4500-NO3

 NITROGEN (NITRATE)*

4500-NO3

 A. Introduction

1. Selection of Method

Determination of nitrate (NO3

) is difficult because of the

relatively complex procedures required, the high probability that

interfering constituents will be present, and the limited concentration ranges of the various techniques.

An ultraviolet (UV) technique (Method B) that measures the

absorbance of NO3

 at 220 nm is suitable for screening uncontaminated water (low in organic matter).

Screen a sample; if necessary, then select a method suitable for

its concentration range and probable interferences. Nitrate may

be determined by ion chromatography (Section 4110), capillary

ion electrophoresis (Section 4140), or the methods shown here.

Applicable ranges are: nitrate electrode method (D), 0.14 to

1400 mg NO3

-N/L; cadmium reduction method (E), 0.01 to

1.0 mg NO3

-N/L; automated hydrazine reduction method (H),

0.01 to 10 mg NO3

-N/L; automated cadmium reduction method

(F), 0.1 to 10 mg NO3

-N/L; cadmium reduction flow injection

method (I), 0.000 25 to 10 mg NO3

-N/L; second-derivative

ultraviolet spectrophotometric method (C), 0.05 to 2 mg NO3

-

N/L. For higher NO3

-N concentrations, dilute into the range of

the selected method.

The colorimetric and UV second-derivative methods require

an optically clear sample. Filter turbid sample through 0.45-m

membrane filter. Test filters for nitrate contamination.

2. Storage of Samples

Start NO3

 determinations promptly after sampling. If storage

is necessary, store for up to 2 d at 4°C; disinfected samples are

stable much longer (at least 14 d) without acid preservation.

NOTE: If nitrite, NO2

, is present, then acid preservation can

cause disproportionation of HNO2 to NO3

 and NO (nitric

oxide). NO can be oxidized and hydrolyzed to nitrate. As a

result, nitrate values may be the sum of nitrate and nitrite.

Therefore, do not acidify samples for nitrate determination.

4500-NO3

 B. Ultraviolet Spectrophotometric Screening Method

1. General Discussion

a. Principle: Use this technique only for screening samples

that have low organic matter contents, i.e., uncontaminated natural waters and potable water supplies. The NO3

 calibration

curve follows Beer’s law up to 11 mg N/L.

Measurement of UV absorption at 220 nm enables rapid determination of NO3

. Because dissolved organic matter also may

absorb at 220 nm and NO3

 does not absorb at 275 nm, a second

measurement made at 275 nm may be used to correct the NO3



value. The extent of this empirical correction is related to the nature

and concentration of organic matter and may vary from one water

to another. Consequently, this method is not recommended if a

significant correction for organic matter absorbance is required,

although it may be useful in monitoring NO3

 levels within a water

body with a constant type of organic matter. Correction factors for

organic matter absorbance can be established by the method of

additions in combination with analysis of the original NO3

 content

by another method. Sample filtration is intended to remove possible

interference from suspended particles. Acidification with 1N HCl is

designed to prevent interference from hydroxide or carbonate concentrations up to 1000 mg CaCO3/L. Chloride has no effect on the

determination.

b. Interference: Dissolved organic matter, surfactants, NO2

,

and Cr6interfere. Various inorganic ions not normally found in

natural water, such as chlorite and chlorate, may interfere. Inorganic substances can be compensated for by independent analysis of their concentrations and preparation of individual correction curves. For turbid samples, see 4500-NO3

A.1.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Spectrophotometer, for use at 220 and 275 nm with matched

silica cells of 1-cm or longer light path.

3. Reagents

a. Nitrate-free water: Use redistilled or distilled, deionized

water of highest purity to prepare all solutions and dilutions.

b. Stock nitrate solution: Dry potassium nitrate (KNO3) in an

oven at 105°C for 24 h. Dissolve 0.7218 g in water and dilute to

1000 mL; 1.00 mL  100 g NO3

-N. Preserve with 2 mL

CHCl3/L. This solution is stable for at least 6 months.

c. Intermediate nitrate solution: Dilute 100 mL stock nitrate

solution to 1000 mL with water; 1.00 mL  10.0 g NO3

-N.

Preserve with 2 mL CHCl3/L. This solution is stable for 6

months.

d. Hydrochloric acid solution, HCl, 1N.

* Approved by Standard Methods Committee, 2000. Editorial revisions, 2011.

Joint Task Group: 21st Edition—(4500-NO3

.C)—Thomas R. Holm (chair),

Martin S. Frant, Karla Alicia Johnson, Scott Stieg; 20th Edition (4500-NO3

.I)—

Scott Stieg (chair), Bradford R. Fisher, Owen B. Mathre, Theresa M. Wright.

1
4. Procedure

a. Treatment of sample: To 50 mL clear sample, filtered if

necessary, add 1 mL HCl solution and mix thoroughly.

b. Preparation of standard curve: Prepare NO3

 calibration

standards in the range 0 to 7 mg NO3

-N/L by diluting to 50 mL

the following volumes of intermediate nitrate solution: 0, 1.00,

2.00, 4.00, 7.00 . . . 35.0 mL. Treat NO3

 standards in same

manner as samples.

c. Spectrophotometric measurement: Read absorbance or

transmittance against redistilled water set at zero absorbance or

100% transmittance. Use a wavelength of 220 nm to obtain

NO3

 reading and a wavelength of 275 nm to determine interference due to dissolved organic matter.

5. Calculation

For samples and standards, subtract two times the absorbance

reading at 275 nm from the reading at 220 nm to obtain absorbance due to NO3

. Construct a standard curve by plotting

absorbance due to NO3

 against NO3-N concentration of standard. Using corrected sample absorbances, obtain sample concentrations directly from standard curve. NOTE: If correction

value is more than 10% of the reading at 220 nm, do not use this

method.

6. Bibliography

HOATHER, R.C. & R.F. RACKMAN. 1959. Oxidized nitrogen and sewage

effluents observed by ultraviolet spectrophotometry. Analyst

84:549.

GOLDMAN, E. & R. JACOBS. 1961. Determination of nitrates by ultraviolet

absorption. J. Amer. Water Works Assoc. 53:187.

ARMSTRONG, F.A.J. 1963. Determination of nitrate in water by ultraviolet

spectrophotometry. Anal. Chem. 35:1292.

NAVONE, R. 1964. Proposed method for nitrate in potable waters.

J. Amer. Water Works Assoc. 56:781.

4500-NO3

 C. Second-Derivative Ultraviolet Spectrophotometric Method (PROPOSED)

1. General Discussion

a. Principle: Even though nitrate strongly absorbs ultraviolet

(UV) light, determining nitrate by measuring the absorbance at

one wavelength is not feasible because natural organic matter

(NOM) and other solutes also absorb UV light. The spectra of

NOM from different water sources usually are different, so the

UV nitrate screening procedure (4500-NO3

.B) may not give

reliable results. The UV spectrum of nitrate is quite different

from that of NOM. In the nitrate spectrum, the absorbance

increases rapidly from 230 to 210 nm, whereas in NOM spectra,

the absorbance in the same wavelength range increases gradually. Computing the second derivative of a sample spectrum

effectively eliminates the background NOM contribution.

b. Interferences: The nitrite UV spectrum is similar to that of

nitrate. However, nitrite concentrations usually are much lower

than nitrate concentrations. Bicarbonate absorbs weakly at wavelengths below 210 nm, but does not affect the second-derivative

signal of nitrate. Bromide interferes at seawater concentrations

(68 mg Br/L, salinity 35‰) so this method cannot be used to

determine nitrate in seawater. Neither Fe nor Cu interferes at

2 mg/L, but both metals seriously interfere at 20 mg/L.1 The

method has been tested only for potable water. Its suitability for

nitrate determination in wastewater has not been tested.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

a. Spectrophotometer, capable of scanning from 250 to 200 nm.

Some spectrophotometers can compute the second derivative of

a spectrum. If the spectrophotometer cannot compute secondderivative spectra, then a computer interface is needed.

b. Cuvettes, matched 1-cm quartz.

c. Pipettor, adjustable 1 to 5 mL. Use a pipettor that clicks into

place for volume adjustment, not one with a sliding scale.*

d. Cuvette mixer.†

e. Computer, cable, and software for transferring data from the

spectrophotometer and computing second-derivative spectra.

The cable and software may be available from the spectrophotometer manufacturer. These items are needed only if the spectrophotometer cannot compute second-derivative spectra.

3. Reagents

a. Nitrate stock solution, 100 mg NO3

-N/L: Prepare as in

4500-NO3

.B.3b or obtain from a commercial source.

b. Nitrate-free water, distilled or deionized. Use to prepare

standards and dilute samples if necessary.

4. Procedure

a. Sample treatment: Filter samples if turbid. Add 1 mL 1M

H2SO4 or 2 mL 1M HCl to 100 mL sample, mix thoroughly.

b. Standards: Dilute 0.5, 1.0, 1.5, 2.0, and 2.5 mL nitrate stock

solution to 100 mL to prepare standards containing 0.5, 1.0, 1.5,

2.0, and 2.5 mg N/L.

c. Measurement: Pipet 3.0 mL standard or sample into sample

cuvette. Discard and pipet another 3.0 mL. Scan from 250 to

200 nm. If absorbance goes off-scale, dilute sample by withdrawing some sample with the macropipet and then pipetting

same volume of nitrate-free water into the cuvette. For dilutions

of 1:3, 1:5, and 1:10, withdraw 2.0, 2.4, and 2.7 mL. Mix with

cuvette mixer. Alternatively, perform dilutions in volumetric

glassware. Record any dilution. Compute second-derivative

* Wheaton pipet sold as Fisher #13-707-61 or VWR #53499-605, or equivalent.

† Bel-Art F37752-0000, or equivalent.

NITROGEN (NITRATE) (4500-NO3

)/Second-Derivative Ultraviolet Spectrophotometric Method

2
spectrum. Find maximum second derivative in the range 230 to

220 nm and record value. If spectrophotometer cannot compute

second-derivative spectra, then set it to read the absorbance every

1.0 nm and transfer the spectra to the computer after each scan.

5. Calculation

a. Calibration: Compute second-derivative spectrum and find

maximum for each sample, blank, or standard if the spectrophotometer cannot do this. Use the simplified least-squares procedure2,3 to simultaneously smooth and differentiate spectra.‡ Perform least-squares linear regression using the second derivatives

of the blank and standard spectra.

S Int  C  Slp

where:

S  maximum second derivative,

C  concentration,

Int  intercept of regression line, and

Slp  slope of regression line.

Thus,

C S  Int

Slp

b. Samples: Calculate the nitrate concentration, C, from the

equation above.

6. Precision and Bias

Nitrate concentrations determined by the UV second-derivative method were compared with concentrations determined by

ion chromatography for 79 potable-water samples with nitrate

concentrations of up to 40 mg NO3

-N/L.4 The slope of the

regression line was 0.94 with a standard error of 0.005. For 65

out of the 79 samples, the difference between nitrate concentrations determined by second-derivative UV spectrophotometry

and ion chromatography was 0.5 mg N/L or less. The maximum

difference for all samples was 1.6 mg NO3

-N/L. The differences between the nitrate concentrations determined by the two

methods appeared to be random, but they were not normally

distributed.

7. References

1. CRUMPTON, W.G., T.M. ISENHARD & P.D. MITCHELL. 1992. Nitrate and

organic N analyses with second-derivative spectroscopy. Limnol.

Oceanogr. 37:907.

2. SAVITSKY, A. & M. GOLAY. 1964. Smoothing and differentiation of

data by simplified least squares procedures. Anal. Chem. 36:1627.

3. STEINIER, J., Y. TERMONIA & J. DELTOUR. 1972. Comments on smoothing and differentiation of data by simplified least squares. Anal.

Chem. 44:1906.

4. HOLM, T.R., W.R. KELLY, L.F. SIEVERS & D.L. WEBB. 1997. A

comparison of ultraviolet spectrophotometry with other methods for

the determination of nitrate in water. Spectroscopy 12:38.

8. Bibliography

SIMAL, J., M.S. LAGE & I. IGLESIAS. 1985. Second derivative ultraviolet

spectroscopy and sulfamic acid method for determination of nitrates

in water. J. Assoc. Offic. Anal. Chem. 68:962.

SUZUKI, N. & R. KURODA. 1987. Direct simultaneous determination of

nitrate and nitrite by ultraviolet second-derivative spectrophotometry. Analyst 112:1077.

4500-NO3

 D. Nitrate Electrode Method

1. General Discussion

a. Principle: The NO3

 ion electrode is a selective sensor that

develops a potential across a thin, porous, inert membrane that

holds in place a water-immiscible liquid ion exchanger. The

electrode responds to NO3

 ion activity between about 105 and

101 M (0.14 to 1400 mg NO3

-N/L). The lower limit of

detection is determined by the small but finite solubility of the

liquid ion exchanger.

b. Interferences: Chloride and bicarbonate ions interfere

when their weight ratios to NO3

-N are 10 or 5, respectively. Ions that are potential interferences but do not normally

occur at significant levels in potable waters are NO2

, CN,

S2, Br, I, ClO3

, and ClO4. Although the electrodes function satisfactorily in buffers over the range pH 3 to 9, erratic

responses have been noted where pH is not held constant. Because the electrode responds to NO3

 activity rather than concentration, ionic strength must be constant in all samples and

standards. Minimize these problems by using a buffer solution

containing Ag2SO4 to remove Cl, Br, I, S2, and CN,

sulfamic acid to remove NO2

, a buffer at pH 3 to eliminate

HCO3

 and to maintain a constant pH and ionic strength, and

Al2(SO4)3 to complex organic acids.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

a. pH meter, expanded-scale or digital, capable of 0.1 mV

resolution.

b. Double-junction reference electrode.* Fill outer chamber

with (NH4)2SO4 solution.

‡ A Microsoft Excel worksheet for this calculation can be obtained from Tom

Holm, Illinois State Water Survey, trholm@illinois.edu.

* Orion Model 90-02, or equivalent.

NITROGEN (NITRATE) (4500-NO3

)/Nitrate Electrode Method

3
c. Nitrate ion electrode:† Carefully follow manufacturer’s

instructions regarding care and storage.

d. Magnetic stirrer: TFE-coated stirring bar.

3. Reagents

a. Nitrate-free water: Prepare as described in 4500-

NO3

.B.3a. Use for all solutions and dilutions.

b. Stock nitrate solution: Prepare as described in 4500-

NO3

.B.3b.

c. Standard nitrate solutions: Dilute 1.0, 10, and 50 mL stock

nitrate solution to 100 mL with water to obtain standard solutions of 1.0, 10, and 50 mg NO3

-N/L, respectively.

d. Buffer solution: Dissolve 17.32 g Al2(SO4)3  18H2O,

3.43 g Ag2SO4, 1.28 g H3BO3, and 2.52 g sulfamic acid

(H2NSO3H), in about 800 mL water. Adjust to pH 3.0 by slowly

adding 0.10N NaOH. Dilute to 1000 mL and store in a dark glass

bottle.

e. Sodium hydroxide, NaOH, 0.1N.

f. Reference electrode filling solution: Dissolve 0.53 g

(NH4)2SO4 in water and dilute to 100 mL.

4. Procedure

a. Preparation of calibration curve: Transfer 10 mL of 1 mg

NO3

-N/L standard to a 50-mL beaker, add 10 mL buffer, and

stir with a magnetic stirrer. Immerse tips of electrodes and record

millivolt reading when stable (after about 1 min). Remove electrodes, rinse, and blot dry. Repeat for 10-mg NO3

-N/L and

50-mg NO3

-N/L standards. Plot potential measurements

against NO3

-N concentration on semilogarithmic graph paper,

with NO3

-N concentration on the logarithmic axis (abscissa)

and potential (in millivolts) on the linear axis (ordinate). A

straight line with a slope of 57  3 mV/decade at 25°C should

result. Recalibrate electrodes several times daily by checking

potential reading of the 10 mg NO3

-N standard and adjusting

the calibration control until the reading plotted on the calibration

curve is displayed again.

b. Measurement of sample: Transfer 10 mL sample to a

50-mL beaker, add 10 mL buffer solution, and stir (for about 1

min) with a magnetic stirrer. Measure standards and samples at

about the same temperature. Immerse electrode tips in sample

and record potential reading when stable (after about 1 min).

Read concentration from calibration curve.

5. Precision

Over the range of the method, precision of 0.4 mV, corresponding to 2.5% in concentration, is expected.

6. Bibliography

LANGMUIR, D. & R.I. JACOBSON. 1970. Specific ion electrode determination of nitrate in some fresh waters and sewage effluents. Environ.

Sci. Technol. 4:835.

KEENEY, D.R., B.H. BYRNES & J.J. GENSON. 1970. Determination of

nitrate in waters with nitrate-selective ion electrode. Analyst

95:383.

MILHAM, P.J., A.S. AWAD, R.E. PAULL & J.H. BULL. 1970. Analysis of

plants, soils and waters for nitrate by using an ion-selective electrode. Analyst 95:751.

SYNNOTT, J.C., S.J. WEST & J.W. ROSS. 1984. Comparison of ionselective electrode and gas-sensing electrode technique for measurement of nitrate in environmental samples. In Pawlowski et al.,

eds., Studies in Environmental Science, No. 23, Chemistry for

Protection of the Environment. Elsevier Press, New York, N.Y.

4500-NO3

 E. Cadmium Reduction Method

1. General Discussion

a. Principle: NO3

 is reduced almost quantitatively to nitrite

(NO2

) in the presence of cadmium (Cd). This method uses

commercially available Cd granules treated with copper sulfate

(CuSO4) and packed in a glass column.

The NO2

 produced thus is determined by diazotizing with

sulfanilamide and coupling with N-(1-naphthyl)-ethylenediamine dihydrochloride to form a highly colored azo dye that is

measured colorimetrically. A correction may be made for any

NO2

 present in the sample by analyzing without the reduction

step. The applicable range of this method is 0.01 to 1.0 mg

NO3

-N/L. The method is recommended especially for NO3

levels below 0.1 mg N/L where other methods lack adequate

sensitivity.

b. Interferences: Suspended matter in the column will restrict

sample flow. For turbid samples, see 4500-NO3

.A.l. Concentrations of iron, copper, or other metals above several milligrams

per liter lower reduction efficiency. Add EDTA to samples to

eliminate this interference. Oil and grease will coat the Cd

surface. Remove by pre-extraction with an organic solvent (see

Section 5520). Residual chlorine can interfere by oxidizing the

Cd column, reducing its efficiency. Check samples for residual

chlorine (see DPD methods in Section 4500-Cl). Remove residual chlorine by adding sodium thiosulfate (Na2S2O3) solution

(Section 4500-NH3.B.3d). Sample color that absorbs at about

540 nm interferes.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

a. Reduction column: Purchase or construct the column* (Figure 4500-NO3

:1) from a 100-mL volumetric pipet by removing

the top portion. The column also can be constructed from two

† Orion Model 93-07, Corning Model 476134, or equivalent.

* Tudor Scientific Glass Co., 555 Edgefield Road, Belvedere, SC 29841, Cat.

TP-1730, or equivalent.

NITROGEN (NITRATE) (4500-NO3

)/Cadmium Reduction Method

4
pieces of tubing joined end to end: join a 10-cm length of

3-cm-ID tubing to a 25-cm length of 3.5-mm-ID tubing. Add a

TFE stopcock with metering valve1 to control flow rate.

b. Colorimetric equipment: One of the following is required:

1) Spectrophotometer, for use at 543 nm, providing a light

path of 1 cm or longer.

2) Filter photometer, with light path of 1 cm or longer and

equipped with a filter having maximum transmittance near 540 nm.

3. Reagents

a. Nitrate-free water: See 4500-NO3

.B.3a. The absorbance

of a reagent blank prepared with this water should not exceed

0.01. Use for all solutions and dilutions.

b. Copper-cadmium granules: Wash 25 g new or used 20- to

100-mesh Cd granules† with 6N HCl and rinse with water. Swirl

Cd with 100 mL 2% CuSO4 solution for 5 min or until blue color

partially fades. Decant and repeat with fresh CuSO4 until a

brown colloidal precipitate begins to develop. Gently flush with

water to remove all precipitated Cu.

c. Color reagent: Prepare as directed in Section 4500-

NO2

B.3b.

d. Ammonium chloride-EDTA solution: Dissolve 13 g NH4Cl

and 1.7 g disodium ethylenediamine tetraacetate in 900 mL

water. Adjust to pH 8.5 with conc NH4OH and dilute to 1 L.

e. Dilute ammonium chloride-EDTA solution: Dilute 300 mL

NH4Cl-EDTA solution to 500 mL with water.

f. Hydrochloric acid, HCl, 6N.

g. Copper sulfate solution, 2%: Dissolve 20 g CuSO4.5H2O in

500 mL water and dilute to 1 L.

h. Stock nitrate solution: Prepare as directed in 4500-

NO3

.B.3b.

i. Intermediate nitrate solution: Prepare as directed in 4500-

NO3

.B.3c.

j. Stock nitrite solution: See Section 4500-NO2

.B.3e.

k. Intermediate nitrite solution: See Section 4500-NO2

.B.3f.

l. Working nitrite solution: Dilute 50.0 mL intermediate nitrite solution to 500 mL with nitrite-free water; 1.00 mL  5 g

NO2

-N.

4. Procedure

a. Preparation of reduction column: Insert a glass wool plug

into bottom of reduction column and fill with water. Add sufficient Cu–Cd granules to produce a column 18.5 cm long. Maintain water level above Cu–Cd granules to prevent entrapment of

air. Wash column with 200 mL dilute NH4Cl-EDTA solution.

Activate column by passing through it, at 7 to 10 mL/min, at

least 100 mL of a solution composed of 25% 1.0 mg NO3

-N/L

standard and 75% NH4Cl-EDTA solution.

b. Treatment of sample:

1) Turbidity removal—For turbid samples, see 4500-

NO3

.A.1.

2) pH adjustment—Adjust pH to between 7 and 9, as necessary, using a pH meter and dilute HCl or NaOH. This ensures a

pH of 8.5 after adding NH4Cl-EDTA solution.

3) Sample reduction—To 25.0 mL sample or a portion diluted to

25.0 mL, add 75 mL NH4Cl-EDTA solution and mix. Pour mixed

sample into column and collect at a rate of 7 to 10 mL/min. Discard

first 25 mL. Collect the rest in original sample flask. There is no

need to wash columns between samples, but if columns are not to

be reused for several hours or longer, pour 50 mL dilute NH4ClEDTA solution on to the top and let it pass through the system.

Store Cu–Cd column in this solution and never let it dry.

4) Color development and measurement—As soon as possible, and not more than 15 min after reduction, add 2.0 mL color

reagent to 50 mL sample and mix. Between 10 min and 2 h

afterward, measure absorbance at 543 nm against a distilled

water-reagent blank. NOTE: If NO3

 concentration exceeds the

standard curve range (about 1 mg N/L), use remainder of reduced sample to make an appropriate dilution and analyze again.

c. Standards: Using the intermediate NO3

-N solution, prepare

standards in the range 0.05 to 1.0 mg NO3

-N/L by diluting the

following volumes to 100 mL in volumetric flasks: 0.5, 1.0, 2.0, 5.0,

and 10.0 mL. Carry out reduction of standards exactly as described

for samples. Compare at least one NO2

 standard to a reduced

NO3

 standard at the same concentration to verify reduction column efficiency. Reactivate Cu–Cd granules as described in 4500-

NO3

.3b above when efficiency of reduction falls below about

75%.

5. Calculation

Obtain a standard curve by plotting absorbance of standards

against NO3

-N concentration. Compute sample concentrations

directly from standard curve. Report as milligrams oxidized N

per liter (the sum of NO3

-N plus NO2-N) unless the concentration of NO2

 † EM Laboratories, Inc., 500 Exec. Blvd., Elmsford, NY, Cat. 2001, or equivalent. -N is separately determined and subtracted.

Figure 4500-NO3

:1. Reduction column.

NITROGEN (NITRATE) (4500-NO3

)/Cadmium Reduction Method

5
6. Precision and Bias

In a single laboratory using wastewater samples at concentrations of 0.04, 0.24, 0.55, and 1.04 mg NO3

 NO2-N/L, the

standard deviations were 0.005, 0.004, 0.005, and 0.01,

respectively. In a single laboratory using wastewater with additions of 0.24, 0.55, and 1.05 mg NO3

 NO2-N/L, the

recoveries were 100%, 102%, and 100%, respectively.2

7. References

1. WOOD, E.D., F.A.J. ARMSTRONG & F.A. RICHARDS. 1967. Determination of nitrate in sea water by cadmium-copper reduction to nitrite.

J. Mar. Biol. Assoc. U.K. 47:23.

2. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1979. Methods for Chemical Analysis of Water and Wastes, Method 353.3. U.S. Environmental Protection Agency, Washington, D.C.

8. Bibliography

STRICKLAND, J.D.H. & T.R. PARSONS. 1972. A Practical Handbook of Sea

Water Analysis, 2nd ed. Bull. No. 167, Fisheries Research Board

Canada, Ottawa, Ont.

NYDAHL, F. 1976. On the optimum conditions for the reduction of nitrate

by cadmium. Talanta 23:349.

AMERICAN SOCIETY FOR TESTING AND MATERIALS. 1987. Annual Book of

ASTM Standards, Vol. 11.01. American Soc. Testing & Materials,

Philadelphia, Pa.

4500-NO3

 F. Automated Cadmium Reduction Method

1. General Discussion

a. Principle: See 4500-NO3

.E.1a.

b. Interferences: Sample turbidity may interfere. Remove by

filtration before analysis. Sample color that absorbs in the photometric range used for analysis also will interfere.

c. Application: Nitrate and nitrite, singly or together in potable, surface, and saline waters and domestic and industrial wastewaters, can be determined over a range of 0.5 to 10 mg N/L.

d. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Automated analytical equipment: An example of the continuous-flow analytical instrument consists of the components shown

in Figure 4500-NO3

:2.

3. Reagents

a. Deionized distilled water: See 4500-NO3

.B.3a.

b. Copper sulfate solution: Dissolve 20 g CuSO4  5H2O in

500 mL water and dilute to 1 L.

c. Wash solution: Use water for unpreserved samples. For

samples preserved with H2SO4, add 2 mL conc H2SO4/L wash

water.

d. Copper-cadmium granules: See 4500-NO3

.E.3b.

e. Hydrochloric acid, HCl, conc.

f. Ammonium hydroxide, NH4OH, conc.

g. Color reagent: To approximately 800 mL water, add, while

stirring, 100 mL conc H3PO4, 40 g sulfanilamide, and 2 g

N-(1-naphthyl)-ethylenediamine dihydrochloride. 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.

h. Ammonium chloride solution: Dissolve 85 g NH4Cl in

water and dilute to 1 L. Add 0.5 mL polyoxyethylene 23 lauryl

ether.*

i. Stock nitrate solution: See 4500-NO3

.B.3b.

j. Intermediate nitrate solution: See 4500-NO3

.B.3c.

k. Standard nitrate solutions: Using intermediate NO3

-N

solution and water, prepare standards for calibration curve in

appropriate nitrate range. Compare at least one NO2

 standard to

a NO3

 standard at the same concentration to verify column

reduction efficiency. To examine saline waters prepare standard

solutions with the substitute ocean water described in Section

4500-NH3.G.3g.

l. Standard nitrite solution: See Section 4500-NO2

.B.3g.

4. Procedure

Set up manifold as shown in Figure 4500-NO3

:2 and follow

general procedure described by the manufacturer.

If sample pH is below 5 or above 9, adjust to between 5 and

9 with either conc HCl or conc NH4OH.

5. Calculation

Prepare standard curves by plotting response of standards

processed through the manifold against NO3

 * Brij-35, available from ICI Americas, Inc., Wilmington, DE, or equivalent. -N concentration

Figure 4500-NO3

:2. Nitrate-nitrite manifold.

NITROGEN (NITRATE) (4500-NO3

)/Automated Cadmium Reduction Method

6
in standards. Compute sample NO3

-N concentration by comparing sample response with standard curve.

6. Precision and Bias

Data obtained in three laboratories with an automated system

based on identical chemical principles but having slightly different configurations are given in the table below. Analyses were

conducted on four natural water samples containing exact increments of inorganic nitrate:

Increment as

NO3

-N

g/L

Standard

Deviation

g N/L

Bias

%

Bias

g N/L

290 12 5.75 17

350 92 18.10 63

2310 318 4.47 103

2480 176 2.69 67

In a single laboratory using surface water samples at concentrations of 100, 200, 800, and 2100 g N/L, the standard deviations were 0, 40, 50, and 50 g/L, respectively, and at

concentrations of 200 and 2200 g N/L, recoveries were 100 and

96%, respectively.

Precision and bias for the system described herein are believed

to be comparable.

7. Bibliography

FIORE, J. & J.E. O’BRIEN. 1962. Automation in sanitary chemistry—

Parts 1 and 2. Determination of nitrates and nitrites. Wastes Eng.

33:128 & 238.

ARMSTRONG, F.A., C.R. STEARNS & J.D. STRICKLAND. 1967. The measurement of upwelling and subsequent biological processes by

means of the Technicon AutoAnalyzer and associated equipment.

Deep Sea Res. 14:381.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1979. Methods for Chemical

Analysis of Water and Wastes. U.S. Environmental Protection

Agency, Washington, D.C.

AMERICAN SOCIETY FOR TESTING AND MATERIALS. 1987. Annual Book of

ASTM Standards, Vol. 11.01. American Soc. Testing & Materials,

Philadelphia, Pa.

4500-NO3

 G. (Reserved)

4500-NO3

 H. Automated Hydrazine Reduction Method

1. General Discussion

a. Principle: NO3

 is reduced to NO2 with hydrazine sulfate. The NO2

(originally present) plus reduced NO3 is determined by diazotization with sulfanilamide and coupling with

N-(1-naphthyl)-ethylenediamine dihydrochloride to form a

highly colored azo dye that is measured colorimetrically.

b. Interferences: Sample color that absorbs in the photometric

range used will interfere. Concentrations of sulfide ion of less

than 10 mg/L cause variations of NO3

 and NO2 concentrations of 10%.

c. Application: NO3

 NO2 in potable and surface water

and in domestic and industrial wastes can be determined over a

range of 0.01 to 10 mg N/L.

d. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Automated analytical equipment: An example of the continuous-flow analytical instrument consists of the components shown

in Figure 4500-NO3

:3.

3. Reagents

a. Color developing reagent: To approximately 500 mL water

add 200 mL conc phosphoric acid and 10 g sulfanilamide. After

sulfanilamide is dissolved completely, add 0.8 g N-(1-naphthyl)-

ethylenediamine dihydrochloride. Dilute to 1 L with water, store

Figure 4500-NO3

:3. Nitrate-nitrite manifold.

NITROGEN (NITRATE) (4500-NO3

)/Automated Hydrazine Reduction Method

7
in a dark bottle, and refrigerate. Solution is stable for approximately 1 month.

b. Copper sulfate stock solution: Dissolve 2.5 g

CuSO4  5H2O in water and dilute to 1 L.

c. Copper sulfate dilute solution: Dilute 20 mL stock solution

to 2 L.

d. Sodium hydroxide stock solution, 10N: Dissolve 400 g

NaOH in 750 mL water, cool, and dilute to 1 L.

e. Sodium hydroxide, 1.0N: Dilute 100 mL stock NaOH solution to 1 L.

f. Hydrazine sulfate stock solution: Dissolve 27.5 g

N2H4  H2SO4 in 900 mL water and dilute to 1 L. This solution

is stable for approximately 6 months. CAUTION: Toxic if ingested. Mark container with appropriate warning.

g. Hydrazine sulfate dilute solution: Dilute 22 mL stock solution to 1 L.

h. Stock nitrate solution: See 4500-NO3

.B.3b.

i. Intermediate nitrate solution: See 4500-NO3

.B.3c.

j. Standard nitrate solutions: Prepare NO3

 calibration standards in the range 0 to 10 mg/L by diluting to 100 mL the

following volumes of stock nitrate solution: 0, 0.5, 1.0, 2.0 . . .

10.0 mL. For standards in the range of 0.01 mg/L use intermediate nitrate solution. Compare at least one nitrite standard to a

nitrate standard at the same concentration to verify the efficiency

of the reduction.

k. Standard nitrite solution: See Sections 4500-NO2

.B.3e, f,

and g.

4. Procedure

Set up manifold as shown in Figure 4500-NO3

:3 and follow

general procedure described by manufacturer. Run a 2.0-mg

NO3

-N/L and a 2.0-mg NO2-N/L standard through the system

to check for 100% reduction of nitrate to nitrite. The two peaks

should be of equal height; if not, adjust concentration of the

hydrazine sulfate solution: If the NO3

 peak is lower than the

NO2

 peak, increase concentration of hydrazine sulfate until

they are equal; if the NO3

 peak is higher than the NO2, reduce

concentration of hydrazine sulfate. When correct concentration

has been determined, no further adjustment should be necessary.

5. Calculation

Prepare a standard curve by plotting response of processed

standards against known concentrations. Compute concentrations of samples by comparing response with standard curve.

6. Precision and Bias

In a single laboratory using drinking water, surface water, and

industrial waste at concentrations of 0.39, 1.15, 1.76, and

4.75 mg NO3

-N/L, the standard deviations were 0.02, 0.01,

0.02, and 0.03, respectively. In a single laboratory using

drinking water at concentrations of 0.75 and 2.97 mg NO3

-N/L,

the recoveries were 99% and 101%.1

7. Reference

1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1979. Methods for Chemical Analysis of Water and Wastes. U.S. Environmental Protection

Agency, Washington, D.C.

8. Bibliography

KAMPHAKE, L., S. HANNAH & J. COHEN. 1967. Automated analysis for

nitrate by hydrazine reduction. Water Res. 1:205.

4500-NO3

 I. Cadmium Reduction Flow Injection Method

1. General Discussion

a. Principle: The nitrate in the sample is reduced quantitatively to nitrite by passage of the sample through a copperized

cadmium column. The resulting nitrite plus any nitrite originally

in the sample is determined as a sum by diazotizing the nitrite

with sulfanilamide followed by coupling with N-(1-naphthyl)

ethylenediamine dihydrochloride. The resulting water-soluble

dye has a magenta color; absorbance of the color at 540 nm is

proportional to the nitrate nitrite in the sample. This sum also

is known as total oxidized nitrogen (TON).

Nitrite alone can be determined by removing the cadmium

column, recalibrating the method, and repeating the sample

analyses. A TON and a nitrite FIA method also can be run in

parallel for a set of samples. In this arrangement, the concentrations determined in the nitrite method can be subtracted from the

corresponding concentrations determined in the TON method to

give the resulting nitrate concentrations of the samples.

Also see Section 4500-NO2

 and Section 4130, Flow Injection Analysis (FIA).

b. Interferences: Remove large or fibrous particulates by filtering sample through glass wool. Guard against nitrate and

nitrite contamination from reagents, water, glassware, and the

sample preservation process.

Residual chlorine can interfere by oxidizing the cadmium reduction column. Samples that contain large concentrations of oil and

grease will coat the surface of the cadmium. Eliminate this interference by pre-extracting sample with an organic solvent.

Low results would be obtained for samples that contain high

concentrations of iron, copper, or other metals. In this method,

EDTA is added to the buffer to reduce this interference.

Also see Sections 4500-NO2

.B.1b and c and 4500-NO3.A.2

and B.1b.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Flow injection analysis equipment consisting of:

a. FIA injection valve with sample loop or equivalent.

NITROGEN (NITRATE) (4500-NO3

)/Cadmium Reduction Flow Injection Method

8
b. Multichannel proportioning pump.

c. FIA manifold (Figure 4500-NO3

:4) with flow cell. Relative flow rates only are shown in Figure 4500-NO3

:4. Tubing

volumes are given as an example only; they may be scaled down

proportionally. Use manifold tubing of an inert material, such as

TFE.

d. Absorbance detector, 540 nm, 10-nm bandpass.

e. Injection valve control and data acquisition system.

3. Reagents

Use reagent water (10 megohm) to prepare carrier and all

solutions. To prevent bubble formation, degas carrier and buffer

with helium. Pass He at 140 kPa (20 psi) through a helium

degassing tube. Bubble He through 1 L solution for 1 min. As an

alternative to preparing reagents by weight/weight, use weight/

volume.

a. Ammonium chloride buffer: CAUTION: Fumes. Use a hood.

To a tared 1-L container add 800.0 g water, 126 g conc hydrochloric acid, HCl, 55.6 g ammonium hydroxide, NH4OH, and

1.0 g disodium EDTA. Shake until dissolved. The pH of this

buffer should be 8.5.

b. Sulfanilamide color reagent: To a tared, dark 1-L container

add 876 g water, 170 g 85% phosphoric acid, H3PO4, 40.0 g

sulfanilamide, and 1.0 g N-(1-naphthyl)ethylenediamine dihydrochloride (NED). Shake until wetted and stir with stir bar for

30 min until dissolved. This solution is stable for 1 month.

c. Hydrochloric acid, HCl, 1M: To a 100-mL container, add

92 g water, then add 9.6 g conc HCl. Stir or shake to mix.

d. Copper sulfate solution, 2%: To a 1-L container, add 20 g

copper sulfate pentahydrate, CuSO4  5H2O, to 991 g water. Stir

or shake to dissolve.

e. Copperized cadmium granules: Place 10 to 20 g coarse

cadmium granules (0.3- to 1.5-mm-diam) in a 250-mL beaker.

Wash with 50 mL acetone, then water, then two 50-mL portions

1M HCl (¶ c above). Rinse several times with water. CAUTION:

Cadmium is toxic and carcinogenic. Collect and store all

waste cadmium. When handling cadmium, wear gloves and

follow the precautions described on the cadmium’s Material

Safety Data Sheet.

Add 100 mL 2% copper sulfate solution (¶ d above) to

cadmium prepared above. Swirl for about 5 min, then decant the

liquid and repeat with a fresh 100-mL portion of the 2% copper

sulfate solution. Continue this process until the blue aqueous

copper color persists. Decant and wash with at least five portions

of ammonium chloride buffer (¶ a above) to remove colloidal

copper. The cadmium should be black or dark gray. The copperized cadmium granules may be stored in a bottle under

ammonium chloride buffer.

f. Stock nitrate standard, 200 mg N/L: In a 1-L volumetric

flask dissolve 1.444 g potassium nitrate, KNO3, in about 600 mL

water. Add 2 mL chloroform. Dilute to mark and invert to mix.

This solution is stable for 6 months.

g. Stock nitrite standard, 200.0 mg N/L: In a 1-L volumetric

flask dissolve 0.986 g sodium nitrite, NaNO2, or 1.214 g potassium nitrite, KNO2, in approximately 800 mL water. Add 2 mL

chloroform. Dilute to mark and invert to mix. Refrigerate.

h. Standard solution: Prepare nitrate or nitrite standards in the

desired concentration range, using the stock standards (¶ f or g

above), and diluting with water.

4. Procedure

Set up a manifold equivalent to that in Figure 4500-NO3

:4

and pack column with copperized cadmium granules. Follow

methods supplied by column and instrument manufacturer or

laboratory’s standard operating procedure for this method.

5. Calculations

Prepare standard curves by plotting absorbance of standards

processed through the manifold versus TON or nitrite concentration. The calibration curve is linear.

If TON includes measurable nitrite concentrations, it is important that the cadmium column be 100% efficient. If the

efficiency is less, the nitrite in the sample will give a positive

percent error equal to the difference from 100%, causing an error

in TON and nitrate determinations. To measure efficiency of the

cadmium column, prepare two calibration curves, one using

nitrate standards and one using equimolar nitrite standards. The

column efficiency is:

Column efficiency 

100%  (slope of nitrate curve/slope of nitrite curve)

Determine column efficiency at least weekly.

6. Precision and Bias

In the studies described below, nitrate was measured. There

was no significant concentration of nitrite in the samples.

a. Recovery and relative standard deviation: Table 4500-

NO3

:I gives results of single-laboratory studies.

b. MDL: A 800-L sample loop was used in the method

described above. Using a published MDL method,1 analysts ran

21 replicates of a 2.00-g N/L standard. These gave a mean of

1.82 g N/L, a standard deviation of 0.098 g N/L, and MDL of

0.25 g N/L. A lower MDL may be obtained by increasing the

sample loop volume and increasing the ratio of carrier flow rate

to reagent flow rate.

Figure 4500-NO3

:4. FIA nitrate nitrite manifold.

NITROGEN (NITRATE) (4500-NO3

)/Cadmium Reduction Flow Injection Method

9
7. Reference

1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1984. Definition and procedure for the determination of method detection limits. Appendix B

to 40 CFR 136 rev. 1.11 amended June 30, 1986. 49 CFR 43430.

TABLE 4500-NO3

:I. RESULTS OF SINGLE-LABORATORY STUDIES WITH

SELECTED MATRICES

Matrix

Sample/Blank

Designation

Known

Addition

mg

NO3

-N/L

Recovery

%

Relative

Standard

Deviation

%

Wastewater

treatment

plant

influent

Reference

sample*

— 102 —

Blank† 0.2 100 —

0.4 100 —

Site A‡ 0 — 1

0.2 101 —

0.4 96 —

Site B‡ 0 — 1

0.2 90 —

0.4 88 —

Site C‡ 0 — 1

0.2 95 —

0.4 95 —

Wastewater

treatment

plant

effluent

Reference

sample*

— 102 —

Blank† 0.2 100 —

0.4 95 —

Site A‡ 0 — 0.9

0.2 95 —

0.4 102 —

Site B‡ 0 — 0.7

0.2 91 —

0.4 101 —

Site C‡ 0 — 0.5

0.2 91 —

0.4 96 —

Landfill

leachate

Reference

sample*

— 98 —

Blank† 0.2 100 —

0.4 98 —

Site A‡ 0 — 1

0.2 104 —

0.4 96 —

Site B‡ 0 — 1

0.2 95 —

0.4 94 —

Site C‡ 0 — 1

0.2 91 1

0.4 93 —

* U.S. EPA QC sample, 1.98 mg N/L.

† Determined in duplicate.

‡ Samples without known additions determined four times; samples with known

additions determined in duplicate. Typical difference between duplicates: influent,

2%; effluent, 1%; leachate, 1%.

NITROGEN (NITRATE) (4500-NO3

)/Cadmium Reduction Flow Injection Method

10