EPA 353.2

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

353.2-1DETERMINATION OF NITRATE-NITRITE NITROGEN BY AUTOMATED

COLORIMETRY

1.0SCOPE AND APPLICATION

1.1This method covers the determination of nitrite singly, or nitrite and nitrate

combined in drinking, ground, surface, domestic and industrial wastes.

1.2The applicable range is 0.05-10.0 mg/L nitrate-nitrite nitrogen. The range may

be extended with sample dilution.

2.0SUMMARY 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-(1-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.

2.2Reduced 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.

2.3Limited performance-based method modifications may be acceptable provided

they are fully documented and meet or exceed requirements expressed in

Section 9.0, Quality Control.

3.0DEFINITIONS

3.1Calibration 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.

3.2Calibration 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.

3.3Instrument 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.

353.2-23.4Laboratory 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.

3.5Laboratory 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.

3.6Laboratory 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.

3.7Linear Calibration Range (LCR) -- The concentration range over which the

instrument response is linear.

3.8Material 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.

3.9Method 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.

3.10Quality 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.

3.11Stock 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.

4.0INTERFERENCES

4.1Build 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.

353.2-34.2Low 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.

4.3Residual chlorine can produce a negative interference by limiting reduction

efficiency. Before analysis, samples should be checked and if required,

dechlorinated with sodium thiosulfate.

4.4Samples 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.

4.5Method interferences may be caused by contaminants in the reagent water,

reagents, glassware, and other sample processing apparatus that bias analyte

response.

5.0SAFETY

5.1The 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.

5.2Each 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.

5.3The following chemicals have the potential to be highly toxic or hazardous,

consult MSDS.

5.3.1Cadmium (Section 7.1)

5.3.2Phosphoric acid (Section 7.5)

5.3.3Hydrochloric acid (Section 7.6)

5.3.4Sulfuric acid (Section 7.8)

5.3.5Chloroform (Sections 7.10 and 7.11)

6.0EQUIPMENT AND SUPPLIES

6.1Balance -- Analytical, capable of accurately weighing to the nearest 0.0001 g.

6.2Glassware -- Class A volumetric flasks and pipets as required.

353.2-46.3Automated continuous flow analysis equipment designed to deliver and react

sample and reagents in the required order and ratios.

6.3.1Sampling device (sampler)

6.3.2Multichannel pump

6.3.3Reaction unit or manifold

6.3.4Colorimetric detector

6.3.5Data recording device

7.0REAGENTS AND STANDARDS

7.1Granulated cadmium: 40-60 mesh (CASRN 7440-43-9). Other mesh sizes may

be used.

7.2Copper-cadmium: The cadmium granules (new or used) are cleaned with

dilute HCl (Section 7.6) and copperized with 2% solution of copper sulfate

(Section 7.7) in the following manner:

7.2.1Wash the cadmium with HCl (Section 7.6) and rinse with distilled

water. The color of the cadmium so treated should be silver.

7.2.2Swirl 10 g cadmium in 100 mL portions of 2% solution of copper

sulfate (Section 7.7) for five minutes or until blue color partially fades,

decant and repeat with fresh copper sulfate until a brown colloidal

precipitate forms.

7.2.3Wash 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.

7.3Preparation 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 (Section 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: 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 Section 10.1.

7.4Reagent water: Because of possible contamination, this should be prepared by

passage through an ion exchange column comprised of a mixture of both

strongly acidic-cation and strongly basic-anion exchange resins. The

353.2-5regeneration of the ion exchange column should be carried out according to

the manufacturer's instructions.

7.5Color reagent: To approximately 800 mL of reagent water, add, while stirring,

100 mL conc. phosphoric acid (CASRN 76-38-2), 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.

7.6Dilute hydrochloric acid, 6N: Add 50 mL of conc. HCl (CASRN 77-01-0) to

reagent water, cool, and dilute to 100 mL.

7.7Copper sulfate solution, 2%: Dissolve 20 g of CuSO5H O (CASRN 7758-99-8)

42

in 500 mL of reagent water and dilute to 1 L.

7.8Wash solution: Use reagent water for unpreserved samples. For samples

preserved with H SO, use 2 mL H SO (CASRN 77-93-9), per liter of wash

2424

water.

7.9Ammonium 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 conc.

ammonium hydroxide (CASRN 1336-21-6) and dilute to 1 L. Add 0.5 mL Brij-

35 (CASRN 9002-92-0).

7.10Stock nitrate solution: Dissolve 7.218 g KNO (CASRN 7757-79-1) and dilute to

3

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 six months. 1 mL

= 1.0 mg NO-N.

3

7.11Stock nitrite solution: Dissolve 6.072 g KNO in 500 mL of reagent water and

2

dilute to 1 L in a volumetric flask. Preserve with 2 mL of chloroform and keep

under refrigeration. 1.0 mL = 1.0 mg NO-N.

2

7.12Standard nitrate solution: Dilute 1.0 mL of stock nitrate solution (Section 7.10)

to 100 mL. 1.0 mL = 0.01 mg NO-N. Preserve with .2 mL of chloroform.

3

Solution is stable for six months.

7.13Standard nitrite solution: Dilute 10.0 mL of stock nitrite (Section 7.11) solution

to 1000 mL. 1.0 mL = 0.01 mg NO-N. Solution is unstable; prepare as

2

required.

8.0SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1Samples should be collected in plastic or glass bottles. All bottles must be

thoroughly cleaned and rinsed with reagent water. Volume collected should

353.2-6

be sufficient to insure a representative sample, allow for replicate analysis (if

required), and minimize waste disposal.

8.2Samples must be preserved with H SO to a pH <2 and cooled to 4°C at the

24

time of collection.

8.3Samples 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.

8.4Samples to be analyzed for nitrate or nitrite only should be cooled to 4°C and

analyzed within 48 hours.

9.0QUALITY CONTROL

9.1Each 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.

9.2INITIAL DEMONSTRATION OF PERFORMANCE

9.2.1The 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.

9.2.2Linear Calibration Range (LCR) -- The LCR must be determined

initially and verified every six 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.

9.2.3Quality 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.

353.2-79.2.4Method Detection Limit (MDL) -- MDLs must be established for all

analytes, using reagent water (blank) fortified at a concentration of two

(6)

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:

where,t =Student's t value for a 99% confidence level and a

standad 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.

9.3ASSESSING LABORATORY PERFORMANCE

9.3.1Laboratory 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.

9.3.2Laboratory Fortified Blank (LFB) -- The laboratory must analyze at least

one LFB with each batch of samples. Calculate accuracy as percent

recovery (Section 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.

9.3.3The 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 recoverymeasurements, 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.

9.3.4Instrument 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

10th 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.

9.4ASSESSING ANALYTE RECOVERY AND DATA QUALITY

9.4.1Laboratory 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.

9.4.2Calculate 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:

where,R =percent recovery

C =fortified sample concentration

s

C = sample background concentration

s = concentration equivalent of analyte added to

sample9.4.3If 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 (Section 9.3), the recovery problem encountered with the LFM is

judged to be either matrix or solution related, not system related.

9.4.4Where 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.

10.0CALIBRATION AND STANDARDIZATION

10.1Prepare a series of at least three standards, covering the desired range, and a

blank by diluting suitable volumes of standard nitrate solution (Section 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.

10.2Set up manifold as shown in Figure 1. Care should be taken not to introduce

air into the reduction column.

10.3Place appropriate standards in the sampler in order of decreasing

concentration and perform analysis.

10.4Prepare 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.

10.5After 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: Condition column by running 1 mg/L standard for 10 minutes if a new

reduction column is being used. Subsequently wash the column with reagents

for 20 minutes.

11.0PROCEDURE

11.1If the pH of the sample is below 5 or above 9, adjust to between 5 and 9 with

either conc. HCl or conc. NH OH.

4

11.2Set up the manifold as shown in Figure 1. Care should be taken not to

introduce air into reduction column.11.3Allow system to equilibrate as required. Obtain a stable baseline with all

reagents, feeding reagent water through the sample line.

11.4Place appropriate nitrate and/or nitrite standards in sampler in order of

decreasing concentration and complete loading of sampler tray.

11.5Switch sample line to sampler and start analysis.

12.0DATA ANALYSIS AND CALCULATIONS

12.l 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.

12.2Report only those values that fall between the lowest and the highest

calibration standards. Samples exceeding the highest standard should be

diluted and reanalyzed.

l2.3Report results in mg/L as nitrogen.

13.0METHOD PERFORMANCE

13.1Three laboratories participating in an EPA Method Study analyzed four natural

water samples containing exact increments of inorganic nitrate, with the

following results:

Increment as Precision as Accuracy as

Nitrate Nitrogen Standard Deviation Bias, Bias,

mg N/L mg N/L % mg N/L

0.290.012+ 5.75+ 0.017

0.350.092+ 18.10+ 0.063

2.310.318+ 4.47+ 0.103

2.480.176- 2.69- 0.067

13.2The interlaboratory precision and accuracy data in Table 1 were developed

using a reagent water matrix. Values are in mg NO-N/L.

3

13.3Single laboratory precision data can be estimated at 50-75% of the

interlaboratory precision estimates.

l4.0POLLUTION PREVENTION

14.1Pollution 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 preventiontechniques to address their waste generation. When wastes cannot be feasibly

reduced at the source, the Agency recommends recycling as the next best

option.

14.2The 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.

14.3For information about pollution prevention that may be applicable to

laboratories and research institutions, consult "Less is Better: Laboratory

Chemical Management for Waste Reduction

Chemical Society's Department of Government Regulations and Science Policy,

1155 16th Street N.W., Washington, D.C. 20036, (202) 872-4477.

15.0WASTE MANAGEMENT

15.1The 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

in Section 14.3.

16.0REFERENCES

1.Fiore, J., and O'Brien, J.E., "Automation in Sanitary Chemistry - Parts 1 & 2:

Determination of Nitrates and Nitrites

(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

(1967).

3.Annual Book of ASTM Standards, Part 31, "Water

(1976).

4.Standard Methods for the Examination of Water and Wastewater, 17th Edition,

pp. 4-91, Method 4500-NO3 F (1992).

5.Chemical Analyses for Water Quality Manual, Department of the Interior,

FWPCA, R.A. Taft Engineering Center Training Program, Cincinnati, Ohio

45226 (January, 1966).

6.Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.

17.0TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA Number of True Standard

Values Value Mean Residual Deviation Residual

Reported(T)(X)for X(S)for S 1630.2500.24790.00070.0200-0.0001

1830.4510.4441-0.00390.02-0.0002

2130.6500.790.00120.03980.0017

1700.9500.95370.00740.0484-0.0031

163 1.90 1.870.00370.0918-0.0024

172 2.20 2.19710.00250.110.0087

183 2.41 2.3732-0.03120.12730.0102

214 3.20 3.20420.01090.1456-0.0070

172 6.50 6.49780.000.31560.0148

2138.007.9814-0.00550.3673-0.0008

1708.508.51350.02730.3635-0.0271

21410.09.9736-0.01060.4353-0.0227 REGRESSIONS: X = 0.999T + 0.002, S = 0.045T + 0.009

353.2-13