperse the solids. Wait until air bubbles disappear then pour the
sample into the turbidimeter tube. Read the turbidity directly from
the instrument scale or from the appropriate calibration curve.
180.1-7
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11 2 Turbidities exceeding 40 units: Dilute the sample with one or more
volumes of turbidity- free water until the turbidity falls below 40
units The turbidity of the original sample is then computed from
the turbidity of the diluted sample and the dilution factor. For
example if 5 volumes of turbidity- free water were added to 1 volume
of sample, and the diluted sample showed a turbidity of 30 units,
then the turbidity of the original sample was 180 units,
11.2.1 Some turbidimeters are equipped with several separate scales.
The higher scales are to be used only as indicators of
required dilution volumes to reduce readings to less than 40
NTU.
NOTE 1: Comparative work performed in the Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati)
indicates a progressive error on sample turbidities in excess
of 40 units.
12.0 DATA ANALYSTS AND CALCULATIONS
12.1 Multiply sample readings by appropriate dilution to obtain final
reading.
12.2 Report results as follows:
0.0 - 1.0
1-10
10-40
40 - 100
100 - 400
400 - 1000
> 1000
13.0 METHOD PERFORMANCE
Record to Nearest:
O-05
0.1
5
1°
50
100
13.1 In a single laboratory (EMSL-Cincinnati), using surface water
samples at levels of 26, 41, 75 and 180 NTU, the standard deviations
were ± 0.60, ± 0.94, ± 1.2 and ± 4.7 units, respectively.
13.2 The interlaboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in NTU.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
180.1-8
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Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management .for Waste Reduction," available from
the American Chemical Society's Department of Government Regulations
and Science Policy, 1155 16th Street N.W., Washington D.C. 20036,
(202)872-4477.
15.0 WASTE MANAGEMENT
15.1 The U.S. Environmental Protection Agency requires that laboratory
waste management practices be conducted consistent with all
applicable rules and regulations. Excess reagents, samples and
method process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water and land by minimizing and controlling all releases from
hoods, and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Annual Book of ASTM Standards, Volume 11.01 Water (1), Standard
D1889-88A, p. 359, (1993).
2. Standard Methods for the Examination of Water and Wastewater, 18th
Edition, pp. 2-9, Method 2130B, (1992).
180.1-9
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17.0 TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
373
374
289
482
484
489
640
487
288
714
641
TRUE
VALUE
(T)
0.450
0.600
0.65
0.910
0.910
1.00
1.36
3.40
4.8
5.60
5.95
MEAN
(X)
0.4864
0.6026
0.6931
0.9244
0.9919
0.9405
1.3456
3.2616
4.5684
5.6984
5.6026
RESIDUAL
FOR X
0.0027
-0.0244
0.0183
0.0013
0.0688
-0.0686
-0.0074
-0.0401
-0.0706
0.2952
-0.1350
STANDARD
DEVIATION
(S)
0.1071
0.1048
0.1301
0.2512
0.1486
0.1318
0.1894
0.3219
0.3776
0.4411
0.4122
RESIDUAL
FOR S
-0.0078
-0.0211
0.0005
0.1024
-0.0002
-0.0236
0.0075
-0.0103
-0.0577
-0.0531
-0.1078
REGRESSIONS: X = 0.955T + 0.54, S = 0.074T + 0.082
180.1-10
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METHOD 300.0
DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
John D. Pfaff
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.1
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
300.0-1
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METHOD 300.0
DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of the following inorganic
anions:
PART A.
Bromide
Chloride
Fluoride
Nitrate
PART B.
Bromate
Chlorate
Nitrite
Ortho-Phosphate-P
Sulfate
Chlorite
1.2 The matrices applicable to each method are shown below:
A. Drinking water, surface water, mixed domestic and industrial
wastewaters, groundwater, reagent waters, solids (after
extraction 11.7), leachates (when no acetic acid is used).
B. Drinking water and reagent waters
1.3 The single laboratory Method Detection Limit (MDL defined in Sect.
3.2) for the above analytes is listed in Tables 1A and IB. The MDL
for a specific matrix may differ from those listed, depending upon
the nature of the sample.
1.4 Method A is recommended for drinking and wastewaters. The
multilaboratory ranges tested for each anion are as follows:
Analyte
Bromide
Chloride
Fluoride
Nitrate-N
Nitrite-N
Ortho-Phosphate-P
Sulfate
0.63
0.78
0.26
0.42
0.36
0.69
2.85
- 21.0
- 26.0
- 8.49
- 14.0
- 12.0
- 23.1
- 95.0
300.0-2
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1.5 This method is recommended for use only by or under the supervision
of analysts experienced in the use of ion chromatography and in the
interpretation of the resulting ion chromatograms.
1.6 When this method is used to analyze unfamiliar samples for any of
the above anions, anion identification should be supported by the
use of a fortified sample matrix covering the anions of interest.
The fortification procedure is described in Sect. 11.6.
1.7 Users of the method data should state the data-quality objectives
prior to analysis. Users of the method must demonstrate the ability
to generate acceptable results with this method, using the
procedures described in Sect. 9.0.
2.0 SUMMARY OF METHOD
2.1 A small volume of sample, typically 2 to 3 ml, is introduced into
an ion chromatograph. The anions of interest are separated and
measured, using a system comprised of a guard column, analytical
column, suppressor device, and conductivity detector.
2.2 The main differences between Parts A and B are the separator columns
and guard columns. Sections 6.0 ,and 7,0 will elicit the
differences.
2.3 An extraction procedure must be performed to use this method for
solids (See 11.7).
2.4 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 FIELD DUPLICATES (FD) Two separate samples collected at the same
time and place under identical circumstances and treated exactly the
same throughout field and laboratory procedures. Analyses of field
duplicates indicate the precision associated with sample collection
preservation and storage, as well as with laboratory procedures.
3.4 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) - A solution of one or
more method analytes, surrogates, internal standards, or other test
300.0-3
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substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.5 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.7 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.8 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.9 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.10 METHOD DETECTION LIMIT (MDL) The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.11 PERFORMANCE EVALUATION SAMPLE (PE) A solution of method analytes
distributed by the Quality Assurance Research Division (QARD),
Environmental Monitoring Systems Laboratory (EMSL-Cincinnati), U. S.
Environmental Protection Agency, Cincinnati, Ohio, to multiple
laboratories for analysis. A volume of the solution is added to a
known volume of reagent water and analyzed with procedures used for
samples. Results of analyses are used by QARD to determine
statistically the accuracy and precision that can be expected when a
method is performed by a competent analyst. Analyte true values are
unknown to the analyst.
3.12 QUALITY CONTROL SAMPLE (QCS) A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
300.0-4
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sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.13 STOCK STANDARD SOLUTION (SSS) A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
4.0 INTERFERENCES
4.1 Interferences can be caused by substances with retention times that
are similar to and overlap those of the anion of interest. Large
amounts of an anion can interfere with the peak resolution of an
adjacent anion. Sample dilution and/or fortification can be, used to
solve most interference problems associated with retention times.
4.2 The water dip or negative peak that elutes near, and can interfere
with, the fluoride peak can usually be eliminated by the addition of
the equivalent of 1 mL of concentrated eluent (7.3 100X) to 100 mL
of each standard and sample.
4.3 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that lead to discrete artifacts or elevated baseline in ion
chromatograms.
4.4 Samples that contain particles larger than 0.45 microns and reagent
solutions that contain particles "larger than 0.20 microns require
filtration to prevent damage to instrument columns and flow systems.
4.5 Any anion that is not retained by the column or only slightly
retained will elute in the area of fluoride and interfere. Known
coelution is caused by carbonate and other small organic anions. At
concentrations of fluoride above 1.5 mg/L, this interference may not
be significant, however, it is the responsibility of the user to
generate precision and accuracy information in each sample matrix.
4.6 The acetate anion elutes early during the chromatographic run. The
retention times of the anions also seem to differ when large amounts
of acetate are present. Therefore, this method is not recommended
for leachates of solid samples when acetic acid is used for pH
adjustment.
4.7 The quantitation of unretained peaks should be avoided, such as low
molecular weight organic acids (formate, acetate, propionate etc.)
which are conductive and coelute with or near fluoride and would
bias the fluoride quantitation in some drinking and most waste
waters.
4.8 Any residual chlorine dioxide present in the sample will result in
the formation of additional chlorite prior to analysis. If any
300.0-5
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concentration of chlorine dioxide is suspected in the sample purge
the sample with an inert gas (argon or nitrogen) for about five
minutes or until no chlorine dioxide remains.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Sulfuric acid (7.4)
6.0 Equipment and Supplies
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Ion chromatograph Analytical system complete with ion chromato-
graph and all required accessories including syringes, analytical
columns, compressed gasses and detectors.
6.2.1 Anion guard column: A protector of the separator column. If
omitted from the system the retention times will be shorter.
Usually packed with a substrate the same as that in the
separator column.
6.2.2 Anion separator column: This column produces the separation
shown in Figures 1 and 2.
6.2.2.1 Anion analytical column (Method A): The
separation shown in Figure 1 was generated using a
Dionex AS4A column (P/N 37041). An optional
column may be used if comparable resolution of
peaks is obtained, and the requirements of Sect.
9.2 can be met.
6.2.2.2 Anion analytical column (Method B). The
separation shown in Figure 2 was generated using a
Dionex AS9 column (P/N 42025). An optional column
may be used if comparable resolution of peaks is
300.0-6
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obtained and the requirements of Sect. 9.2 can be
met.
6.2.3 Anion suppressor device: The data presented in this method
were generated using a Dionex anion micro membrane
suppressor (P/N 37106).
6.2.4 Detector Conductivity cell: approximately 1.25 /zL
internal volume, (Dionex, or equivalent) capable of
providing data as required in Sect. 9.2.
6.3 The Dionex AI-450 Data Chromatography Software was used to generate
all the data in the attached tables. Systems using a stripchart
recorder and integrator or other computer based data system may
achieve approximately the same MDL's but the user should demonstrate
this by the procedure outlined in Sect. 9.2.
7.0 Reagents and Standards
7.1 Sample bottles: Glass or polyethylene of sufficient volume to
allow replicate analyses of anions of interest.
7.2 Reagent water: Distilled or deionized water, free of the anions of
interest. Water should contain particles no larger than 0.20
microns.
7.3 Eluent solution (Method A and Method B): Sodium bicarbonate (CASRN
144-55-8) 1.7 mM, sodium carbonate (CASRN 497-19-8) 1.8 mM.
Dissolve 0.2856 g sodium bicarbonate (NaHCO,) and 0.3816 g of sodium
carbonate (Na2C03) in reagent water (7.2) and dilute to 2 L.
7.4 Regeneration solution (micro membrane suppressor): Sulfuric acid
(CASRN-7664-93-9) 0.025N. Dilute 2.8 ml cone, sulfuric acid
(H2S04) to 4 L with reagent water.
7.5 Stock standard solutions, 1000 mg/L (1 mg/mL): Stock standard
solutions may be purchased as certified solutions or prepared from
ACS reagent grade materials (dried at 105°C for 30 min) as listed
below.
7.5.1 Bromide (Br~) 1000 mg/L: Dissolve 1.2876 g sodium bromide
(NaBr, CASRN 7647-15-6) in reagent water and dilute to 1 L.
7.5.2 Bromate (Br03') 1000 mg/L: Dissolve 1.1798g of sodium
bromate (NaBr03, CASRN 7789-38-0) in reagent water and
dilute to 1 L.
7.5.3 Chlorate (C103~) 1000 mg/L: Dissolve 1.2753g of sodium
chlorate (NaC103, CASRN 7775-09-9) in reagent water and
dilute to 1 L.
300.0-7
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7.5.4 Chloride (CL~) 1000 mg/L: Dissolve 1.6485 g sodium
chloride (NaCl, CASRN 7647-14-5) in reagent water and
dilute to 1 L.
7.5.5 Chlorite (C102") 1000 mg/L: Dissolve 1.3410g of sodium
chlorite (NaC102, CASRN 7758-19-2) in reagent water and
dilute to 1 L.
7.5.6 Fluoride (F") 1000 mg/L: Dissolve 2.2100g sodium fluoride
(NaF, CASRN 7681-49-4) in reagent water and dilute to 1 L.
7.5.7 Nitrate (NO",-N) 1000 mg/L: Dissolve 6.0679 g sodium
nitrate (NaN03, CASRN 7631-99-4) in reagent water and
dilute to 1 L.
7.5.8 Nitrite (NO~,-N) 1000 mg/L: Dissolve 4.9257 g sodium
nitrite (NaN02, CASRN 7632-00-0) in reagent water and
dilute to 1 L.
7.5.9 Phosphate (PO=,-P) 1000 mg/L: Dissolve 4.3937 g potassium
phosphate (KH2PO,, CASRN 7778-77-0) in reagent water
and dilute to 1 L.
7.5.10 Sulfate (S04=) 1000 mg/L: Dissolve 1.8141 g potassium
sulfate (K,S04, CASRN 7778-80-5) in reagent water and
dilute to 1 L.
NOTE: Stability of standards: Stock standards (7.5) are
stable for at least 1 month when stored at 4°C.
Except for the chlorite standard which is only stable
for two weeks. Dilute working standards should be
prepared weekly, except those that contain nitrite
and phosphate should be prepared fresh daily.
7.6 Ethylenediamine preservation solution: Dilute 10 mL of
ethylenediamine (99%) (CASRN 107-15-3) to 200 mL with reagent
water. Use 1 mL of this dilution to each 1 L of sample taken.
8.0 Sample Collection. Preservation and Storage
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis, if required, and ;minimize
waste disposal.
8.2 Sample preservation and holding times for the anions that can be
determined by this method are as follows:
Analvte Preservation Holding Time
Bromate None required 28 days
300.0-8
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Bromide None required 28 days
Chlorate None required 28 days
Chloride None required 28 days
Chlorite Cool to 4°C immediately
Fluoride None required 28 days
Nitrate-N Cool to 4°C 48 hours
Combined cone. H2SO, 28 days
(Nitrate/Nitrite) to a pH < 2
Nitrite-N Cool to 4°C 48 hours
0-Phosphate-P Cool to 4°C 48 hours
Sulfate Cool to 4°C 28 days
NOTE: If the determined value for the combined
nitrate/nitrite exceeds 0.5 mg/L as N", a resample
must be analyzed for the individual concentrations
of nitrate and nitrite.
8.3 The method of preservation and the holding time for samples
analyzed by this method are determined by the anions of interest.
In a given sample, the anion that requires the most preservation
treatment and the shortest holding time will determine the preser-
vation treatment. It is recommended that all samples be cooled to
4°C and held for no longer than 28 days for Method A and analyzed
immediately in Method B.
NOTE: If the sample cannot be analyzed for chlorite within < 10
minutes, the sample may be preserved by adding 1 ml of the
ethylenediamine (EDA) preservation solution (7.6) to 1 L
of sample. This will preserve the concentration of the
chlorite for up to 14 days. This addition of EDA has no
effect on bromate or chlorate, so they can also be
determined in a sample preserved with EDA. Residual
chlorine dioxide should be removed from the sample
(per 4.8) prior to the addition of EDA.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
300.0-9
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(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
300.0-10
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9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
establish an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required) and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift, the instrument recalibrated. All samples following
300.0-11
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the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.1.1 If the concentration of fortification is less than
25% of the background concentration of the matrix
the matrix recovery should not be calculated.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculated using the
following equation:
II
C. - C
X 100
where, R ~ percent recovery.
Cs = fortified sample concentration.
C - sample background concentration.
s » concentration equivalent of analyte added to
sample.
9.4.3 Until sufficient data becomes available (usually a minimum
of 20 to 30 analysis), assess laboratory performance against
recovery limits for method A of 80 to 120% and 75 to 125%
for method B. When sufficient internal performance data
becomes available develop control limits from percent mean
recovery and the standard deviation of the mean recovery.
9.4.4 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.5 Where reference materials are available, they should be
analyzed to provide additional performance data. The
300.0-12
4»
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analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
9.4.6 In recognition of the rapid advances occurring in chromatog-
raphy, the analyst is permitted certain options, such as the
use of different columns and/or eluents, to improve the
separations or lower the cost of measurements. Each time
: such modifications to the method are made, the analyst is
required to repeat the procedure in Sect. 9.2.
9.4.7 It is recommended that the laboratory adopt additional
: . quality assurance practices for use with this method. The
specific practices that are most productive depend upon the
needs of the laboratory and the nature of the samples.
Field duplicates may be analyzed to monitor the precision of
the sampling technique. When doubt exists over the
identification of a peak in the chromatogram, confirmatory
techniques such as sample dilution and fortification, must
be used. Whenever possible, the laboratory should perform
analysis of quality control check samples and participate in
relevant performance evaluation sample studies.
9.4.8 At least quarterly, replicates of LFBs should be analyzed to
determine the precision of the laboratory measurements. Add
these results to the on-going control charts to document
data quality.
9.4.9 When using Part B, the analyst should be aware of the purity
of the reagents used to prepare standards. Allowances must
be made when the solid materials are less than 99% pure.
10.0 Calibration and Standardization
10.1 Establish ion chromatographic operating parameters equivalent to
those indicated in Tables 1A or IB.
,10.2 For each analyte of interest, prepare calibration standards at a
minimum of three concentration levels and a blank by adding
: accurately measured volumes of one or more stock standards (7.5) to
a volumetric flask and diluting to volume with reagent water. If
a sample analyte concentration exceeds the calibration range the
sample may be diluted to fall within the'range. If this is not
possible then three new calibration concentrations must be chosen,
two of which must bracket the concentration of the sample analyte of
interest. Each attenuation range of the instrument used to analyze
a sample must be calibrated individually.
10.3 Using injections of 0.1 to 1.0 ml (determined by injection loop
volume) of each calibration standard, tabulate peak height or area
responses against the concentration. The results are used to
prepare a calibration curve for each analyte. During this pro-
cedure, retention times must be recorded.
300.0-13
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10.4 The calibration curve must be verified on each working day, or
whenever the anion eluent is changed, and after every 20
samples. If the response or retention time for any analyte varies
from the expected values by more than ± 10%, the test must be
repeated, using fresh calibration standards. If the results are
still more than ± 10%, a new calibration curve must be prepared
for that analyte.
10.5 Nonlinear response can result when the separator column capacity is
exceeded (overloading). The response of the detector to the sample
when diluted 1:1, and when not diluted, should be compared. If the
calculated responses are the same, samples of this total anionic
concentration need not be diluted.
11.0 Procedure
11.1 Tables 1A and IB summarize the recommended operating conditions for
the ion chromatograph. Included in these tables are estimated
retention times that can be achieved by this method. Other columns,
chromatographic conditions, or detectors may be used if the
requirements of Sect. 9.2 are met.
11.2 Check system calibration daily and, if required, recalibrate as
described in Sect. 10.
11.3 Load and inject a fixed amount of well mixed sample. Flush
injection loop thoroughly, using each new sample. Use the same size
loop for standards and samples. Record the resulting peak size in
area or peak height units. An automated constant volume injection
system may also be used.
11.4 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for each analyte. However, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
11.5 If the response for the peak exceeds the working range of the
system, dilute the sample with an appropriate amount of reagent
water and reanalyze.
11.6 If the resulting chromatogram fails to produce adequate resolution,
or if identification of specific anions is questionable, fortify the
sample with an appropriate amount of standard and reanalyze.
NOTE: Retention time is inversely proportional to concentration.
Nitrate and sulfate exhibit the greatest amount of change,
although all anions are affected to some degree. In some
cases this peak migration may produce poor resolution or
identification.
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11.7 The following extraction should be used for solid materials. Add an
amount of reagent water equal to ten times the weight of dry solid
material taken as a sample. This slurry is mixed for ten minutes
using a magnetic stirring device., Filter the resulting slurry
before injecting using a 0.45 /z membrane type filter. This can be
the type that attaches directly to the end of the syringe. Care
should be taken to show that good recovery and identification of
peaks is obtained with the user's matrix through the use of
fortified samples.
11.8 It has been reported that lower detection limits for bromate
(«7 /jg/L) can be obtained using a. borate based eluent(7). The use
of this eluent or other eluents that improve method performance may
be considered as a minor modification of the method and as such
still are acceptable.
11.9 Should more complete resolution be needed between peaks the eluent
(7.3) can be diluted. This will spread out the run but will also
cause the later eluting anions to be retained longer. The analyst
must determine to what extent the eluent is diluted. This dilution
should not be considered a deviation from the method.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve for each analyte by plotting instrument
response against standard concentration. Compute sample
concentration by comparing sample response with the standard curve
Multiply answer by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg/L.
12.4 Report NO ~ as N
NO ' as N
HP04= as P
13.0 METHODS PERFORMANCE
13.1 Tables 1A and 2A give the single laboratory (EMSL-Cincinnati) MDL
for each anion included in the method under the conditions listed.
13.2 Tables 2A and 2B give the single laboratory (EMSL-Cincinnati)
standard deviation for each anion included in the method in a
variety of waters for the listed conditions.
13.3 Multiple laboratory accuracy and bias data (St) and estimated single
operator values (S0) for reagent, drinking and waste water using
300.0-15
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method A are given for each anion in Tables 3 through 9.
19 laboratories were used for this data.
Data from
13.4 Some of the bias statements, for example chloride and sulfate, may
be misleading due to spiking small increments of the anion into
large naturally occurring concentrations of the same anion.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 Quantity of the chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess reagents, samples and method
process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal
restrictions. For further information on waste management consult
the "Waste Management Manual for Laboratory Personnel," available
from the American Chemical Society at the address listed in Sect.
14.3.
300.0-16
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16.0 REFERENCES -f
1. "Determination of Inorganic Disinfection By-Products by Ion
Chromatography", J. Pfaff, C. Brockhoff. J. Am. Water Works Assoc.,
Vol 82, No. 4, pg 192.
2. Standard Methods for the Examination of Water and Wastewater,
Method 4110B, "Anions by Ion Chromatography", 18th Edition of
Standard Methods (1992).
3. Dionex, System 4000 Operation and Maintenance Manual, Dionex
Corp., Sunnyvale, California 94086, 1988.
4. Method Detection Limit (MDL) as described in "Trace Analyses for
Wastewater," J. Glaser, D. Foerst, G. McKee, S. Quave, W. Budde,
Environmental Science and Technology, Vol. 15, Number 12, page
1426, December, 1981.
5. American Society for Testing and Materials. Test Method for Anions
in Water by Chemically-Suppressed Ion Chromatography D4327-91.
Annual Book of Standards, Vol 11.01 (1993).
6. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
7. Hautman, D.P. & Bolyard, M. Analysis of Oxyhalide Disinfection By-
products and other Anions of Interest in Drinking Water by Ion
Chromatography. Jour, of Chromatog., 602, (1992), 65-74.
300.0-17
-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1A. CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS
IN REAGENT WATER (PART A)
ANALYTE
* RETENTION
PEAK # TIME(MIN) MDL
mg/L
Fluoride
Chloride
Nitrite-N
Bromide
Nitrate-N
o-Phosphate-P
Sulfate
1
2
3
4
5
6
7
1.2
1.7
2.0
2.9
3.2
5.4
6.9
0.01
0.02
0.004
0.01
0.002
0.003
0.02
Standard Conditions:
Columns: as specified in 6.2.2.1
Detector: as specified in 6.2.4
Eluent: as specified in 7.3
Pump Rate: 2.0 mL/min.
Sample Loop: 50 /uL
MDL calculated from data system using a y-axis selection of
1000 ns and with a stripchart recorder with an attenuator
setting of 1 uMHO full scale.
* See Figure 1
300.0-18
-------
TABLE IB. CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS
IN REAGENT WATER (PART B)
ANALYTE
Chlorite
Bromate
Chlorate
*
PEAK #
1
2
4
RETENTION
TIME(MIN)
2.8
3.2
7.1
MDL
mg/L
0.01
0.02
0.003
Standard Conditions:
Column: as specified in 6.2.2.2
Detector: as specified in 6.2.4
Eluent: as specified in 7.3
* See Figure 2
Pump Rate: 1.0 mL/min,
Sample Loop: 50 #L
Attentuation - 1
y-axis - 500 ns
300.0-19
-------
TABLE 2A. SINGLE-OPERATOR ACCURACY AND BIAS OF STANDARD ANIONS
(METHOD A)
ANALYTE
Bromide
Chloride
Fluoride
Nitrate- N
Nitrite- N
o-Phosphate- P
SAMPLE
TYPE
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
KNOWN NUMBER MEAN
CONC. OF RECOVERY
fma/L} REPLICATES %
5.0
5.0
5.0
5.0
5.0
2.0
20.0
20.0
10.0
20.0
20.0
20.0
2.0
1.0
1.0
1.0
0.4
5.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
5.0
5.0
10.0
2.0
10.0
10.0
10.0
10.0
10.0
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
99
105
95
105
92
82
96
108
86
101
114
90
91
92
73
87
95
101
103
104
93
101
97
82
97
121
92
91
96
98
99
99
98
106
95
STANDARD
DEVIATION
Ona/n
0.08
0.10
0.13
0.34
0.34
0.06
0.35
1.19
0.33
5.2
1.3
0.32
0.05
0.06
0.05
0.07
0.07
0.35
0.21
0.27
0.17
0.82
0.47
0.28
0.14
0.25
0.14
0.50
0.35
0.08
0.17
0.26
0.22
0.85
0.33
300.0-20
-------
TABLE 2A (CONT'D)
Sulfate RW 20.0 7 99 0.40
DW 50.0 7 105 3.35
SW 40.0 7 95 1.7
WW 40.0 7 102 6.4
GW 40.0 7 112 3.2
RW = Reagent Water WW = Mixed Domestic and Industrial Wastewater
DW = Drinking Water GW = Groundwater
SW = Surface Water SD = USEPA QC Solid (shale)
300.0-21
-------
TABLE 2B. SINGLE-OPERATOR ACCURACY AND BIAS OF BY-PRODUCT
(PART B)
ANALYTE
NUMBER MEAN STANDARD
SAMPLE SPIKE OF RECOVERY DEVIATION
TYPE (mg/L) REPLICATES % (mg/L)
Bromate RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
Chlorate RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
Chlorite RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
103
98
155
122
95
85
98
98
101
97
100
119
101
115
121
110
100
98
86
94
96
100
76
96
0.07
0.04
0.005
0.01
0.04
0.02
0.005
0.005
0.06
0.01
0.01
0.05
0.04
0.01
0.005
0.01
0.04
0.01
0.01
0.01
0.03
0.02
0.00
0.01
RW s Reagent Water
DW = Drinking Water
300.0-22
-------
TABLE 3. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR FLUORIDE
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.26
0.34
2.12
2.55
6.79
8.49
0.26
0.34
2.12
2.55
6.79
8.49
0.26
0.34
2.12
2.55
6.79
8.49
AM'T FOUND
mg/L
0.25
0.29
2.12
2.48
6.76
8.46
0.24
0.34
2.09
2.55
6.84
8.37
0.25
0.32
2.13
2.48
6.65
8.27
st
0.08
0.11
0.07
0.14
0.20
0.30
0.08
0.11
0.18
0.16
0.54
0.75
0.15
0.08
0.22
0.16
0.41
0.36
S0
0.11
0.12
0.19
0.05
0.06
0.25
0.06
0.15
0.20
BIAS
%
-3.8
-14.7
0.0
-2.7
-0.4
-0.4
-7.7
0.0
-1.4
0.0
+0.7
-1.4
-3.8
-5.9
+0.5
-2.7
-2.1
-2.6
300.0-23
-------
TABLE 4. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR CHLORIDE
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.78
1.04
6.50
7.80
20.8
26.0
0.78
1.04
6.50
7.80
20.8
26.0
0.78
1.04
6.50
7.80
20.8
26.0
AM'T FOUND
mg/L
0.79
1.12
6.31
7.76
20.7
25.9
0.54
0.51
5.24
6.02
20.0
24.0
0.43
0.65
4.59
5.45
18.3
23.0
st
0.17
0.46
0.27
0.39
0.54
0.58
0.35
0.38
1.35
1.90
2.26
2.65
0.32
0.48
1.82
2.02
2.41
2.50
S0
0.29
0.14
0.62
0.20
1.48
1.14
0.39
0.83
1.57
BIAS
%
+1.3
+7.7
-2.9
-0.5
-0.5
-0.4
-30.8
-51.0
-19.4
-22.8
-3.8
-7.7
-44.9
-37.5
-29.4
-30.1
-11.8
-11.5
300.0-24
-------
TABLE 5. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR NITRITE - NITROGEN
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.36
0.48
3.00
3.60
9.60
12.0
0.36
0.48
3.00
3.60
9.60
12.0
0.36
0.48
3.00
3.60
9.60
12.0
AM'T FOUND
mg/L
0.37
0.48
3.18
3.83
9.84
12.1
0.30
0.40
3.02
3.62
9.59
11.6
0.34
0.46
3.18
3.76
9.74
12.0
V
0.04
0.06
0.12
0.12
0.36
0.27
0.13
0.14
0.23
0.22
0.44
0.59
0.06
0.07
0.13
0.18
0.49
0.56
So
0.04
0.06
0.26
0.03
0.12
0.28
0.04
0.10
0.26
BIAS
%
+2.8
0.0
+6.0
+6.4
+2.5
+0.6
-16.7
-16.7
+0.7
+0.6
-0.1
-3.1
-5.6
-4.2
+6.0
+4.4
+1.5
+0.3
300.0-25
-------
TABLE 6. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR BROMIDE
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.63
0.84
5.24
6.29
16.8
21.0
0.63
0.84
5.24
6.29
16.8
21.0
0.63
0.84
5.24
6.29
16.8
21.0
AM'T FOUND St S0 BIAS
mg/L %
0.69
0.85
5.21
6.17
17.1
21.3
0.63
0.81
5.11
6.18
17.0
20.9
0.63
0.85
5.23
6.27
16.6
21.1
0.11
0.12
0.22
0.35
0.70
0.93
0.13
0.13
0.23
0.30
0.55
0.65
0.15
0.15
0.36
0.46
0.69
0.63
0.05
0.21
0.36
0.04
0.13
0.57
0.09
0.11
0.43
+9.5
+1.2
-0.6
-1.9
+1.6
+1.5
0.0
-3.6
-2.5
-1.7
+0.9
-0.4
0.0
+1.2
-0.2
-0.3
-1.0
+0.3
300.0-26
-------
TABLE 7. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR NITRATE - NITROGEN
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.42
0.56
3.51
4.21
11.2
14.0
0.42
0.56
3.51
4.21
11.2
14.0
0.42
0.56
3.51
4.21
11.2
14.0
AM'T FOUND
mg/L
0.42
0.56
3.34
4.05
11.1
14.4
0.46
0.58
3.45
4.21
11.5
14.2
0.36
0.40
3.19
3.84
10.9
14.1
st
0.04
0.06
0.15
0.28
0.47
0.61
0.08
0.09
0.27
0.38
0.50
0.70
0.07
0.16
0.31
0.28
0.35
0.74
So
0.02
0.08
0.34
0.03
0.10
0.48
0.06
0.07
0.51
BIAS
%
0.0
0.0
-4.8
-3.8
-1.1
+2.6
+9.5
+3.6
-1.7
0.0
+2.3
+1.6
-14.6
-28.6
-9.1
-8.8
-3.0
+0.4
300.0-27
-------
TABLE 8. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR ORTHO-PHOSPHATE
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
0.69
0.92
5.77
6.92
18.4
23.1
0.69
0.92
5.77
6.92
18.4
23.1
0.68
0.92
5.77
6.92
18.4
23.1
AM'T FOUND
mg/L
0.69
0.98
5.72
6.78
18.8
23.2
0.70
0.96
5.43
6.29
18.0
22.6
0.64
0.82
5.18
6.24
17.6
22.4
st
0.06
0.15
0.36
0.42
1.04
0.35
0.17
0.20
0.52
0.72
0.68
1.07
0.26
0.28
0.66
0.74
2.08
0.87
S0
0.06
0.18
0.63
0.17
0.40
0.59
0.09
0.34
1.27
BIAS
%
0.0
+6.5
-0.9
-2.0
+2.1
+0.4
+1.4
+4.3
-5.9
-9.1
-2.2
-2.0
-7.2
-10.9
-10.2
-9.8
-4.1
-3.0
300.0-28
-------
TABLE 9. MULTIPLE LABORATORY (n=19)
DETERMINATION OF BIAS FOR SULFATE
WATER
Reagent
Drinking
Waste
AM'T ADDED
mg/L
2.85
3.80
23.8
28.5
76.0
95.0
2.85
3.80
23.8
28.5
76.0
95.0
2.85
3.80
23.8
28.5
76.0
95.0
AM'T FOUND
mg/L
2.83
3.83
24.0
28.5
76.8
95.7
1.12
2.26
21.8
25.9
74.5
92.3
1.89
2.10
20.3
24.5
71.4
90.3
st
0.32
0.92
1.67
1.56
3.42
3.59
0.37
0.97
1.26
2.48
4.63
5.19
0.37
1.25
3.19
3.24
5.65
6.80
S0
0.52
0.68
2.33
0.41
0.51
2.70
0.24
0.58
3.39
BIAS
%
-0.7
+0.8
+0.8
-0.1
+1.1
+0.7
-60.7
-40.3
-8.4
-9.1
-2.0
-2.8
-33.7
-44.7
-14.7
-14.0
-6.1
-5.0
300.0-29
-------
Method A
Ret. Time
1.17
1.73
2.02
2.95
3.20
5.38
6.92
02468
Minutes
Figure 1. Chromatogram showing separation using the AS4A column
Ion
F-
ci-
N02-
Br
N03-
HP042-
so42-
mg/L
2
20
2
2
10
2
60
Method B
3
1 2
W
Peak Ret. Time Ion mg/L
1 2.75 CI02- 0.1
2 3.23 Br03- 0.1
3 3.63 Cl- 0.1
4 7.08 CI03- 0.1
4
A^
i I u w v
I
0 2 46 8
Minutes
Rgure 2. Chromatogram showing separation using the AS9 column
300.0-30
-------
METHOD 335.4
DETERMINATION OF TOTAL CYANIDE BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 1.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AMD DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
335.4-1
-------
METHOD 335.4
DETERMINATION OF TOTAL CYANIDE BY SEMI-AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of cyanide in drinking, ground,
surface, and saline waters, domestic and industrial wastes.
1.2 The applicable range is 5 to 500 /zg/L.
2.0 SUMMARY OF METHOD
2.1 The cyanide as hydrocyanic acid (HCN) is released from cyanide
complexes by means of a manual reflux-distillation operation and
absorbed in a scrubber containing sodium hydroxide solution. The
cyanide ion in the absorbing solution is converted to cyanogen
chloride by reactions with chloramine-T, that subsequently reacts
with pyridine and barbituric acid to give a red-colored complex.
2.2 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.2 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
335.4-2
-------
methodology is In control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
4.0 INTERFERENCES
4.1 Several interferences are encountered with this method. Some of the
known interferences are aldehydes, nitrate-nitrite, oxidizing
agents, such as chlorine, thiocyanate, thiosulfate and sulfide.
Multiple interferences may require the analysis of a series of
laboratory fortified sample matrices (LFM) to verify the suitability
of the chosen treatment. Some interferences are eliminated or
reduced by the distillation.
335.4-3
-------
4.2 Sulfides adversely affect the procedure by producing hydrogen
sulfide during distillation. If a drop of the sample on lead
acetate test paper indicates the presence of sulfide, treat 25 ml
more of the stabilized sample (pH £ 12) than that required for the
cyanide determination with powdered cadmium carbonate. Yellow
cadmium sulfide precipitates if the sample contains sulfide. Repeat
this operation until a drop of the treated sample solution does not
darken the lead acetate test paper. Filter the solution through a
dry filter paper into a dry beaker, and from the filtrate, measure
the sample to be used for analysis. Avoid a large excess of cadmium
and a long contact time in order to minimize a loss by cotnplexation
or occlusion of cyanide on the precipitated material.
4.3 High results may be obtained for samples that contain nitrate and/or
nitrite. During the distillation nitrate and nitrite will form
nitrous acid that will react with some organic compounds to form
oximes. These oximes will decompose under test conditions to
generate HCN. The interference of nitrate and nitrite is eliminated
by pretreatment with sulfamic acid.
4.4 Oxidizing agents, such as chlorine, decompose most of the cyanides.
Test a drop of the sample with potassium iodide-starch paper (KI-
starch paper) at time of collection; a blue color indicates the need
for treatment. Add ascorbic acid, a few crystals at a time, until a
drop of sample produces no color on the indicator paper; then add an
additional 0.06 g of ascorbic acid for each liter of sample volume.
Sodium arsenite has also been employed to remove oxidizing agents.
4.5 Other compatible procedures for the removal or suppression of
interferences may be employed provided they do not adversely effect
the overall performance of the method.
4.6 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been fully established. Each chemical should be regarded as
a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
335.4-4
-------
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Hydrochloric acid (7.5)
5.3.2 Silver nitrate (7.9)
5.3.3 Potassium cyanide (7.10)
5.3.4 Sulfuric acid (7.14)
5.4 Because of the toxicity of evolved hydrogen cyanide (HCN),
distillation should be performed in a well vented hood.
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware Class A volumetric flasks and pi pets as required.
6.3 Midi reflux distillation apparatus including boiling flask
condenser, and absorber as shown in Figure 1.
6.4 Heating mantel or heating block as required.
6.5 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.5.1 Sampling device (sampler)
6.5.2 Multichannel pump
6.5.3 Reaction unit or manifold
6.5.4 Colorimetric detector
6.5.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the analyte of
interest. ASTM type II or equivalent.
7.2 Ascorbic acid: Crystal (CASRN-50-81-7)
7.3 Chloramine-T: Dissolve 2.0 g of chloramine-T (CASRN-127-65-1) in
500 mL of reagent water.
7.4 Magnesium Chloride Solution: Weigh 510 g of MgCl2.6H20 (CASRN-7786-
30-3) into a 1000 ml flask, dissolve and dilute to 1 1 with reagent
water.
335.4-5
-------
7.5
7.6
7.7
7.8
Pyridine Barbituric Acid Reagent: Place 15 g of barbituric acid
(CASRN-67-52-7) in a 1 L beaker. Wash the sides of the beaker with
about 100 ml of reagent water. Add 75 ml of pyridine (CASRN-110-86-
1) and mix. Add 15 ml of cone. HC1 (CASRN-7647-01-0) and mix.
Dilute to 900 ml with reagent water and mix until all the barbituric
acid has dissolved. Transfer the solution to a 1-L flask and dilute
to the mark.
Sodium dihydrogenphosphate buffer, 1 M: Dissolve 138 g of
NaH2P04.H20 (CASRN-10049-21-5) in 1 L of reagent water. Refrigerate
this solution.
Sodium Hydroxide Solution, 1.25 N: Dissolve 50 g of NaOH (CASRN-
1310-73-2) in reagent water, and dilute to 1 L with reagent water.
Sodium Hydroxide, 0.25 N: Dilute 200 ml of 1.25 N Sodium hydroxide
solution (7.7) to 1 L with reagent water.
7.9 Standard Silver Nitrate Solution, 0.0192 N: Prepare by crushing
approximately 5 g AgNO, (CASRN-7761-88-8) crystals and drying to
constant weight at 40°t. Weigh out 3.2647 g of dried AgN03,
dissolve in reagent water, and dilute to 1000 ml (1 mL = 1 mg CN).
7.10 Stock Cyanide Solution: Dissolve 2.51 g of KCN (CASRN-151-50-8) and
2 g KOH (CASRN-1310-58-3) in 900 mL of reagent water. Standardize
with 0.0192 N AgNO, (7.9). Dilute to appropriate concentration so
that 1 ml = 1 mg CN.
7.11 Standard Cyanide Solution, intermediate: Dilute 10.0 ml of stock (1
ml - 1 mg CN) (7.10) to 100.0 with reagent water (1 ml = 100.0 //g
CN.
7.12 Working Standard Cyanide Solution: Prepare fresh daily by diluting
20.0 ml of intermediate cyanide solution (7.11) to 200.0 ml with
reagent water and store in a glass stoppered bottle. 1 ml = 10.0 /jg
CN.
7.13 Sulfamic Acid: (CASRN-212-57-3).
7.14 Sulfuric Acid, 18N: Slowly add 500 ml of concentrated H2S04 (CASRN-
5329-14-6) to 500 ml of reagent water.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
335.4-6
-------
8.2 If the sample contains chlorine or hydrogen sulfide, see Sect. 4.0
for treatment.
8.3 Samples must be preserved with sodium hydroxide pH > 12 and cooled
to 4°C at the time of collection.
8.4 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 14 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, the periodic analysis of laboratory reagent blanks,
fortified blanks, and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding on with the initial determination
of MDLs or continuing with on-going analyses.
335.4-7
-------
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit/45 To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t - Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
becomes available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
335.4-8
-------
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
establish an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations, the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required), and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case, the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculate using the
following equation:
R = GS ' C x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
335.4-9
-------
s = concentration equivalent of analyte added to
sample.
943 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10 1 Prepare a series of at least 3 standards, covering the desired
range, and a blank by pipetting suitable volumes of working standard
solution (7.12) into 100 ml volumetric flasks. To each standard
(except those to be distilled) add 20 mL of 1.25 N sodium hydroxide
and dilute to 100 ml with reagent water.
10.2 It is not imperative that all standards be distilled in the same
manner as the samples. It is recommended that at least two
standards (a high and low) and a blank be distilled and compared to
similar values on the standard curve to insure that the distillation
technique is reliable. If distilled standards do not agree within
± 10% of the undistilled standards the analyst should find the cause
of the apparent error before proceeding. Before distillation,
standards should contain 4 ml 0.25N NaOH (7.8) per 50 ml.
10.3 Set up the manifold as shown in Figure 2 in a hood or a well-
ventilated area.
10.4 Allow the instrument to warm up as required. Pump all reagents,
with 0.25N NaOH in the sample line, until a stable baseline is
achieved.
10.5 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10 6 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
the measured value of the calibration solution and the "true value
concentration.
10 7 After the calibration has been established, it must be verified by
the analysis of a suitable QCS. If measurements exceed ± 10% of the
established QCS value, the analysis should be terminated and the
335.4-10
-------
instrument recalibrated. The new calibration must be verified
before continuing analysis. Periodic reanalysis of the QCS is
recommended as a continuing calibration check.
11.0 PROCEDURE
11.1 Pipet 50 ml of sample or an aliquot diluted to 50 ml into the MIDI
distillation boiling flask. Add boiling chips as required. Pipet
50 ml of sodium hydroxide 0.25 N (7.8) into the absorbing tube.
Connect the boiling flask, condenser, and absorber in the train as
shown in Figure 1.
11.2 Start a slow stream of air entering the boiling flask by adjusting
the vacuum source to maintain about 3 bubbles per minute.
11.3 If samples contain N03 and/or NO., add 0.2 g of sulfamic acid (7.13)
after the air rate is set through the air inlet tube. Mix for 3 min
prior to addition of H2S04.
11.4 Slowly add 5 ml 18 N sulfuric acid (7.14) through the air inlet
tube. Rinse the tube with distilled water and allow the airflow to
mix the flask contents for 3 min. Pour 2 ml of magnesium chloride
(7.4) into the air inlet and wash down with a stream of water.
11.5 Heat the solution to boiling. Reflux for one and one half hours.
Turn off heat and continue the airflow for at least 15 min. After
cooling the boiling flask, disconnect absorber and close off the
vacuum source and remove absorber tube.
11.6 Fill and connect reagent containers and start system. Allow the
instrument to warm up as required. Pump all reagents, with 0.25N
NaOH in the sample line, until a stable baseline is achieved.
11.7 Place standards, distilled standards and unknown samples (ALL in
0.25N NaOH) in sampler tray. Calibrate instrument and begin
analysis. .
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response against
standard concentration. Compute sample concentration by comparing
sample response with the standard curve. Multiply answer by
appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg/L.
335.4-11
-------
13.0 METHOD PERFORMANCE
13.1 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg CN/L.
13.2 Single laboratory precision data can be estimated at 50 to 75% of
the inter!aboratory precision estimates.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The USEPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202)872-4477.
15.0 WASTE MANAGEMENT
15.1 The U.S. Environmental Protection Agency requires that laboratory
waste management practices conducted be consistent with all
applicable rules and regulations. Excess Reagents, samples, and
method process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water and land by minimizing and controlling all releases from
hoods, and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal
restrictions. For further information on waste management consult
the "Waste Management Manual for Laboratory Personnel," available
from the American Chemical Society at the address listed in Sect.
14.3.
335.4-12
-------
16.0 REFERENCES
1. Technicon AutoAnalyzer II Methodology, Industrial Method No. 315-74
WCUV digestion and distillation, Technicon Industrial Systems,
Tarrytown, NY 10591, (1974).
2. Goulden, P.O., Afghan, B.K. and Brooksbank, P., Anal. 44. 1845
(1972).
3. USEPA Contract Laboratory Program, Document Number ILMO 1.0, Method
for Total Cyanide Analysis by MIDI Distillation #335.2 CLP-M.
4. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
335.4-13
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
126
94
158
118
148
92
132
119
148
94
92
158
TRUE
VALUE
(T)
0.020
0.055
0.090
0.110
0.180
0.270
0.530
0.540
0.610
0.700
0.800
0.970
MEAN
(X)
0.0182
0.0501
0.0843
0.1045
0.1683
0.2538
0.5019
0.5262
0.5803
0.6803
0.7726
0.9508
RESIDUAL
FOR X
0.0002
-0.0014
-0.0008
0.0003
-0.0030
-0.0038
-0.0049
0.0098
-0.0032
0.0105
0.0069
0.0222
STANDARD
DEVIATION
(S)
0.0055
0.0092
0.0171
0.0165
0.0236
0.0275
0.0775
0.0679
0.0851
0.1082
0.0880
0.1464
RESIDUAL
FOR S
0.0000
-0.0007
0.0027
-0.0004
-0.0023
-0.0099
0.0069
-0.0039
0.0043
0.0159
-0.0170
0.0197
REGRESSIONS: X = 0.959T - 0.001, S = 0.128T + 0.003
335.4-14
-------
CONDENSER
1
DISTILLATION
UNIT
SUCTION
$- ABSORBER
FIGURE 1. NIDI DISTILLATION APPARATUS
335.4-15
-------
1
8
in
CQ
0
Ill
I
o
EC
8
O
UT
8
11
Q.
I
" i
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in -i
I
335.4-16
-------
1
METHOD 350,,1
DETERMINATION OF AMMONIA NITROGEN BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
350.1-1
-------
METHOD 350.1
DETERMINATION OF AMMONIA NITROGEN BY SEMI-AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of ammonia in drinking, ground,
surface, and saline waters, domestic and industrial wastes.
1 2 The applicable range is 0.01 to 2.0 mg/L NH3 as N. Higher
concentrations can be determined by sample dilution. Approximately
60 samples per hour can be analyzed.
1.3 This method is described for macro glassware; however, micro
distillation equipment may also be used.
2.0 SUMMARY OF METHOD
2 1 The sample is buffered at a pH of 9.5 with a borate buffer in order
to decrease hydrolysis of cyanates and organic nitrogen compounds,
and is distilled into a solution of boric acid. Alkaline phenol and
hypochlorite react with ammonia to form indophenol blue that is
proportional to the ammonia concentration. The blue color formed is
intensified with sodium nitroprusside and measured colorimetrically.
2.3 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2 4 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) -- A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
350.1-2
-------
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
, matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) -- The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
350.1-3
-------
4.0 INTERFERENCES
4.1 Cyanate, which may be encountered in certain industrial effluents,
will hydrolyze to some extent even at the pH of 9.5 at which
distillation is carried out.
4 2 Residual chorine must be removed by pretreatment of the sample with
sodium thiosulfate or other reagents before distillation.
4 3 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5 1 The toxicity or carcinogenicity of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5 2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Sulfuric acid (7.6)
5.3.2 Phenol (7.7)
5.3.3 Sodium nitroprusside (7.10)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware - Class A volumetric flasks and pipets as required.
6.3 An all-glass distilling apparatus with an 800-1000-mL flask.
6.4 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.4.1 Sampling device (sampler)
6.4.2 Multichannel pump
350.1-4
-------
6.4.3 Reaction unit or manifold
6.4.4 Colorimetric detector
6.4.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Reagent water - Ammonia free: Such water is best prepared by
passage through an ion exchange column containing a strongly acidic
cation exchange resin mixed with a strongly basic anion exchange
resin. Regeneration of the column should be carried out according
to the manufacturer's instructions.
NOTE 1: All solutions must be made with ammonia-free water.
7.2 Boric acid solution (20 g/L): Dissolve 20 g H3B03 (CASRN 10043-35-
3) in reagent water and dilute to 1 L.
7.3 Borate buffer: Add 88 ml of 0.1 N NaOH (CASRN 1310-73-2) solution
to 500 ml of 0.025 M sodium tetraborate solution (5.0 g anhydrous
Na2B40, [CASRN 1330-43-4] or 9.5 g Na2B40/10H20 [CASRN 1303-96-4] per
L) ana dilute to 1 L with reagent water.
7.4 Sodium hydroxide, 1 N: Dissolve 40 g NaOH in reagent water and
dilute to 1 L.
7.5 Dechlorinating reagents: A number of dechlorinating reagents may be
used to remove residual chlorine prior to distillation. These
include:
7.5.1 Sodium thiosulfate: Dissolve 3.5 g Na2S203'5H20 (CASRN
10102-17-7) in reagent water and dilute to 1 L. One ml of
this solution will remove 1 mg/L of residual chlorine in 500
ml of sample.
7.5.2 Sodium sulfite: Dissolve 0.9 g Na2S03 (CASRN 7757-83-7) in
reagent water and dilute to 1 L. One ml removes 1 mg/L Cl
per 500 ml of sample.
7.6 Sulfuric acid 5 N: Air scrubber solution. Carefully add 139 ml of
cone, sulfuric acid (CASRN 7664-93-9) to approximately 500 ml of
reagent water. Cool to room temperature and dilute to 1 L with
reagent water.
7.7 Sodium phenolate: Using a 1-L Erlenmeyer flask, dissolve 83 g
phenol (CASRN 108-95-2) in 500 ml of distilled water. In small
increments, cautiously add with agitation, 32 g of NaOH.
Periodically cool flask under water faucet. When cool, dilute to
1 L with reagent water.
350.1-5
-------
7.8 Sodium hypochlorite solution: Dilute 250 ml of a bleach solution
containing 5.25% NaOCl (CASRN 7681-52-9) (such as "Clorox") to 500
ml with reagent water. Available chlorine level should approximate
2% to 3%. Since "Clorox" is a proprietary product, its formulation
is subject to change. The analyst must remain alert to detecting
any variation in this product significant to its use in this
procedure. Due to the instability of this product, storage over an
extended period should be avoided.
7.9 Disodium ethylenediamine-tetraacetate (EDTA) (5%): Dissolve 50 g of
EDTA (disodium salt) (CASRN 6381-92-6) and approximately six pellets
of NaOH in 1 L of reagent water.
7.10 Sodium nitroprusside (0.05%): Dissolve 0.5 g of sodium
nitroprusside (CASRN 14402-89-2) in 1 L of reagent water.
7.11 Stock solution: Dissolve 3.819 g of anhydrous ammonium chloride,
NH4C1 (CASRN 12125-02-9), dried at 105°C, in reagent water, and
dilute to 1 L. 1.0 ml = 1.0 mg NH3-N.
7.12 Standard Solution A: Dilute 10.0 ml of stock solution (7.11) to 1 L
with reagent water. 1.0 ml = 0.01 mg NH3-N.
7.13 Standard Solution B: Dilute 10.0 ml of standard solution A (7.12)
to 100.0 ml with reagent water. 1.0 mL = 0.001 mg NH3-N.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
8.2 Samples must be preserved with H2S04 to a pH < 2 and cooled to 4°C
at the time of collection.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 28 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
350.1-6
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9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
350.1-7
-------
MDLs should be determined every 6 months, when a new
operator begins work or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent Mm
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
established an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required) and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
350.1-8
-------
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift, the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculate using the
following equation:
R = Cs C x 100
where, R = percent recovery.
Cs - fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFH is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
350.1-9
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10.0 CALIBRATION AND STANDARDIZATION
10.1 Prepare a series of at least 3 standards, covering the desired
range, and a blank by diluting suitable volumes of standard
solutions (7.12, 7.13) to 100 ml with reagent water.
10.2 Process standards and blanks as described in Sect. 11, Procedure.
10.3 Set up manifold as shown in Figure 1.
10.4 Prepare flow system as described in Sect. 11, Procedure.
10.5 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10.6 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
the measured value of the calibration solution and the "true value"
concentration.
10.7 After the calibration has been established, it must be verified by
the analysis of a suitable QCS. If measurements exceed ± 10% of the
established QCS value, the analysis should be terminated and the
instrument recalibrated. The new calibration must be verified
before continuing analysis. Periodic reanalysis of the QCS is
recommended as a continuing calibration check.
11.0 PROCEDURE
11.1 Preparation of equipment: Add 500 ml of reagent water to an 800-mL
Kjeldahl flask. The addition of boiling chips that have been
previously treated with dilute NaOH will prevent bumping. Steam out
the distillation apparatus until the distillate shows no trace of
ammonia.
11.2 Sample preparation: Remove the residual chorine in the sample by
adding dechlorinating agent (7.5) equivalent to the chlorine
residual. To 400 ml of sample add 1 N NaOH (7.4), until the pH is
9.5, check the pH during addition with a pH meter or by use of a
short range pH paper.
11.3 Distillation: Transfer the sample, the pH of which has been
adjusted to 9.5, to an 800-mL Kjeldahl flask and add 25 ml of the
borate buffer (7.3). Distill 300 ml at the rate of 6-10 mL/min.
into 50 ml of 2% boric acid (7.2) contained in a 500-mL Erlenmeyer
flask.
NOTE 4: The condenser tip or an extension of the condenser tip must
extend below the level of the boric acid solution.
350.1-10
-------
11.4 Since the intensity of the color used to quantify the concentration
is pH dependent, the acid concentration of the wash water and the
standard ammonia solutions should approximate that of the samples.
11.5 Allow analysis system to warm up as required. Feed wash water
through sample line.
11.6 Arrange ammonia standards in sampler in order of decreasing
concentration of nitrogen. Complete loading of sampler tray with
unknown samples.
11.7 Switch sample line from reagent water to sampler and begin analysis.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg NH3-N/L.
13.0 METHOD PERFORMANCE
13.1 In a single laboratory (EMSL-Cincinnati), using surface water
samples at concentrations of 1.41, 0.77, 0.59 and 0.43 mg NH3-N/L,
the standard deviation was ± 0.005.
13.2 In a single laboratory (EMSL-Cincinnati), using surface water
samples at concentrations of 0.16 and 1.44 mg NH3-N/L, recoveries
were 107% and 99%, respectively.
13.3 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg NH3-N/L.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
350.1-1.1
-------
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202)872-4477.
15.0 WASTE MANAGEMENT
15.1 The U.S. Environmental Protection Agency requires that laboratory
waste management practices be conducted consistent with all
applicable rules and regulations. Excess reagents, samples and
method process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water and land by minimizing and controlling all releases from
hoods, and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal
restrictions. For further information on waste management consult
the "Waste Management Manual for Laboratory Personnel," available
from the American Chemical Society at the address listed in Sect.
14.3.
16.0 REFERENCES
1. Hiller, A., and Van Slyke, D., "Determination of Ammonia in Blood,"
J. Biol. Chem. 102. p. 499 (1933).
2. O'Connor, B., Dobbs, R., Villiers, B., and Dean. R., "Laboratory
Distillation of Municipal Waste Effluents," JWPCF 39, R 25 (1967).
3. Fiore, J., and O'Brien, J.E., "Ammonia Determination by Automatic
Analysis," Wastes Engineering 33, p. 352 (1962).
4. A Wetting Agent Recommended and Supplied by the Technicon
Corporation for Use in AutoAnalyzers.
5. ASTM "Manual on Industrial Water and Industrial Waste Water," 2nd
Ed., 1966 printing, p. 418.
6. Booth, R.L., and Lobring. L.B., "Evaluation of the AutoAnalyzer II:
A Progress Report" in Advances in Automated Analysis: 1972
Technicon International Congress, Vol. 8, p. 7-10, Mediad
Incorporated, Tarrytown, N.Y., (1973).
350.1-12
-------
7. Standards Methods for the Examination of Water and Wastewater, 18th
Edition, p. 4-77, Methods 4500 NH3 B arid H (1992).
8. Annual Book of ASTM Standards, Part 31, "Water," Standard D1426-
79(C).
9. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
350.1-13
-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
134
157
136
195
142
159
156
200
196
156
142
199
TRUE
VALUE
(T)
0.270
0.692
1.20
1.60
3.00
3.50
3.60
4.20
8.76
11.0
13.0
18.0
MEAN
(X)
0.2670
0.6972
1.2008
1.6095
3.0128
3.4991
3.5955
4.2271
8.7257
11.0747
12.9883
17.9727
RESIDUAL
FOR X
-0.0011
0.0059
0.0001
0.0076
0.0069
-0.0083
-0.0122
0.0177
-0.0568
0.0457
-0.0465
-0.0765
STANDARD
DEVIATION
(S)
0.0342
0.0476
0.0698
0.1023
0.1677
0.2168
0.1821
0.2855
0.4606
0.5401
0.6961
1.1635
RESIDUAL
FOR S
0.0015
-0.0070
-0.0112
0.0006
-0.0067
0.0165
-0.0234
0.0488
-0.0127
-0.0495
0.0027
0.2106
REGRESSIONS: X = 1.003T - 0.003, S = 0.052T + 0.019
350.1-14
-------
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350.1-15
-------
-------
METHOD 351.2
DETERMINATION OF TOTAL KJELDAHL NITROGEN BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
351.2-1
-------
METHOD 351.2
DETERMINATION OF TOTAL KOELDAHL NITROGEN BY SEMI-AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1
This method covers the determination of total Kjeldahl nitrogen in
drinking, ground, and surface waters, domestic and industrial
wastes. The procedure converts nitrogen components of biological
origin such as amino acids, proteins and peptides to ammonia, but
may not convert the nitrogenous compounds of some industrial wastes
such as amines, nitro compounds, hydrazones, oximes, semicarbazones
and some refractory tertiary amines.
1.2
The applicable range is 0.1 to 20 mg/L TKN.
extended with sample dilution.
The range may be
2.0 SUMMARY OF METHOD
2.1
2.2
2.3
2.4
2.5
for two
The sample is heated in the presence of sulfuric acid, HpSO, for
and one half hours. The residue is cooled, diluted to 25 ml and
analyzed for ammonia. This digested sample may also be used for
phosphorus determination.
Total Kjeldahl nitrogen is the sum of free-ammonia and organic
nitrogen compounds which are converted to ammonium sulfate
(NH4)2S04, under the conditions of digestion described.
Organic Kjeldahl nitrogen is the difference obtained by subtracting
the free-ammonia value from the total Kjeldahl nitrogen value.
Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
351.2-2
-------
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT. (MDL) -- The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
351.2-3
-------
4.0 INTERFERENCES
4.1 High nitrate concentrations (10X or more than the TKN level) result
in low TKN values. If interference is suspected, samples should be
diluted and reanalyzed.
4.2 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Mercury (7.2, 7.3)
5.3.2 Sulfuric acid (7.2, 7.3, 7.4)
5.3.3 Sodium nitroprusside (7.9)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware - Class A volumetric flasks and pi pets as required.
6.3 Block digester with tubes.
6.4 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.4.1 Sampling device (sampler)
6.4.2 Multichannel pump
6.4.3 Reaction unit or manifold
351.2-4
-------
6.4.4 Colorimetric detector
6.4.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Ammonia free distilled or deionized water, free of
the analyte of interest. ASTM type II or equivalent.
7.2 Mercuric sulfate: Dissolve 8 g red mercuric oxide (HgO) (CASRN
21908-53-2) in 50 mL of 1:4 sulfuric acid (10 ml cone. H2S04: [CASRN
7664-93-9] 40 ml reagent water) and dilute to 100 ml with reagent
water.
7.3 Digestion solution: (Sulfuric acid-mercuric sulfate-potassium
sulfate solution): Dissolve 133 g of K2SO, (CASRN 7778-80-5) in 700
mL of reagent water and 200 mL of cone. H2S04. Add 25 mL of
mercuric sulfate solution (7.1) and dilute to 1 L.
NOTE 1: An alternate mercury-free digestion solution can be
prepared by dissolving 134 g K2S04 and 7.3 g CuSO, in 800 mL reagent
water and then adding 134 mL cone. H2S04 and diluting to 1 L. Use
10 mL solution per 25 mL of sample.
7.4 Sulfuric Acid solution (4%): Add 40 ml. of cone, sulfuric acid to
800 mL of reagent water, cool and dilute to 1 L.
NOTE 2: If alternate mercury-free digestion solution is used,
adjust the above solution to equal the acid concentration of the
digested sample (11.6).
7.5 Stock Sodium Hydroxide (20%): Dissolve 200 g of sodium hydroxide
(CASRN 1310-73-2) in 900 mL of reagent water and dilute to 1 L.
7.6 Stock Sodium Potassium Tartrate solution (20%): Dissolve 200 g
sodium potassium tartrate (CASRN 6381-59-5) in about 800 mL of
reagent water and dilute to 1 L.
7.7 Stock Buffer solution: Dissolve 134.0 g of sodium phosphate,
dibasic (Na2HP04) (CASRN 7558-79-4) in about 800 mL of reagent
water. Add 20 g of sodium hydroxide and dilute to 1 L.
7.8 Working Buffer solution: Combine the reagents in the stated order,
add 250 mL of stock sodium potassium tartrate solution (7.6) to 200
mL of stock buffer solution (7.7) and mix. Add xx mL sodium
hydroxide solution (7.5) and dilute to 1 L. See concentration
ranges, Table 2, for composition of working buffer.
7.9 Sodium Sal icy!ate/Sodium Nitroprusside solution: Dissolve 150 g of
sodium salicylate (CASRN 54-21-7) and 0.3 g of sodium nitroprusside
(CASRN 13755-38-9 or 14402-89-2) in about 600 mL of reagent water
and dilute to 1 L.
351.2-5
-------
7.10 Sodium Hypochlorite solution: Dilute 6.0 ml sodium hypochlorite
solution (CASRN 7681-52-9) (Clorox) to 100 ml with reagent water.
7.11 Ammonium chloride, stock solution: Dissolve 3.819 g NH4C1 (CASRN
12125-02-9) in reagent water and bring to volume in a 1 L volumetric
flask. 1 ml = 1.0 mg NH3-N.
7.12 Teflon boiling chips.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
8.2 Samples must be preserved with H2S04 to a pH < 2 and cooled to 4°C
at the time of collection.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 28 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of linear
calibration ranges and analysis of QCS) and laboratory
performance (determination of MDLs) prior to performing
analyses by this method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
351.2-6
-------
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis, or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) -- MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every six months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
351.2-7
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control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20^-30 data points.
Also, the standard deviation (S) data should be used to
establish an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required), and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
351.2-8
-------
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculated using the
Prt 1 1 r\i»i*i nn r-n-iii-»4--irt«.
following equation:
R = l x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
9'4'3 ,ILthe recovery of any analyte falls outside the designated
LFM recovery range and the 1aboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDI7ATTOM
10.1 Prepare a series of at least 3 standards, covering the desired
range and a blank by diluting suitable volumes of standard solution
(7.11) with reagent water.
10.2 Process standards and blanks as described in Sect. 11, Procedure.
10.3 Set up manifold as shown in Figure 1 and Table 2.
10.4 Prepare flow system as described in Sect. 11, Procedure.
10.5 Place appropriate standards in the sampler in order of decreasina
concentration and perform analysis.
10.6 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
351.2-9
-------
the measured value of the calibration solution and the "true value"
concentration.
10.7 After the calibration has been established, it must be verified by
the analysis of a suitable quality control sample (QCS). If
measurements exceed ± 10% of the established QCS value, the analysis
should be terminated and the instrument recalibrated. The new
calibration must be verified before continuing analysis. Periodic
reanalysis of the QCS is recommended as a continuing calibration
check.
11.0 PROCEDURE
11.1 Pipet 25.0 ml of sample, standard or blank in the digestor tube.
11.2 Add 5 ml of digestion solution (7.3) and mix with a vortex mixer
(See Note 1).
11.3 Add 4-8 Teflon boiling chips (7.12). CAUTION: An excess of Teflon
chips may cause the sample to boil over.
11.4 Place tubes in block digestor preheated to 160°C and maintain
temperature for 1 h.
11.5 Reset temperature to 380°C and continue to heat for one and one half
hour.
(380°C MUST BE MAINTAINED FOR 30 MIN.)
11.6 Remove digestion tubes, cool and dilute to 25 ml with reagent water.
11.7 Excluding the salicylate line, place all reagent lines in their
respective containers, connect the sample probe to the sampler and
start the pump.
11.8 Flush the sampler wash receptacle with about 25 ml of 4% sulfuric
acid (7.4) (See Note 2).
11.9 When reagents have been pumping for at least 5 min, place the
salicylate line in its respective container and allow the system to
equilibrate. If a precipitate forms after the addition of
salicylate, the pH is too low. Immediately stop the proportioning
pump and flush the coils with water using a syringe. Before
restarting the system, check the concentration of the sulfuric acid
solutions and/or the working buffer solution.
11.10 To prevent precipitation of sodium salicylate in the waste tray,
which can clog the tray outlet, keep the nitrogen flowcell pump tube
and the nitrogen Colorimeter "To Waste" tube separate from all other
lines or keep tap water flowing in the waste tray.
11.11 After a stable baseline has been obtained, start the sampler and
perform analysis.
351.2-10
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12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg N/L.
13.0 METHOD PERFORMANCE
13.1 In a single laboratory (EMSL-Cincinnati) using sewage samples at
concentrations of 1.2, 2.6, and 1.7 mg N/L, the precision was ±
0.07, ± 0.03, and ± 0.15, respectively.
13.2 In a single laboratory (EMSL-Cincinnati) using sewage samples at
concentrations 4.7 and 8.74 mg N/L, the recoveries were 99% and 99%,
respectively.
13.3 The interlaboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg N/L.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation.volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science.Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
351.2-11
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15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess Reagents and samples and method
process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult "The Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1.
McDaniel, W.H., Hemphill, R.N. and Donaldson, W.T., "Automatic
Determination of total Kjeldahl Nitrogen in Estuarine Water,"
Technicon Symposia, pp. 362-367, Vol. 1, 1967.
2. Gales, M.E. and Booth, R.L., "Evaluation of Organic Nitrogen
Methods," EPA Office of Research and Monitoring, June, 1972.
3. Gales, M.E. and Booth, R.L., "Simultaneous and Automated
Determination of Total Phosphorus and Total Kjeldahl Nitrogen,"
Methods Development and Quality Assurance Research Laboratory, May
1974.
4. Technicon "Total Kjeldahl Nitrogen and Total Phosphorus BD-40
Digestion Procedure for Water," August 1974.
5. Gales, M.E., and Booth, R.L., "Evaluation of the Technicon Block
Digester System for the Measurement of Total Kjeldahl Nitrogen and
Total Phosphorus," EPA-600/4-78-015, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, 1978.
6. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
351.2-12
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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
115
134
127
164
138
115
175
121
120
127
164
175
TRUE
VALUE
(T)
0.380
0.451
1.00
3.10
3.50
5.71
7.00
8.00
15.0
21.0
25.0
26.9
MEAN
(X)
0.3891
0.4807
1.0095
3.0992
3.4765
5.6083
6.9246
7.9991
15.0213
20.4355
24.7157
26.1464
RESIDUAL
FOR X
-0.0091
0.0125
-0.0000
0.0191
0.0020
-0.0452
-0.0008
0.0877
0.2080
-0.2937
0.0426
-0.4000
STANDARD
DEVIATION
(S)
0.0750
0.1181
0.1170
0.2821
0.3973
0.4869
0.6623
0.6283
1.2495
1.7267
2.0147
2.9743
RESIDUAL
FOR S
-0.0135
0.0238
-0.0227
-0.0310
0.0512
-0.0417
0.0272
-0.0894
-0.0462
-0.0644
-0.1067
0.6960
REGRESSIONS: X - 0.986T + 0.024, S = 0.083T + 0.057
351.2-13
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TABLE 2. CONCENTRATION RANGES
Range Pump mL/min ml NaOH
mg/LN Sample Resample Buffer (7.7)
0-1.5 0.80 0.32 250
0-5.0 0.16 0.32 120
0-10.0 0.16 0.16 80
351.2-14
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8
^
4
8
&
UJ
CO
B
E
1
9
8
.
LL o
351.2-15
-------
-------
METHOD 353.2
DETERMINATION OF NITRATE-NITRITE NITROGEN BY AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
353.2-1
-------
METHOD 353.2
DETERMINATION OF NITRATE-NITRITE NITROGEN BY AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of nitrite singly, or nitrite
and nitrate combined in drinking, ground, surface, domestic and
industrial wastes.
1.2 The applicable range is 0.05 to 10.0 mg/L nitrate-nitrite nitrogen.
The range may be extended with sample dilution.
2.0 SUMMARY OF METHOD
2.1 A filtered sample is passed through a column containing granulated
copper-cadmium to reduce nitrate to nitrite. The nitrite (that was
originally present plus reduced nitrate) is determined by
diazotizing with sulfanilamide and coupling with N-(l-naphthyl)-
ethylenediamine dihydrochloride to form a highly colored azo dye
which is measured colorimetrically. Separate, rather than combined
nitrate-nitrite, values are readily obtained by carrying out the
procedure first with, and then without, the Cu-Cd reduction step.
2.2 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.3 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
353.2-2
-------
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
4.0 INTERFERENCES
4.1 Build up of suspended matter in the reduction column will restrict
sample flow. Since nitrate and nitrite are found in a soluble
state, samples may be pre-filtered,
353.2-3
-------
4 2 Low results might be obtained for samples that contain high
concentrations of iron, copper or other metals. EDTA is added to
the samples to eliminate this interference.
4 3 Residual chlorine can produce a negative interference by limiting
reduction efficiency. Before analysis, samples should be checked
and if required, dechlorinated with sodium thiosulfate.
4.4 Samples that contain large concentrations of oil and grease will
coat the surface of the cadmium. This interference is eliminated by
pre-extracting the sample with an organic solvent.
4 5 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5 1 The toxicity or carcinogenicity of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Cadmium (7.1)
5.3.2 Phosphoric acid (7.5)
5.3.3 Hydrochloric acid (7.6)
5.3.4 Sulfuric acid (7.8)
5.3.5 Chloroform (7.10, 7.11)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware -- Class A volumetric flasks and pi pets as required.
353.2-4
-------
6.3 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.3.1 Sampling device (sampler)
6.3.2 Multichannel pump
6.3.3 Reaction unit or manifold
6.3.4 Colorimetric detector
6.3.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Granulated cadmium: 40-60 mesh (CASRN 7440-43-9). Other mesh sizes
may be used.
7.2 Copper-cadmium: The cadmium granules (new or used) are cleaned with
dilute HC1 (7.6) and copperized with 2% solution of copper sulfate
(7.7) in the following manner:
7.2.1 Wash the cadmium with HC1 (7.6) and rinse with distilled
water. The color of the cadmium so treated should be
silver.
7.2.2 Swirl 10 g cadmium in 100 ml portions of 2% solution of
copper sulfate (7.7) for 5 min or until blue color partially
fades, decant and repeat with fresh copper sulfate until a
brown colloidal precipitate forms.
7.2.3 Wash the copper-cadmium with reagent water (at least 10
times) to remove all the precipitated copper. The color of
the cadmium so treated should be black.
7.3 Preparation of reduction column. The reduction column is a U-
shaped, 35 cm length, 2 mm I.D. glass tube (Note 1). Fill the
reduction column with distilled water to prevent entrapment of air
bubbles during the filling operations. Transfer the copper-cadmium
granules (7.2) to the reduction column and place a glass wool plug
in each end. To prevent entrapment of air bubbles in the reduction
column, be sure that all pump tubes are filled with reagents before
putting the column into the analytical system.
NOTE 1: Other reduction tube configurations, including a 0.081 I.D.
pump tube, can be used in place of the 2-mm glass tube, if checked
as in 10.1.
7.4 Reagent water: Because of possible contamination, this should be
prepared by passage through an iori exchange column comprised of a
mixture of both strongly acidic-cation and strongly basic-anion
353.2-5
-------
exchange resins. The regeneration of the ion exchange column should
be carried out according to the manufacturer's instructions.
7.5 Color reagent: To approximately 800 ml of reagent water, add, while
stirring, 100 ml cone, phosphoric acid (CASRN 7664-38-2), 40 g
sulfanilamide (CASRN 63-74-1) and 2 g N-1-naphthylethylenediamine
dihydrochloride (CASRN 1465-25-4). Stir until dissolved and dilute
to 1 L. Store in brown bottle and keep in the dark when not in use.
This solution is stable for several months.
7.6 Dilute hydrochloric acid, 6N: Add 50 mL of cone. HC1 (CASRN 7647-
01-0) to reagent water, cool and dilute to 100 ml.
7.7 Copper sulfate solution, 2%: Dissolve 20 g of CuS(y5H20 (CASRN
7758-99-8) in 500 ml of reagent water and dilute to 1 L.
7.8 Wash solution: Use reagent water for unpreserved samples. For
samples preserved with H2S04, use 2 ml H2S04 (CASRN 7764-93-9), per
liter of wash water.
7.9 Ammonium chloride-EDTA solution: Dissolve 85 g of reagent grade
ammonium chloride (CASRN 12125-02-9) and 0.1 g of disodium
ethylenediamine tetracetate (CASRN 6381-92-6) in 900 ml of reagent
water. Adjust the pH to 9.1 for preserved or 8.5 for non-preserved
samples with cone, ammonium hydroxide (CASRN 1336-21-6) and dilute
to 1 L. Add 0.5 ml Brij-35 (CASRN 9002-92-0).
7.10 Stock nitrate solution: Dissolve 7.218 g KN03 (CASRN 7757-79-1) and
dilute to 1 L in a volumetric flask with reagent water. Preserve
with 2 ml of chloroform (CASRN 67-66-3) per liter. Solution is
stable for 6 months. 1 mL = 1.0 mg N03-N.
7.11 Stock nitrite solution: Dissolve 6.072 g KN02 in 500 mL of reagent
water and dilute to 1 L in a volumetric flask. Preserve with 2 mL
of chloroform and keep under refrigeration. 1.0 mL = 1.0 mg N02-N.
7.12 Standard nitrate solution: Dilute 1.0 mL of stock nitrate solution
(7.10) to 100 mL. 1.0 mL = 0.01 mg N03-N. Preserve with .2 mL of
chloroform. Solution is stable for 6 months.
7.13 Standard nitrite solution: Dilute 10.0 mL of stock nitrite (7.11)
solution to 1000 mL. 1.0 mL = 0.01 mg N02-N. Solution is unstable;
prepare as required.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
353.2-6
-------
8.2
8.3
8.4
Samples must be preserved with H2S04
at the time of collection.
to a pH < 2 and cooled to 4°C
Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 28 days.
Samples to be analyzed for nitrate or nitrite only should be cooled
to 4°C and analyzed within 48 hours.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory capability
and the periodic analysis of laboratory reagent blanks, fortified
blanks, and other laboratory solutions as a continuing check on
performance. The laboratory is required to maintain performance
records that define the quality of the data that are generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCR
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
353.2-7
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9.2.4 Method Detection Limit (MDL) ~ MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
353.2-8
-------
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
established an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) -- For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required), and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift, the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case, the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4,.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculate using the
following equation:
R = Cs,~ C x 100
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
353.2-9
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9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10.1 Prepare a series of at least 3 standards, covering the desired
range, and a blank by diluting suitable volumes of standard nitrate
solution (7.12). At least one nitrite standard should be compared to
a nitrate standard at the same concentration to verify the
efficiency of the reduction column.
10.2 Set up manifold as shown in Figure 1. Care should be taken not to
introduce air into the reduction column.
10.3 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10.4 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
the measured value of the calibration solution and the "true value"
concentration.
10.5 After the calibration has been established, it must be verified by
the analysis of a suitable quality control sample (QCS). If
measurements exceed ± 10% of the established QCS value, the analysis
should be terminated and the instrument recalibrated. The new
calibration must be verified before continuing analysis. Periodic
reanalysis of the QCS is recommended as a continuing calibration
check.
NOTE 3: Condition column by running 1 mg/L standard for 10 min if a
new reduction column is being used. Subsequently wash the column
with reagents for 20 min.
11.0 PROCEDURE
11.1 If the pH of the sample is below 5 or above 9, adjust to between 5
and 9 with either cone. HC1 or cone. NH4OH.
11.2 Set up the manifold as shown in Figure 1. Care should be taken not
to introduce air into reduction column.
353.2-10
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11.3 Allow system to equilibrate as required. Obtain a stable baseline
with all reagents, feeding reagent water through the sample line.
11.4 Place appropriate nitrate and/or nitrite standards in sampler in
order of decreasing concentration and complete loading of sampler
tray.
11.5 Switch sample line to sampler and start analysis.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg/L as nitrogen.
13.0 METHOD PERFORMANCE
13.1 Three laboratories participating in an EPA Method Study analyzed
four natural water samples containing exact increments of inorganic
nitrate, with the following results:
Accuracy as
Increment as Precision as
Nitrate Nitrogen Standard Deviation Bias, Bias,
mq N/liter mq N/liter % mq N/liter
0.29 0.012 + 5.75 + 0.017
0.35 0.092 . + 18.10 + 0.063
2.31 0.318 +4.47 + 0.103
2.48 0.176 - 2.69 - 0.067
13.2 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg N03-N/L.
13.3 Single laboratory precision data can be estimated at 50% to 75% of
the inter!aboratory precision estimates.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
353.2-11
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Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess reagents, samples, and method
process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Fiore, 0., and O'Brien, J.E., "Automation in Sanitary Chemistry -
Parts 1 & 2: Determination of Nitrates and Nitrites," Wastes
Engineering 33, 128 &238 (1962).
2. Armstrong, F.A., Stearns, C.R., and Strickland, J.D., "The
Measurement of Upwelling and Subsequent Biological Processes by
Means of the Technicon AutoAnalyzer and Associated Equipment," Deep
Sea Research 14, pp. 381-389 (1967).
3. Annual Book of ASTM Standards, Part 31, "Water," Standard D1254, p.
366 (1976).
4. Standard Methods for the Examination of Water and Wastewater, 17th
Edition, pp. 4-91, Method 4500-N03 F (1992).
353.2-12
-------
Chemical Analyses for Water Quality Manual, Department of the
Interior, FWPCA, R.A. Taft Engineering Center Training Program,
Cincinnati, Ohio 45226 (January, 1966).
Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
353.2-13
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17.0 TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
163
183
213
170
163
172
183
214
172
213
170
214
TRUE
VALUE
(T)
0.250
0.451
0.650
0.950
1.90
2.20
2.41
3.20
6.50
8.00
8.50
10.0
MEAN
(X)
0.2479
0.4441
0.6479
0.9537
1.8987
2.1971
2.3732
3.2042
6.4978
7.9814
8.5135
9.9736
RESIDUAL
FOR X
0.0007
-0.0039
0.0012
0.0074
0.0037
0.0025
-0.0312
0.0109
0.0089
-0.0055
0.0273
-0.0106
STANDARD
DEVIATION
CS)
0.0200
0.0289
0.0398
0.0484
0.0918
0.1164
0.1273
0.1456
0.3156
0.3673
0.3635
0.4353
RESIDUAL
FOR S
-0.0001
-0.0002
0.0017
-0.0031
-0.0024
0.0087
0.0102
-0.0070
0.0148
-0.0008
-0.0271
-0.0227
REGRESSIONS: X = 0.999T + 0.002, S = 0.045T + 0.009
353.2-14
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353.2-15
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METHOD 365.1
DETERMINATION OF PHOSPHORUS BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
365.1-1
-------
METHOD 365.1
DETERMINATION OF PHOSPHORUS BY AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of specified forms of
phosphorus in drinking, ground, and surface waters, and domestic and
industrial wastes.
1.2 The methods are based on reactions that are specific for the
orthophosphate ion. Thus, depending on the prescribed pretreatment
of the sample, the various forms of phosphorus that may be
determined are defined in Section 3 and given in Figure 1.
1.2.1 Except for in-depth and detailed studies, the most commonly
measured forms are total and dissolved phosphorus, total and
dissolved orthophosphate. Hydrolyzable phosphorus is
normally found only in sewage-type samples. Insoluble forms
of phosphorus are determined by calculation.
1.3 The applicable range is 0.01 to 1.0 mg P/L. Approximately 20-30
samples per hour can be analyzed.
2.0 SUMMARY OF METHOD
2.1 Ammonium molybdate and antimony potassium tartrate react in an acid
medium with dilute solutions of phosphorus to form an antimony-
phospho-molybdate complex. This complex is reduced to an intensely
blue-colored complex by ascorbic acid. The color is proportional to
the phosphorus concentration.
2.2 Only orthophosphate forms a blue color in this test. Polyphosphates
(and some organic phosphorus compounds) may be converted to the
orthophosphate form by manual sulfuric acid hydrolysis. Organic
phosphorus compounds may be converted to the orthophosphate form by
manual persulfate digestion.5 The developed color is measured
automatically.
2.3 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.4 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
365.1-2
-------
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) -- A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.,
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) -- The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
365.1-3
-------
3.10 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) ~ A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source
3.12 TOTAL PHOSPHORUS (P) All of the phosphorus present in the sample
regardless of forms, as measured by the persulfate digestion
procedure.
3.12.1 TOTAL ORTHOPHOSPHATE (P-ortho) Inorganic phosphorus
[(PO*)"3] in the sample as measured by the direct
colorimetric analysis procedure.
3.12.2 TOTAL HYDROLYZABLE PHOSPHORUS (P-hydro) Phosphorus in the
sample as measured by the sulfuric acid hydrolysis
procedure, and minus predetermined orthophosphates. This
hydrolyzable phosphorus includes polyphosphates [(P207)~ ,
(P3010)"5, etc.] plus some organic phosphorus.
3.12.3 TOTAL ORGANIC PHOSPHORUS (P-org) -- Phosphorus (inorganic
plus oxidizable organic) in the sample as measured by the
persulfate digestion procedure, and minus hydrolyzable
phosphorus and orthophosphate.
3.13 DISSOLVED PHOSPHORUS (P-D) -- All of the phosphorus present in the
filtrate of a sample filtered through a phosphorus-free filter of
0.45 micron pore size and measured by the persulfate digestion
procedure.
3.13.1 DISSOLVED ORTHOPHOSPHATE (P-D ortho) ~ As measured by he
direct colorimetric analysis procedure.
3.13.2 DISSOLVED HYDROLYZABLE PHOSPHORUS (P-D, hydro) As
measured by the sulfuric acid hydrolysis procedure and minus
predetermined dissolved orthophosphates.
3.13.3 DISSOLVED ORGANIC PHOSPHORUS (P-D, org) ~ As measured by
the persulfate digestion procedure, and minus dissolved
hydrolyzable phosphorus and orthophosphate.
3.14 The following forms, when sufficient amounts of phosphorus are
present in the sample to warrant such consideration, may be
calculated:
3.14.1 INSOLUBLE PHOSPHORUS (P-I) = (P)-(P-D).
365.1-4
-------
3.14.1.1 INSOLUBLE ORTHOPHOSPHATE (P-I, ortho) = (P, ortho)
- (P-D, ortho).
3.14.1.2 INSOLUBLE HYDROLYZABLE PHOSPHORUS (P-I, hydro) =
(P, hydro) - (P-D, hydro).
3.14.1.3 INSOLUBLE ORGANIC PHOSPHORUS (P-I, org) = (P,
org) -(P-D, org).
3.15 All phosphorus forms shall be reported as P, mg/L, to the third
place.
4.0 INTERFERENCES
4.1 No interference is caused by copper, iron, or silicate at
concentrations many times greater than their reported concentration
in seawater. However, high iron concentrations can cause
precipitation of, and subsequent loss, of phosphorus.
4.2 The salt error for samples ranging from 5% to 20% salt content was
found to be less than 1%.
4.3 Arsenate is determined similarly to phosphorus and should be
considered when present in concentrations higher than phosphorus.
However, at concentrations found in sea water, it does not
interfere.
4.4 Sample turbidity must be removed by filtration prior to analysis for
orthophosphate. Samples for total or total hydrolyzable phosphorus
should be filtered only after digestion. Sample color that absorbs
in the photometric range used for analysis will also interfere.
4.5 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
, have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
365.1-5
-------
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Sulfuric acid (7.2, 7.7)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware -- Class A volumetric flasks and pi pets as required.
6.3 Hot plate or autoclave'.
6.4 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.4.1 Sampling device (sampler)
6.4.2 Multichannel pump
6.4.3 Reaction unit or manifold
6.4.4 Colorimetric detector
6.4.5 Data recording device
6.5 Acid-washed glassware: All glassware used in the determination
should be washed with hot 1:1 HC1 and rinsed with distilled water.
The acid-washed glassware should be filled with distilled water and
treated with all the reagents to remove the last traces of
phosphorus that might be adsorbed on the glassware. Preferably,
this glassware should be used only for the determination of
phosphorus and after use it should be rinsed with distilled water
and kept covered until needed again. If this is done, the treatment
with 1:1 HC1 and reagents is only required occasionally. Commercial
detergent should never be used.
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the analyte of
interest. ASTM type II or equivalent.
7.2 Sulfuric acid solution, 5N: Slowly add 70 ml of cone. H-,SO, (CASRN
7664-93-9) to approximately 400 ml of distilled water. Cool to, room
temperature and dilute to 500 mL with distilled water.
7.3 Antimony potassium tartrate solution: Weight 0.3 g
K(SbO)C4H,06.l/2H20 (CASRN 28300-74-5), dissolved in 50 ml distilled
water in 100-mL volumetric flask, dilute to volume. Store at 4°C in
a dark, glass-stoppered bottle.
365.1-6
-------
7.4 Ammonium molybdate solution: Dissolve 4 g (NH,),Mo70P,.4H,0 (CASRN
12027-67-7) in 100 ml reagent water. Store in a plastic-Wtle at
T" L »
7.5 Ascorbic acid, 0.1M: Dissolve 1.8 g of ascorbic acid (CASRN 50-81-
7) in 100 ml of reagent water. The solution is stable for about a
week if prepared with water containing no more than trace amounts of
heavy metals and stored at 4°C.
7.6 Combined reagent: Mix the above reagents in the following
proportions for 100 ml of the mixed reagent: 50 ml of 5N H?SO,
(7.2), 5 ml of antimony potassium tartrate solution (7.3), 15 ml of
ammonium molybdate solution (7.4), and 30 ml of ascorbic acid
solution (7.5). Mix after addition of each reagent. All reagents
must reach room temperature before they are mixed and must be mixed
in the order given. If turbidity forms in the combined reagent,
shake and let stand for a few minutes until the turbidity disappears
before processing. This volume is sufficient for 4 h operation.
Since the stability of this solution is limited, it must be freshly
prepared for each run.
NOTE 1: A stable solution can be prepared by not including the
ascorbic acid in the combined reagent. If this is done, the mixed
reagent (molybdate, tartrate, and acid) is pumped through the
distilled water line and the ascorbic acid solution (30 ml of 7.5
diluted to 100 ml with reagent water) through the original mixed
reagent line.
7.7 Sulfuric acid solution, 11 N: Slowly add 155 ml cone. HPSO, to 600
mL reagent water. When cool, dilute to 500 ml.
7.8 Ammonium persulfate (CASRN 7727-54-0).
7.9 Acid wash water: Add 40 mL of sulfuric acid solution (7.7) to 1 L
of reagent water and dilute to 2 L. (Not to be used when only
orthophosphate is being determined).
7.-10 Phenolphthalein indicator solution (5 g/L): Dissolve 0 5 g of
phenolphthalein (CASRN 77-09-8) in a solution of 50 ml of isopropyl
alcohol (CASRN 67-63-0) and 50 ml of reagent water.
7.11 Stock phosphorus solution: Dissolve 0.4393 g of predried (105°C for
1 h) Potassium phosphate monobasic: KH2PQ, (CASRN 7778-77-0) in
reagent water and dilute to 1000 ml. 1.0 ml = 0.1 mg P.
7.12 Standard phosphorus solution: Dilute 10.0 ml of stock solution
(7.11) to 100 ml with reagent water. 1.0 ml = 0.01 mg P.
7.13 Standard phosphorus solution: Dilute 10.0 ml of standard solution
(7.12) to 100 ml with reagent water. 1.0 mL - 0.001 mg P.
365.1-7
-------
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleaned and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
8.2 Samples must be preserved with H2S04 to a pH < 2 and cooled to 4°C
at the time of collection.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 28 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
365.1-8
-------
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit.(5) To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
365.1-9
-------
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
establish an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required) and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculate using the
following equation:
365.1-10
-------
R = f x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10.1 Prepare a series of at least 3 standards, covering the desired
range, and a blank by pipetting and diluting suitable volumes of
working standard solutions (7.12 or 7.13) into 100 ml volumetric
flasks. Suggested ranges include 0.00 to 0.10 and 0.20 to 1.00
mg/L.
10.2 Process standards and blanks as described in Sect. 11, Procedure.
10.3 Set up manifold as shown in Figure 2.
10.4 Prepare flow system as described in Sect. 11, Procedure.
10.5 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10.6 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
the measured value of the calibration solution and the "true value"
concentration.
10.7 After the calibration has been established, it must be verified by
the analysis of a suitable quality control sample (QCS). If
measurements exceed ± 10% of the established QCS value, the analysis
should be terminated and the instrument recalibrated. The new
calibration must be verified before continuing analysis. Periodic
365.1-11
-------
reanalysis of the QCS is recommended as a continuing calibration
check.
11.0 PROCEDURE
11.1 Phosphorus
11.1.1 Add 1 ml of sulfuric acid.solution (7.7) to a 50 ml sample
and/or standard in a 125-mL Erlenmeyer flask.
11.1.2 Add 0.4 g of ammonium persulfate (7.8).
11.1.3 Boil gently on a pre-heated hot plate for approximately 30-
40 min or until a final volume of about 10 ml is reached.
Do not allow sample to go to dryness. Alternately, heat for
30 min in an autoclave at 121°C (15-20 psi).
11.1.4 Cool and dilute the sample to 50 ml. If sample is not clear
at this point, filter.
11.1.5 Determine phosphorus as outlined (11.3.2) with acid wash
water (7.9) in wash tubes.
11.2 Hydrolyzable Phosphorus
11.2.1. Add 1 mL of sulfuric acid solution (7.7) to a 50 ml sample
and/or standard in a 125 ml Erlenmeyer flask.
11.2.2 Boil gently on a pre-heated hot plate for 30-40 min until a
final volume of about 10 ml is reached. Do not allow sample
to go to dryness. Alternatively, heat for 30 min in an
autoclave at 121°C (15-20 psi).
11.2.3 Determine phosphorus as outlined (11.3.2) with acid wash
water (7.9) in wash tubes.
11.3 Orthophosphate
11.3.1 Add 1 drop of phenolphthalein indicator solution (7.10) to
approximately 50 ml of sample. If a red color develops, add
sulfuric acid solution (7.7) drop-wise to just discharge the
color. Acid samples must be neutralized with 1 N sodium
hydroxide (40 g NaOH/L).
11.3.2 Set up manifold as shown in Figure 1.
11.3.3 Allow system to equilibrate as required. Obtain a stable
baseline with all reagents, feeding reagent water through
the sample line.
11.3.4 Place standards in sampler in order of decreasing
concentration, and complete filling of sampler tray.
365.1-12
-------
11.3.5 Switch sample line from reagent water to Sampler and begin
analysis.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lotoest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed. Any sample whose
computed value is less than 5% of its immediate predecessor must be
rerun.
12.3 Report results in mg P/L.
13.0 METHOD PERFORMANCE
13.1 Six laboratories (using Technicon AAI equipment) participating in an
EPA Method Study, analyzed four natural water samples containing
exact increments of orthophosphate, with the following results:
Increment as Precision as Accuracy as
Orthophosphate Standard Deviation Bias Bias
mg P/liter mo P/liter _% mq P/liter
0.04 0.019 +16.7 +0.007
0.04 0.014 -8.3 -0.003
0.29 0.087 -15.5 -0.05
0.30 0.066 -12.8 -0.04
13.2 In a single laboratory (EMSL), using surface water samples at
concentrations of 0.04, 0.19, 0.35, and 0.84 mg P/L, standard
deviations were ±0.005, ±0.000, ±0.003, and ±0.000, respectively.
13.3 In a single laboratory (EMSL), using surface water samples at
concentrations of 0.07 and 0.76 mg P/L, recoveries were 99% and
100%, respectively.
13.4 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg P04-P/L.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
365.1-13
-------
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government Regulations
and Science Policy, 1155 16th Street N.W., Washington D.C. 20036,
(202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess reagents, samples and method
process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods, and bench operations, complying with the letter and spirit of
any waster discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Murphy, J. and Riley, J., "A Modified Single Solution for the
Determination of Phosphate in Natural Waters." Anal. Chim. Acta.,
27, 31 (1962).
2. Gales, M., Jr., Julian, E., and Kroner, R., "Method for Quantitative
Determination of Total Phosphorus in Water." Jour. AWWA, 58. No.
10, 1363 (1966).
3. Lobring, L.B. and Booth, R.L., "Evaluation of the AutoAnalyzer II; A
Progress Report," Technicon International Symposium, June, 1972,
New York, N.Y.
4. Standard Methods for the Examination of Water and Wastewater, 18th
Edition, p. 4-116, Method 4500-P F (1992).
5. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
365.1-14
-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
54
69
88
87
57
69
53
87
64
57
88
63
TRUE
VALUE
(T)
0.150
0.351
0.625
1.80
2.50
2.75
3.50
3,60
4.00
7.01
8.20
9.00
MEAN
(X)
0.1530
0.3670
0.6090
1.7374
2.4867
2.8344
3.5619
3.4957
3.8523
6.9576
8.0995
8.6717
RESIDUAL
FOR X
-0.0017
0.0140
-0.0141
-0.0444
0.0146
0.1158
0.1038
-0.0610
-0.0989
0.0383
0.0068
-0.2099
STANDARD
DEVIATION
(S) i
0.0128
0.0368
0.0413
0.1259
0.1637
0.2019
0.2854
0.2137
0.3158
0.5728
0.5428
0.6770
RESIDUAL
FOR S
-0.0010
0.0084
-0.0069
-0.0072
-0.0200
0.0002
0.0295
-0.0495
0.0237
0.0632
-0.0528
0.0236
REGRESSIONS: X - 0.986T + 0.007, S = 0.072T + 0.003
365.1-15
-------
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METHOD 375.2
DETERMINATION OF SULFATE BY AUTOMATED COLORIMETRY
Edited by James W. O'De'll
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
375.2-1
-------
METHOD 375.2
DETERMINATION OF SULFATE IN WATER BY AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This automated method is applicable to drinking, ground and surface
water, domestic and industrial wastes.
1.2 The applicable range is 3 to 300 mg S04/L. The sensitivity of the
method can be increased by a minor modification to analyze samples
in the range of 0.5 to 30 mg S04/L. Approximately 30 samples per
hour can be analyzed.
2.0 SUMMARY OF METHOD
2.1 The sample is first passed through a sodium form cation-exchange
column to remove multivalent metal ions. The sample containing
sulfate is then reacted with an alcohol solution of barium chloride
and methyl thymol blue (MTB) at a pH of 2.5-3.0 to form barium
sulfate. The combined solution is raised to a pH of 12.5-13.0 so
that excess barium reacts with MTB. The uncomplexed MTB color is
gray; if it is all chelated with barium, the color is blue.
Initially, the barium and MTB are equimolar and equivalent to 300 mg
S04/L; thus the amount of uncomplexed MTB is equal to the sulfate
present.
2.2 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.3 Limited performance-based method mpdifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
375.2-2
-------
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) -- Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
\
3.9 METHOD DETECTION LIMIT (MDL) -- The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
375.2-3
-------
4.0 INTERFERENCES
4.1 The ion exchange column eliminates interferences from multivalent
cations. A mid-scale sulfate standard containing Ca+* should be
analyzed periodically to insure that the column is functioning
properly.
4.2 Samples with pH below 2 should be neutralized because high acid
concentrations elute cations from the ion exchange resin.
4.3 Turbid samples should be filtered or centrifuged.
4.4 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Barium chloride (7.2)
5.3.2 Hydrochloric acid (7.3)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware Class A volumetric flasks and pi pets as required.
6.3 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.3.1 Sampling device (sampler)
6.3.2 Multichannel pump
375.2-4
-------
6.3.3 Reaction unit or manifold
6.3.4 Colorimetric detector
. 6.3.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the analyte of
interest. ASTM type II or equivalent.
7.2 Barium chloride: Dissolve 0.7630 g of barium chloride dihydrate
(BaCl2-2H20) (CASRN 10326-27-9) in 250 ml of reagent water and
dilute to 500 ml.
7.3 Methyl thymol blue: Dissolve 0.1182 g of methyl thymol blue (3'3-bis-
N,N-biscarboxymethyl)-amino methyl thymolsulfone-phthalein
pentasodium salt) (CASRN 1945-77-3) in 25 ml of barium chloride
solution (7.2). Add 4 ml of 1.0 N hydrochloric acid (CASRN 7647-01-
0) which changes the color to bright orange. Add 71 ml of reagent
water and dilute to 500 ml with ethanol. The pH of this solution is
2.6. This reagent should be prepared the day before and stored in a
brown plastic bottle in the refrigerator.
7.4 Buffer, pH 10.5 ± 0.5: Dissolve 6.75 g of ammonium chloride (CASRN
12125-02-9) in 500 ml of reagent water. Add 57 ml of concentrated
ammonium hydroxide (CASRN 1336-21-6) and dilute to 1 L with
distilled water.
7.5 Buffered EDTA: Dissolve 20 g of tetrasodium EDTA (CASRN 64-02-8) in
J pH 10.5 buffer (7.4), and dilute to 500 ml with buffer.
7.6 Sodium hydroxide solution (50%): Dissolve 250 g NaOH (CASRN 1310-
73-2) in 300 ml of reagent water, cool, and dilute to 500 ml.
7.7 Sodium hydroxide, 0.18N: Dilute 7.2 ml of sodium hydroxide solution
(7.6) to 500 ml.
7.8 Ion exchange resin: Bio-Rex 70, 20-50 mesh, sodium form, Bio-Rad
Laboratories, Richmond, California. Free from fines by stirring
with several portions of reagent water and decant the supernate
before settling is complete.
7.9 Dilution Water: Add 0.75 ml of sulfate stock solution (7.10) and 3
drops of Brij-35 (CASRN 9002-92-0) to 2 L of reagent water.
7.10 Sulfate stock solution, 1 ml = 1 mg SO,: Dissolve 1.479. g of dried
(105°C) Na2S04 (CASRN 7757-82-6) in reagent water and dilute to 1 L.
7.11 Dilute sulfate solution, 1 ml = 0.1 mg S04: Dilute 50 ml of sulfate
stock solution (7.10) to 500 ml with reagent water.
375.2-5
-------
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleansed and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
8.2 No chemical preservation required. Cool sample to 4°C.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, samples maintained at 4°C may be held for up
to 28 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
375.2-6
-------
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with, on-going analyses.
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit.c ' To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates],
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) -- The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) -- The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
375.2-7
-------
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
established an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required) and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case, the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculate using the
following equation:
375.2-8
-------
R = Cs " C x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10.1 Set up the manifold for high (0-300 mg S04/L) or low (0-30 mg S04/L)
level samples as described in Figure 1.
10.1.1 High level working standards, 10-300 mg/L: As a minimum,
prepare high level working standards by diluting the
following volumes of stock standard (7.10) to 100 ml.
ml Stock mq/L SO^
1 10
5 50
10 100
15 150
25 250
30 300
10.1.2 Low level working standards, 1-30 mg/L: Prepare at least
this number of low level working standards by diluting the
following volumes of dilute sulfate solution (7.11) to 100
mL.
375.2-9
-------
mL Stock
1
5
10
15
25
30
mq/L SO,
1.0
5.0
10.0
15.0
25.0
30.0
10.2 The ion exchange column is prepared by pulling a slurry of the resin
into a piece of glass tubing 7.5 inches long, 2.0 mm ID and 3.6 mm
OD. This is conveniently done by using a pipet and a loose fitting
glass wool plug in the tubing. Care should be taken to avoid
allowing air bubbles to enter the column. If air bubbles become
trapped, the column should be prepared over again. The column can
exchange the equivalent of 35 mg of calcium. For the high level
manifold this corresponds to about 900 samples with 200 mg/L Ca.
The column should be prepared as often as necessary to assure that
no more than 50% of its capacity is used up.
10.3 Allow the instrument to warm up as required.
until a stable baseline is achieved.
Pump all reagents
10.4 Analyze all working standards in duplicate at the beginning of a run
to develop a standard curve. Control standards are analyzed every
hour to assure that the system remains properly calibrated. Since
the chemistry is non-linear, data recording devices should be
adjusted accordingly.
10.5 Prepare standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based on regression curve fitting techniques. Acceptance
or control limits should be established using the difference the
measured value of the calibration solution and the "true value"
concentration.
10.6 After the calibration has been established, it must be verified by
the analysis of a suitable quality control sample (QCS). If
measurements exceed ± 10% of the established QCS value, the analysis
should be terminated and the instrument recalibrated. The new
calibration must be verified before continuing analysis. Periodic
reanalysis of the QCS is recommended as a continuing calibration
check.
11.0 PROCEDURE
11.1 Set up instrument as specified under calibration and standardization
(10.0).
375.2-10
-------
11.2 Fill and connect reagent containers and start system. Allow the
system to equilibrate as required. Pump all reagents until a stable
baseline is achieved.
11.3 Place standards and samples in sampler tray. Calibrate instrument,
and begin analysis.
11.4 At the end of each day, the system should be washed with the
buffered EDTA solution (7.5). This is done by placing the
methyl thymol blue line and the sodium hydroxide line in reagent
water for a few minutes and then in the buffered EDTA solution for
10 min. Wash the system with reagent water for 15 min before
shutting down.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg/L.
13.0 METHOD PERFORMANCE
13.1 In a single laboratory the estimated standard deviation, calculated
from duplicate analyses of 26 surface and wastewaters at a mean
concentration of 100 mg/L was ±1.6 mg/L.
13.2 The mean recovery from 24 surface and wastewaters was 102%.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
375.2-11
-------
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess Reagents and samples and method
process wastes should be characterized and disposed of in an
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods and bench operations, complying with the letter and spirit of
any waster discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Lazrus, A.L., Hill, K.C. and Lodge, J.P., "Automation in Analytical
Chemistry," Technicon Symposia, 1965.
2. Coloros, E., Panesar, M.R. and Parry, F.P., "Linearizing the ,
Calibration Curve in Determination of Sulfate by the Methyl thymol
Blue Method," Anal. Chem. 48, 1693 (1976).
3. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
375.2-12
-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
»
N WATER
8
8
LU
§
Li
e
in
G5
S
2
Si
6 8 1
in
.Q
3
03
i
03
-3
§
.2
3
375.2-13
-------
-------
METHOD 410.4
THE DETERMINATION OF CHEMICAL OXYGEN DEMAND
BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2,,0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
410.4-1
-------
METHOD 410.4
THE DETERMINATION OF CHEMICAL OXYGEN DEMAND
BY SEMI-AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of chemical oxygen demand (COD)
in ground and surface waters, domestic and industrial wastes.
1.2 The applicable range is 3-900 mg/L.
2.0 SUMMARY OF METHOD
2.1 Sample, blanks, and standards in sealed tubes are heated in an oven
or block digestor in the presence of dichromate at 150°C. After two
hours, the tubes are removed from the oven or digester, cooled, and
measured spectrophotometrically at 600 nm. The colorimetric
determination may also be performed manually.
2.2 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.3 Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) -- A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
410.4-2
-------
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) -- The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
4.0 INTERFERENCES
4.1 Chlorides are quantitatively oxidized by dichromate and represent a
positive interference. Mercuric sulfate is added to the digestion
tubes to complex the chlorides.
4.2 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
410.4-3
-------
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been fully established. Each chemical should be regarded as
a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Mercuric sulfate (7.2)
5.3.2 Potassium dichromate (7.2)
5.3.3 Sulfuric acid (7.2, 7.3, 7,4)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware Class A volumetric flasks and pi pets as required.
6.3 Block digestor or drying oven capable of maintaining 150°C.
6.4 Muffle furnace capable of 500°C.
6.5 Culture tube with Teflon-lined screw cap, 16 x 100 mm or 25 x 150
mm.
6.6 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.6.1 Sampling device (sampler)
6.6.2 Multichannel pump
6.6.3 Reaction unit or manifold
6.6.4 Colorimetric detector
6.6.5 Data recording device
410.4-4
-------
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the analyte of
interest. ASTM type II or equivalent.
7.2 Digestion solution: Add 5.1 g potassium dichromate K2Cr207 (CASRN
7778-50-9), 84 ml cone, sulfuric acid H,S04 (CASRN 8014-95-7) and
16.7 g mercuric sulfate HgS04 (CASRN 7783-35-9) to 250 ml of reagent
water, cool and dilute to 500 ml. CAUTION: CAN BE VERY HOT!
7.3 Catalyst solution: Add 22 g silver sulfate Ag2S04 (CASRN 10294-26-
5) to a 4.09 kg bottle of cone. H2S04. Stir until dissolved.
7.4 Sampler wash solution: Add 250 mL of cone. H2S04 to 250 ml of
reagent water. CAUTION: PREPARE CAREFULLY, HIGH HEAT GENERATION!
7.5 Stock potassium hydrogen phthalate standard: Dissolve 0.425 g KHP
(CASRN 877-24-7) in 400 mL of reagent water and dilute to 500 mL.
1 mL = 1 mg COD.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All
bottles must be thoroughly cleansed and rinsed with reagent water.
Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize
waste disposal.
8.2 Samples must be preserved with H2S04 to a pH < 2 and cooled to 4°C
at the time of collection.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples maintained at 4°C may be held
for up to 28 days.
9.0 QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks, and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of linear
calibration ranges and analysis of QCS) and laboratory
410.4-5
-------
performance (determination of MDLs) prior to performing
analyses by this method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) -- MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit.<2) To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL - (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
410.4-6
-------
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) ~ The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
established an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) -- For all
determinations, the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required), and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift, the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
410.4-7
-------
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case, the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample, and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculated using the
following equation:
R = f _ x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C - sample background concentration.
s = concentration equivalent of analyte added to
sample.
9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARDIZATION
10.1 Prepare a series of at least 3 standards, covering the desired
range, by diluting appropriate volumes of the stock standard
(7.5)and a blank.
10.2 Process standards and blanks as described under Procedure (11.0).
10.3 Set up manifold as shown in Figure 1.
410.4-8
-------
10.4 Allow the instrument to warm up as required. Pump all reagents
until a stable baseline is achieved.
10.5 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10.6 Prepare a standard curve by plotting instrument response against
concentration values. A calibration curve may be fitted to the
calibration solutions concentration/response data using computer or
calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between
the measured value of the calibration solution and the "true value"
concentration.
10.7 After the calibration has been established, it must be verified by
the analysis of a suitable QCS. If measurements exceed ± 10% of the
established QCS value, the analysis should be terminated and the
instrument recalibrated. The new calibration must be verified
before continuing analysis. Periodic reanalysis of the QCS is
recommended as a continuing calibration check.
11.0 PROCEDURE
11.1 Wash all culture tubes and screw caps with 20% H2SO, before their
first use to prevent contamination. Trace contamination may be
removed from the tubes by igniting them in a muffle furnace at 500°C
for 1 h.
11.2 Pi pet 2.5 mL of sample, standard or blank, into 16 x 100 mm tubes or
10 mL into 25 x 100 mm tubes.
11.3 Add 1.5 ml of digestion solution (7.2) to the 16 x 100 mm tubes or
6.0 ml to the 25 x 150 mm tubes and mix.
11.4 Add 3.5 ml of catalyst solution (7.3) carefully down the side of the
16 x 100 mm tubes or 14.0 ml to the 25 x 150 mm tubes.
11.5 Cap tubes tightly and shake to mix layer. CAUTION: Tubes are hot.
11.6 Place tubes into a block digester or oven at 150°C and heat for 2 h.
11.7 Remove, mix, and cool tubes. Allow any precipitate to settle.
11.8 Fill and connect reagent containers and start system. Allow the
instrument to warm up as required., Pump all reagents until a stable
baseline is achieved.
11.9 Place standards, blanks, and samples in sampler tray. Calibrate
instrument, and begin analysis.
410.4-9
-------
12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in mg/L.
13.0 METHOD PERFORMANCE
13.1 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg COD/L.
13.2 Single laboratory precision data can be estimated at 50 to 75% of
the inter!aboratory precision estimates.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess reagents, samples, and method
process wastes should be characterized and disposed of in an
410.4-10
-------
1
acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Jirka, A.M., and M.J. Carter, "Micro-Semi-Automated Analysis of
Surface and Wastewaters for Chemical Oxygen Demand." Anal. Chem.
47:1397, (1975).
2. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
410.4-11
-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TflRI F 1 . INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
241
144
140
112
261
181
262
182
141
250
144
113
TRUE
VALUE
(T)
18.2
26.3
28.5
43.5
46.6
50.0
65.4
76.2
91.7
121
201
229
MEAN
(X)
18.9398
26.1454
32.7275
42.8360
45.3034
49.4740
63.2876
75.7960
94.0772
117.7424
196.9391
221.8109
RESIDUAL
FOR X
-0.4220
-1.0445
3.4115
-0.9763
-1.5049
-0.6201
-1.6894
0.3816
3.6833
-0.9678
0.9151
-1.2730
STANDARD
DEVIATION
(S)
5.2026
5.6142
6.2230
6.4351
6.7677
7.0494
7.6041
8.4490
7.9289
9.6197
14.6995
17.3403
RESIDUAL
FOR S
-0.0964
-0.0888
0.4103
-0.1257
0.0523
0.1644
-0.0489
0.2573
-1.0358
-0.8063
0.2837
1.5280
REGRESSIONS: X - 0.966T - 1.773, S = 0.050T + 4.391
410.4-12
-------
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410.4-13
-------
-------
METHOD 420.4
DETERMINATION OF TOTAL RECOVERABLE PHENOLICS
BY SEMI-AUTOMATED COLORIMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 1.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
420.4-1
-------
METHOD 420.4
DETERMINATION OF TOTAL RECOVERABLE PHENOLICS
BY SEMI-AUTOMATED COLORIMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of phenolic materials in
drinking, ground, surface, and saline waters, and domestic and
industrial wastes.
1.2 The applicable range is from 2 to 500 /zg/L. The working ranges are
2 to 200 /zg/L and 10 to 500 /zg/L.
2.0 SUMMARY OF METHOD
2.1 This semi-automated method is based on the distillation of phenol
and subsequent reaction of the distillate with alkaline ferricyanide
and 4-aminoantipyrine to form a red complex which is measured at 505
or 520 nm.
2.2 Color response of phenolic materials with 4-aminoantipyrine is not
the same for all compounds. Because phenolic type wastes usually
contain a variety of phenols, it is not possible to duplicate a
mixture of phenols to be used as a standard. For this reason,
phenol has been selected as a standard and any color produced by the
reaction of other phenolic compounds is reported as phenol. This
value will represent the minimum concentration of phenolic compounds
present in the sample.
2.3 Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.
2.4 Limited performance based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0 DEFINITIONS
3.1 CALIBRATION BLANK (CB) A volume of reagent water fortified with
the same matrix as the calibration standards, but without the
analytes, internal standards, or surrogate analytes.
3.2 CALIBRATION STANDARD (CAL) ~ A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
420.4-2
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3.3 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.4 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
other blank matrices to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3,6 LABORATORY REAGENT BLANK (LRB) An aliquot of reagent water or
other blank matrices that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the
apparatus.
3.7 LINEAR CALIBRATION RANGE (LCR) -- The concentration range over which
the instrument response is linear.
3.8 MATERIAL SAFETY DATA SHEET (MSDS) Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical
properties, fire, and reactivity data including storage, spill, and
handling precautions.
3.9 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99%
confidence that the analyte concentration is greater than zero.
3.10 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations that is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.11 STOCK STANDARD SOLUTION (SSS) A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
420.4-3
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4.0 INTERFERENCES
4.1 Interferences from sulfur compounds are eliminated by acidifying the
sample to a pH of 4.0 and aerating briefly by stirring.
4.2 Oxidizing agents such as chlorine, detected by the liberation of
iodine upon acidification in the presence of potassium iodide, are
removed immediately after sampling by the addition of an excess of
ferrous ammonium sulfate (7.11). If chlorine is not removed, the
phenolic compounds may be partially oxidized and the results may be
low.
4.3 Background contamination from plastic tubing and sample containers
is eliminated by filling the wash receptacle by siphon (using Kel-F
tubing) and using glass tubes for the samples and standards.
4.4 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus
that bias analyte response.
5.0 SAFETY
5.1 The toxicity or carcinogen!city of each reagent used in this method
have not been fully established. Each chemical should be regarded
as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel
involved in the chemical analysis. The preparation of a formal
safety plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or
hazardous, consult MSDS.
5.3.1 Potassium ferricyanide (7.2)
5.3.2 Phenol (7.5)
5.3.3 Sulfuric acid (7.10)
6.0 EQUIPMENT AND SUPPLIES
6.1 Balance Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.2 Glassware Class A volumetric flasks and pipets as required.
420.4-4
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6.3 Distillation apparatus, all glass consisting of a 1-L pyrex
distilling apparatus with Graham condenser. Reduced volume
apparatus also may be used.
6.4 pH meter with electrodes.
6.5 Automated continuous flow analysis equipment designed to deliver and
react sample and reagents in the required order and ratios.
6.5.1 Sampling device (sampler)
6.5.2 Multichannel pump
6.5.3 Reaction unit or manifold
6.5.4 Colorimetric detector
6.5.5 Data recording device
7.0 REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the analyte of
interest. ASTM type II or equivalent.
7.2 Buffered potassium ferricyanide: Dissolve 1.0 g potassium
ferricyanide (CSRN 13746-66-2), 1.55 g boric acid (CASRN 10043-35-
3), and 1.875 g potassium chloride (CASRN 7447-40-7) in 400 ml of
reagent water. Adjust to pH of 10.3 with 1 N sodium hydroxide
(CASRN 1310-73-2) (7.3) and dilute to 500 ml. Add 0.25 mL of Brij-
35 (CASRN 9002-92-0). Prepare fresh weekly.
7.3 Sodium hydroxide (IN): Dissolve 20 g NaOH in 250 ml of reagent
water, cool and dilute to 500 ml.
7.4 4-Aminoantipyrine: Dissolve 0.13 g of 4-aminoantipyrine (CASRN 83-
07-8) in 150 ml of reagent water and dilute to 200 mL. Prepare
fresh each day.
7.5 Stock phenol: Dissolve 0.50 g phenol (CASRN 108-95-2) in 500 ml of
reagent water and dilute to 500 ml. Add 0.25 ml cone. H2SO, (CASRN
7664-93-9) as preservative. 1.0 mL = 1.0 mg phenol.
7.6 Standard phenol solution A: Dilute 1.0 ml of stock phenol solution
(7.5) to 100 ml with reagent water. 1.0 ml = 0.01 mg phenol.
7.7 Standard phenol solution B: Dilute 10.0 ml of standard phenol
solution A (7.6) to 100 ml with reagent water. 1.0 mL = 0.001 mg
phenol.
7.8 Standard solution C: Dilute 10.0 mL of standard phenol solution B
(7.7) to 100 mL with reagent water. 1.0 mL = 0.0001 mg phenol.
420.4-5
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7.9 Sodium hydroxide, 1+9: Dilute 10 ml of IN NaOH (7.3) to 100 mL with
reagent water.
7 10 Sulfuric acid, 1+9 : Slowly add 10 ml cone. H2S04 (CASRN 7764-93-9)
to 70 ml of reagent water. Cool and dilute to 100 ml with reagent
water.
7.11 Ferrous ammonium sulfate: Dissolve 0.55 g ferrous ammonium sulfate
in 250 ml reagent water containing 0.5 ml H2S04 and dilute to 500 mL
with freshly boiled and cooled reagent water.
8.0 SAMPLE COLLECTION. PRESERVATION AND STORAGE
8 1 Samples should be collected in glass bottles only. All bottles must
be thoroughly cleansed and rinsed with reagent water. Volume
collected should be sufficient to insure a representative sample,
allow for replicate analysis (if required), and minimize waste
disposal.
8.2 Samples must be preserved at time of collection with H2S04 to a pH
of < 2 and cooled to 4°C.
8.3 Samples should be analyzed as soon as possible after collection. If
storage is required, preserved samples are maintained at 4°C and may
be held up to 28 days.
9.0 QUALITY CONTROL
9.1
Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks,
fortified blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to maintain per-
formance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of LCRs
and analysis of QCS) and laboratory performance
(determination of MDLs) prior to performing analyses by this
method.
9.2.2 Linear Calibration Range (LCR) The LCR must be determined
initially and verified every 6 months or whenever a
significant change in instrument response is observed or
expected. The initial demonstration of linearity must use
sufficient standards to insure that the resulting curve is
linear. The verification of linearity must use a minimum of
a blank and three standards. If any verification data
420.4-6
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exceeds the initial values by ± 10%, linearity must be
reestablished. If any portion of the range is shown to be
nonlinear, sufficient standards must be used to clearly
define the nonlinear portion.
9.2.3 Quality Control Sample (QCS) When beginning the use of
this method, on a quarterly basis or as required to meet
data-quality needs, verify the calibration standards and
acceptable instrument performance with the preparation and
analyses of a QCS. If the determined concentrations are not
within ± 10% of the stated values, performance of the
determinative step of the method is unacceptable. The
source of the problem must be identified and corrected
before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.4 Method Detection Limit (MDL) MDLs must be established for
all analytes, using reagent water (blank) fortified at a
concentration of two to three times the estimated instrument
detection limit.(4) To determine MDL values, take seven
replicate aliquots of the fortified reagent water and
process through the entire analytical method. Perform all
calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
MDLs should be determined every 6 months, when a new
operator begins work or whenever there is a significant
change in the background or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) The laboratory must
analyze at least one LRB with each batch of samples. Data
produced are used to assess contamination from the
laboratory environment. Values that exceed the MDL indicate
laboratory or reagent contamination should be suspected and
corrective actions must be taken before continuing the
analysis.
9.3.2 Laboratory Fortified Blank (LFB) The laboratory must
analyze at least one LFB with each batch of samples.
Calculate accuracy as percent recovery (Sect. 9.4.2). If
the recovery of any analyte falls outside the required
420.4-7
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control limits of 90-110%, that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to assess
laboratory performance against the required control limits
of 90-110%. When sufficient internal performance data
become available (usually a minimum of 20-30 analyses),
optional control limits can be developed from the percent
mean recovery (x) and the standard deviation (S) of the mean
recovery. These data can be used to establish the upper and
lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each five to
ten new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
established an on-going precision statement for the level of
concentrations included in the LFB. These data must be kept
on file and be available for review.
9.3.4 Instrument Performance Check Solution (IPC) -- For all
determinations the laboratory must analyze the IPC (a mid-
range check standard) and a calibration blank immediately
following daily calibration, after every tenth sample (or
more frequently, if required), and at the end of the sample
run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the
instrument is within ± 10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is
still within ± 10%. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms calibration
to be outside the limits, sample analysis must be
discontinued, the cause determined and/or in the case of
drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must
be kept on file with the sample analyses data.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) -- The laboratory
must add a known amount of analyte to a minimum of 10% of
the routine samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis. The
analyte concentration must be high enough to be detected
above the original sample and should not be less than four
' 420.4-8
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times the MDL. The added analyte concentration should be
the same as that used in the laboratory fortified blank.
9.4.2 Calculate the percent recovery for each analyte, corrected
for concentrations measured in the unfortified sample and
compare these values to the designated LFM recovery range
90-110%. Percent recovery may be calculated usinq the
following equation:
R = GS " C x 100
s
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte added to
sample.
9.4.3 If the recovery of any analyte falls outside the designated
LFM recovery range and the laboratory performance for that
analyte is shown to be in control (Sect. 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related.
9.4.4 Where reference materials are available, they should be
analyzed to provide additional performance data. The
analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method acceptably.
10.0 CALIBRATION AND STANDARnT7flTTnM
10.1 Prepare a series of at least 3 standards, covering the desired
JolSinS /? blank by pipetting suitable volumes of working standard
solutions (7.6, 7.7, 7.8) into 100-mL volumetric flasks. Suggested
ranges include 1 to 5, 10 to 100, and 200 to SOO'jag/L. iu"ested
10'2 2«nS "Ot perat1ye thaj a11 standards be distilled in the same
IX ThiS It s??pl?fi J* 1s recommenc|ed that at least one standard
and a blank be distilled and compared to similar values on the
CUTfW'??U!;e !hai the ^Illation technique is
proceeding. Before distillation, standards
a pH of 4 with H2S04.
10.3 Set up the manifold as shown in Figure 1 in a hood or a well-
' ventilated area.
10.4 Allow the instrument to warm up as required. Pump all reagents
until a stable baseline is achieved.
420.4-9
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10.5 Place appropriate standards in the sampler in order of decreasing
concentration and perform analysis.
10.6 Prepare standard curve by plotting instrument response concentration
values. A calibration curve may be fitted to the calibration
solutions concentration/response data using computer or calculator
based regression curve fitting techniques. Acceptance or control
limits should be established using the difference between the
measured value of the calibration solution and the "true value"
concentration.
10.7 After the calibration has been established, it must be verified by
the analysis of a suitable quality control sample (QCS). If
measurements exceed ± 10% of the established QCS value, the analysis
should be terminated and the instrument recalibrated. The new
calibration must be verified before continuing analysis. Periodic
reanalysis of the QCS is recommended as a continuing calibration
check.
11.0 PROCEDURE
11.1 Distillation
11.1.1 Measure 500 ml sample into a beaker. Adjust the pH to
approximately 4 with 1+9 NAOH (7.9) or 1+9 H2S04 (7.10), and
transfer to the distillation apparatus.
11.1.2 Distill 450 ml of sample, stop the distillation, and when
boiling ceases add 50 ml of warm reagent water to the flask
and resume distillation until 500 mL have been collected.
11.1.3 If the distillate is turbid, filter through a prewashed
membrane filter.
11.2 Set up the manifold as shown in Figure 1.
11.3 Fill the wash receptacle by siphon with reagent water. Use Kel-F
tubing with a fast flow (1 L/h).
11.4 Allow the instrument to warm up as required. Run a baseline with
all reagents, feeding reagent water through the sample line. Use
polyethylene tubing for sample line. When new tubing is used, about
2 hours may be required to obtain a stable baseline. This two hour
time period may be necessary to remove the residual phenol from the
tubing.
11.5 Place appropriate phenol standards in sampler in order of decreasing
concentration. Complete loading of sampler tray with unknown
samples, using glass tubes.
11.6 Switch sample line from reagent water to sampler and begin analysis.
420.4-10
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12.0 DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve by plotting instrument response
against standard concentration. Compute sample concentration by
comparing sample response with the standard curve. Multiply answer
by appropriate dilution factor.
12.2 Report only those values that fall between the lowest and the
highest calibration standards. Samples exceeding the highest
standard should be diluted and reanalyzed.
12.3 Report results in /jg/L.
13.0 METHOD PERFORMANCE
13.1 The inter!aboratory precision and accuracy data in Table 1 were
developed using a reagent water matrix. Values are in mg Phenol/L.
13.2 Single laboratory precision data can be estimated at 50 to 75% of
the inter!aboratory precision estimates.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or
eliminates the quantity or toxicity of waste at the point of
generation. Numerous opportunities for pollution prevention exist
in laboratory operation. The EPA has established a preferred
hierarchy of environmental management techniques that places
pollution prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When
wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 The quantity of chemicals purchased should be based on expected
usage during its shelf life and disposal cost of unused material.
Actual reagent preparation volumes should reflect anticipated usage
and reagent stability.
14.3 For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better:
Laboratory Chemical Management for Waste Reduction," available from
the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations. Excess Reagents and samples and method
process wastes should be characterized and disposed of in an
420.4-11
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acceptable manner. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all releases from
hoods, and bench operations, complying with the letter and spirit of
any waste discharge permit and regulations, and by complying with
all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste
Management Manual for Laboratory Personnel," available from the
American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
1. Technicon AutoAnalyzer II Methodology, Industrial Method No. 127-
71W, Mil.
2 Standard Methods for the Examination of Water and Wastewater, 14th
Edition, p. 574, Method 510 (1975).
3. Gales, M.E. and Booth, R.L., "Automated 4 AAP Phenolic Method," AWWA
68, 540 (1976).
4. Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
420.4-12
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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
99
87
76
110
89
107
86
62
76
89
61
110
TRUE
VALUE
(T)
0.020
0.250
0.400
0.545
0.604
0.660
0.800
0.817
0.970
2.96
4.18
4.54
MEAN
(X)
0.0149
0.1443
0.2352
0.3364
0.3610
0.3959
0.4627
0.4692
0.5680
1.7734
2.3916
2.7150
RESIDUAL
FOR X
0.0000
-0.0052
-0.0021
0.0142
0.0043
0.0064
-0.0087
-0.0122
-0.0029
0.0377
-0.0582
0.0545
STANDARD
DEVIATION
(S)
0.0074
0.0268
0.0422
0.0681
0.0625
0.0894
0.0806
0.0776
0.1017
0.3065
0.4044
0.5382
RESIDUAL
FOR S
0.0000
-0.0038
-0.0036
0.0076
-0.0039
0.0173
-0.0057
-0.0104
-0.0017
0.0018
-0.0237
0.0737
REGRESSIONS: X = 0.585T + 0.003, S = 0.101T + 0.005
420.4-13
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420.4-14
U.S. GOVERNMENT PRINTING OFFICE 1993750 -002/80267
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