Extract from manual:
U. S. Environmental Protection Agency. 1983.
Nitrogen, Ammonia. Method 350.1 (Colorimetric, Automated,
Phenate). pp.350-1.1 -- 350-1.4. In Methods for Chemical
Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A.,
Cincinnati, Ohio, USA.
Method 350.1 (Colorimetric, Automated Phenate)
1. Scope and Application
2. Summary of Method
1.1 This method covers the determination of ammonia in
drinking, surface, and saline waters, domestic and industrial wastes
in the range of 0.01 to 2.0 mg/l NH3 as N. This range is for
photometric measurements made at 630-660 nm in a 15 mm or 50 mm
tubular flow cell. Higher concentrations can be determined by sample
dilution. Approximately 20 to 60 samples per hour can be analyzed.
3. Sample Handling and Preservation
2.1 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
3.1 Preservation by addition of 2 ml conc. H2SO4 per liter
and refrigeration at 4 degrees C.
4.1 Calcium and magnesium ions may be present in
concentration sufficient to cause precipitation problems during
analysis. A 5% EDTA solution is used to prevent the precipitation of
calcium and magnesium ions from river water and industrial waste.
For sea water a sodium potassium tartrate solution is used.
4.2 Sample turbidity and color may interfere with this
method. Turbidity must be removed by filtration prior to analysis.
Sample color that absorbs in the photometric range used will also
5.1 Technicon AutoAnalyzer Unit (AAI or AAII) consisting of:
5.1.2 Manifold (AAI) or Analytical Cartridge (AAII).
5.1.3 Proportioning pump.
5.1.4 Heating bath with double delay coil (AAI).
5.1.5 Colorimeter equipped with 15 mm tubular flow cell
and 630-660 nm filters.
5.1.7 Digital printer for AAII (optional).
6.1 Distilled water: Special precaution must be taken to
insure that distilled water is free of ammonia. Such water is
prepared by passage of distilled water through an ion exchange column
comprised of a mixture of both strongly acidic cation and strongly
basic anion exchange resins. The regeneration of the ion exchange
column should be carried out according to the instruction of the
NOTE 1: All solutions must be made using ammonia-free water.
6.2 Sulfuric acid 5N: Air scrubber solution. Carefully add 139
ml of conc. sulfuric acid to approximately 500 ml of ammonia-free
distilled water. Cool to room temperature and dilute to 1 liter with
ammonia-free distilled water.
6.3 Sodium phenolate: Using a 1 liter Erlenmeyer flask,
dissolve 83 g phenol 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 liter with
6.4 Sodium hypochlorite solution: Dilute 250 ml of a bleach
solution containing 5.25% NaOCl (such as "Clorox") to 500 ml with
distilled 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
6.5 Disodium ethylenediamine-tetraacetate (EDTA) (5%):
Dissolve 50 g of EDTA (disodium salt) and approximately six pellets
of NaOH in 1 liter of distilled water.
NOTE 2: On salt water samples where EDTA solution does not
prevent precipitation of cations, sodium potassium tartrate solution
may be used to advantage. It is prepared as follows:
6.5.1 Sodium potassium tartrate solution: 10% NaKC4H4O6 x
4H2O. To 900 ml of distilled water add 100 g sodium potassium
tartrate. Add 2 pellets of NaOH and a few boiling chips, boil
gently for 45 minutes. Cover, cool, and dilute to 1 liter with
ammonia-free distilled water. Adjust pH to 5.2 +/-.05 with H2SO4.
After allowing to settle overnight in a cool place, filter to
remove precipitate. Then add 1/2 ml Brij-35 (note 4) (available
from Technicon Corporation) solution and store in stoppered
6.6 Sodium nitroprusside (0.05%): Dissolve 0.5 g of sodium
nitroprusside in 1 liter of distilled water.
6.7 Stock solution: Dissolve 3.819 g of anhydrous ammonium
chloride, NH4CI, dried at 105 degrees C, in distilled water, and
dilute to 1000 ml. 1.0 ml = 1.0 mg NH3-N.
6.8 Standard Solution A: Dilute 10.0 ml of stock solution
(6.7) to 1000 ml with distilled water. 1.0 ml = 0.01 mg NH3-N.
6.9 Standard solution B: Dilute 10.0 ml of standard solution A
(6.8) to 100.0 ml with distilled water. 1.0 ml = 0.001 mg NH3-N.
6.10 Using standard solutions A and B. prepare the following
standards in 100 ml volumetric flasks (prepare fresh daily):
ml Standard Solution/100 ml NH3-N, mg/l
NOTE 3: When saline water samples are arnlyzed, Substitute
Ocean Water (SOW) should be used for preparing the above standards
used for the calibration curve; otherwise, distilled water is used.
If SOW is used, subtract its blank background response from the
standards before preparing the standard curve.
Substitute Ocean Water (SOW)
NaCl 24.53 g/l NaHCO3 0.20 g/l
MgCl2 5.20 g/l KBr 0.10 g/l
Na2SO4 4.09 g/l H3BO3 0.03 g/l
CaCl2 1.16 g/l SrCl2 0.03 g/l
KCl 0.70 g/l NaF 0.003 g/l
7.1 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. For example, if the samples have been preserved with 2
ml conc. H2SO4/liter, the wash water and standards should also
contain 2 ml conc. H2SO4/liter.
7.2 For a working range of 0.01 to 2.00 mg NH3-N/l (AAI), set
up the manifold as shown in Figure 1. For a working range of .01 to
1.0 mg NH3-N/l (AAII), set up the manifold as shown in Figure 2.
Higher concentrations may be accommodated by sample dilution.
7.3 Allow both colorimeter and recorder to warm up for 30
minutes. Obtain a stable baseline with all reagents, feeding
distilled water through sample line.
7.4 For the AAI system, sample at a rate of 20/hr, 1:1. For
the AAII use a 60/hr 6:1 cam with a common wash.
7.5 Arrange ammonia standards in sampler in order of
decreasing concentration of nitrogen. Complete loading of sampler
tray with unknown samples.
7.6 Switch sample line from distilled water to sampler and
9. Precision and Accuracy
8.1 Prepare appropriate standard curve derived from
processing ammonia standards through manifold. Compute concentration
of samples by comparing sample peak heights with standard curve.
9.1 In a single laboratory (EMSL), 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.
9.2 In a single laboratory (EMSL), using surface water
samples at concentrations of 0.16 and 1.44 mg NH3-N/l, recoveries
were 107% and 99%, respectively.
1. Hiller, A., and D. Van Slyke. 1933.
Determination of ammonia in blood. J. Biol. Chem. v.102, p.
2. O'Connor, B., R. Dobbs, B. Villiers, and R. Dean. 1967.
Laboratory distillation of municipal waste effluents. JWPCF
3. Fiore, J., and J. E. O'Brien. 1962.
Ammonia determination by automatic analysis. Wastes
Engineering v.33 p.352.
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. 1966.
2nd ed. p.418.
6. Booth, R. L., and L. B. Lobring 1973.
Evaluation of the AutoAnalyzer II: a progress report. In Advances
in Automated Analysis: 1972 Technicon International Congress,
v. 8, p.7-10, Mediad Incorporated, Tarrytown, N.Y.
7. Standard Methods for the Examination of Water and Wastewater. 1975.
14th ed. p.616, Method 604.