WATER QUALITY: Analytical methods--Methods for analysis of organic nitrogen and ammonium plus organic (Kjeldahl) nitrogen 

                                                 April 19, 1976

QUALITY OF WATER BRANCH TECHNICAL MEMORANDUM NO. 76.16

Subject: WATER QUALITY: Analytical methods--Methods for analysis 
         of organic nitrogen and ammonium plus organic (Kjeldahl) 
         nitrogen

Attached are three methods for the analysis of organic and 
ammonium plus organic (Kjeldahl) nitrogen in water and sediment. 
They represent methodology approved for use in our Central 
Laboratories System as of April 1, 1976.

The three methods now have provisional status and should be given 
appropriate distribution to cooperators and WRD field offices. Any 
questions about the attached methods or other water-quality 
methods should be directed to the Quality of Water Branch.

                              R. J. Pickering
                              Chief, Quality of Water Branch

Attachments

WRD Distribution: A, B, FO-L, PO

     Nitrogen, dissolved organic and nitrogen, dissolved ammonia
                plus dissolved organic (Kjeldahl)

                    Kjeldahl block digestion

Automated salicylate-hypochlorite colorimetric method (I-2552-76)

Parameters & codes: Nitrogen, organic, dissolved (mg/l as N):00607
                    Nitrogen ammonia plus organic,
                       dissolved (mg/l as N):                00636
                    Nitrogen, Kjeldahl, dissolved (mg/l 
                       as N):                                00623

Application

1.1 Concentrations of O to 10 mg/l nitrogen can be determined by 
this method. Concentrations greater than 10 mg/l must be reduced 
by dilution.

2. Summary of method

2.1 Organic nitrogen compounds are reduced to the ammonium ion by 
digestion in a block digestor with sulfuric acid in the presence 
of mercuric sulfate, which acts as a catalyst, and potassium 
sulfate. The ammonium ion produced by this digestion, as well as 
the ammonium ion which was originally present, is determined as 
Kjeldahl nitrogen by the reaction with sodium salicylate, sodium 
nitroprusside, and sodium hypochlorite in an alkaline media. The 
resulting green color is directly proportional to the 
concentration of ammonia present. Organic nitrogen is determined 
by subtracting ammonia as determined in "Method I-2522" from 
Kjeldahl nitrogen.

3. Interferences

3.1 A comparison study of results obtained by this method with 
those from the digestion-distillation and indophenol methods 
(Methods I-1550 and I-2522) indicated the absence of 
interferences.

3. 2 This determination is easily contaminated by the presence of 
ammonia in the laboratory atmosphere. The digestion process should 
be performed inside a fume hood which is operating properly and 
which is located in the most ammonia-free area of the laboratory. 
Other laboratory manipulations should be performed in and near 
this hood only if there is no possibility of ammonia contamination 
from these manipulations.

4. Apparatus

4.1 Technicon block digestor, Model BD-40 with 75-ml digestion 
tubes.

4.2 With this equipment, the following operating conditions have 
been found satisfactory:

Mode switch - Automatic
Low temp. regulator - 160 deg. C
High temp. regulator - 370 deg. C
Low temp. timer - 1-1/2 hr
Total cycle time - 3-1/2 hr

4.3 Technicon AutoAnalyzer II, consisting of sampler, cartridge 
manifold, proportioning pump, water bath, colorimeter, voltage 
stabilizer, recorder, and printer.

4.4 With this equipment, the following operating conditions have 
been found satisfactory.

Absorption cell              15 mm
Wavelength                  660 nm
Cam                          60 (6/1)
Water-bath temperature       37 deg. C

     Note 1. Two different concentrations ranges of O to
3 mg/l and 2 to 10 mg/l as N are obtained by using different
STD CAL settings.

5. Reagents

5.1 Ammonium chloride standard solution I, 1.00 ml = 1.00 mg NH3 
as N: Dissolve 3.819 g NH4Cl, dried overnight over sulfuric acid, 
in ammonia-free water and dilute to 1,000 ml.

5.2 Ammonium chloride standard solution II, 1.00 ml = 0.010 mg NH3 
as N: Dilute 10.00 ml NH4Cl standard solution I to 1,000 ml with 
ammonia-free water. Prepare fresh daily.

5.3 Buffer stock solution, 71 g/l: Dissolve 71 g anhydrous Na2HP04 
in approx 800 ml demineralized water. Add 100 ml 5M NaOH, dilute 
to 1,000 ml with ammonia-free water, and mix thoroughly.

5.4 Buffer working solution: Add, with stirring, 250 ml of stock 
potassium sodium tartrate solution to 200 ml of stock buffer 
solution. Slowly, with stirring, add 120 ml 5M NaOH. Dilute to 
1,000 ml with ammonia-free water, add 1 ml BRIJ-35 solution and 
mix thoroughly.

5.5 Mercuric sulfate solution, 10.4 g/100 ml: Dissolve 8 g red HgO 
in 50 ml 3.6M H2S04 and dilute to 100 ml with ammonia-free water.

5.6 Potassium sodium tartrate solution, 149 g/l: Dissolve 200 g 
NaKC4H406.4H20 in approx 600 ml ammonia-free water. Dilute to 
1,000 ml.

5.7 Sodium hydroxide solution 5M: Add, with cooling and stirring, 
200 g NaOH to approx 800 ml ammonia-free water. Cool and dilute to 
1,000 ml.

5.8 Sodium hypochlorite solution: Dilute 6.0 ml sodium 
hypochlorite solution to 100 ml with ammonia-free water. Add 
0.1 ml BRIJ-35. A commercial bleach solution containing 5.25 
percent available chlorine is satisfactory. Prepare fresh daily.

5.9 Sodium salicylate-sodium nitroprusside solution: Dissolve 
150 g sodium salicylate and 0.30 g sodium nitroprusside [Na 
Fe(CN)5NO. 2H2O] in approx 600 ml ammonia free water. Filter 
through Whatman 41 filter paper, or equivalent, and dilute to 
1,000 ml. Add 1.0 ml BRIJ-35 and store in a light-resistant 
container.

5.10 Sulfuric acid, concentrated (sp gr 1.84).

5.11 Sulfuric acid, 0.20M : Cautiously add 11 ml concentrated H2SO 
(sp gr 1.84) to ammonia-free water and dilute to 1,000 ml.

5.12 Sulfuric acid, 3.6M: Cautiously add 200 ml concentrated H2S04 
(sp gr 1.84) to approx 700 ml ammonia-free water. Cool and dilute 
to 1,000 ml with ammonia-free water.

5.13 Sulfuric acid-mercuric sulfate-potassium sulfate solution: 
Dissolve 267 g K SO in approx 1,300 ml ammonia-free water. 
Cautiously add 400 ml conc H2S04 (sp gr 1. 84) and 50 ml mercuric 
sulfate solution (5.5). Cool and dilute to 2,000 ml with ammonia-
free water.

6. Procedure

Samples should be collected according to instructions given in 
Part III, Section 1, "Sample Preparation".

All glassware should be rinsed with ammonia-free water.

6.1 Pipet a volume of sample containing less than 0. 2 mg ammonia 
plus organic nitrogen (as N) into a digestor tube (20. 0 ml 
maximum) and adjust the volume to 20 ml with ammonia-free water.

6.2 Prepare an ammonia-free water blank and at least five 
standards containing either from 0. 00 to 0. 06 or 0. 00 to 0. 20 
mg ammonium (as N), depending upon use of O to 3 mg/l or 2 to 10 
mg/l concentration range of interest. The standards and blank must 
also undergo the digestion process.

6.3 Add 4. 0 ml sulfuric acid-mercuric sulfate-potassium sulfate 
solution (5.13) and two BOILING CHIPS. Mix well before placing in 
digestor (NOTES 2 and 3).

Note 2. Protective eye glasses and clothing are to be considered 
mandatory for this entire procedure because the use of sulfuric 
acid and high temperature solutions is very hazardous.

Note 3. Teflon boiling chips, available from Cole-Parmer 
Instrument Co., are preferable. Before use they should be soaked 
in dilute HCl (approximately 6M), rinsed several times in ammonia-
free water, and dried at 180 C. In order to avoid contamination by 
laboratory fumes, they should then be stored in a tightly-
stoppered container in a relatively ammonia-free area of the 
laboratory.

6.4 Digest under a hood for 3.5 hr using the listed conditions . 
It is imperative that the heating block cool to 150 C before 
subsequent batches of samples are placed in the digestor. Extreme 
splattering will occur if this direction is not followed.

6.5 Cautiously remove tubes from the digestor and allow to cool 
for approximately 15 minutes in a hood. Quickly add approximately 
50 ml ammonia-free water to each tube with vigorous agitation and 
extreme caution (NOTE 4). Allow to cool briefly before making the 
final dilution to the calibration mark. Stopper the tubes and 
invert several times until well mixed. (NOTE 5). If it is 
necessary to dilute a portion of this solution to remain within 
the designated concentration range, this dilution must be made 
with 0.20M H2S04.

Note 4. The precipitation of salts is minimized by this procedure. 
A vortex mixer is useful for agitating the water-acid mixture.

Note 5. Allow any precipitate which has formed or any boiling chip 
flakes to settle before filling the sample cups in sec. 6.8.

6.6 Set up manifold (fig. ). If the laboratory air is contaminated 
with ammonia, it must be passed through a scrubber containing 2.5M 
H2S04 before it enters the air-manifold tube.

6.7 Allow the colorimeter, recorder, and water bath to warm up for 
at least 30 min. Adjust the baseline to read zero scale divisions 
on the recorder with all reagents, but with 0.20M H2S04 in the 
sample line. The solution remaining in the wash reservoir from 
previous runs may become contaminated; therefore this reservoir 
should be emptied and rinsed with fresh wash solution before 
proceeding.

Place all reagent lines except salicylate into their respective 
containers, allow at least 5 min for the introduction of these 
reagents, and then place the salicylate line into its reagent 
container. If a precipitate forms after the addition of the 
salicylate, the pH is too low.

6.8 Beginning with the most concentrated working standard, place a 
complete set of standards in the first positions of the first 
sample tray (NOTE 6). Place individual standards of differing 
concentrations in every eighth position of the remainder of this 
and subsequent sample trays. Fill the remainder of each sample 
tray with unknown samples.

Note 6. To avoid possible contamination of the sample cups, they 
should remain sealed in their packages until just prior to use. If 
a contamination problem still is present, it may be necessary to 
soak the cups in ammonia-free water for several hours prior to 
use.

6.9 Begin analysis. When the peak from the most concentrated 
working standard appears on the recorder, adjust the STD CAL 
control until the flat portion of the peak reads full scale.

7. Calculations

7.1 Prepare an analytical curve by plotting the height of each 
standard peak versus its respective nitrogen concentration.

7.2 Compute the concentration of NH3 as N (mg/l) in each sample by 
comparing its peak-height to the analytical curve. Any baseline 
drift that may occur must be taken into account when computing the 
height of a sample or standard peak. Likewise, the amount of NH3 
as N present from the addition of H2S04, as indicated by the 
concentration of NH as N in the blank, must be subtracted from the 
total concentration of NH3 as N in each sample.

7.2.A  Dissolved nitrogen, dissolved ammonia and dissolved organic 
(dissolved Kjeldahl) as N is equal to NH3 as N (mg/l) as 
determined in paragraph 7.2.

7.2.B   Nitrogen, dissolved organic as N is equal to the nitrogen, 
dissolved ammonia and dissolved organic as N minus dissolved 
ammonia as N as determined in "Method I-2522".

8. Report

8.1 Report dissolved organic nitrogen (00607) or dissolved ammonia 
plus organic nitrogen (00636) as N, concentrations as follows: 
Less than 1.0 mg/l, one decimal; 1.0 mg/l and above, two 
significant figures.

9. Precision

9.1 Analysis of five test samples for ammonia by the 
salicylatehypochlorite method without the digestion, 10 times each 
by one laboratory, resulted in mean values of 0.048, 0.439, 1.471, 
2.529, and 3 . 449 mg/l and standard deviations of 0. 011, 0. 007, 
0. 022, 0. 041, and 0.125 mg/l, respectively.

      Nitrogen, total organic and nitrogen, total ammonia plus
                  total organic (total Kjeldahl)

         Kjeldahl block digestion method (I-4552-76)

Parameters and codes: Nitrogen, organic, total (mg/l as N):  00605
                      Nitrogen, ammonia plus organic total
                        (mg/l as N):                         00635
                      Nitrogen, Kjeldahl, total (mg/l as N): 00625

1. Application

1.1 This method may be used to analyze samples of water-suspended 
sediment mixtures containing O to 10 mg/l as nitrogen.

2. Summary of method

2.1 This method is identical to "Nitrogen, dissolved organic and 
nitrogen, dissolved ammonia, plus dissolved organic (Method I-
2552) ," except that a portion of an unfiltered, well-mixed sample 
is taken for analysis.

3. Interferences

3.1 A comparison study of results obtained by this method with 
those from the digestion-distillation and indophenol methods 
(Methods I1550 and I-2522) indicated the absence of interferences.

4. Apparatus

4.1 Stirrer, blender with glass container.

4. 2 For additional items of required apparatus, see "Nitrogen, 
dissolved organic and nitrogen, dissolved ammonia plus dissolved 
organic (Method I-2552)."

5. Reagents

5.1 See "Nitrogen, dissolved organic and nitrogen, dissolved 
ammonia plus dissolved organic and (Method I-2552)."

6. Procedure

Collect samples according to instructions given in Part III, 
Section 1, "Sample Preparation".

All glassware should be rinsed with ammonia-free water.

6.1 Transfer the entire contents of the sample bottle to the 
blender. Mix at approx 4500 rpm for 15 sec and, while continuing 
to mix, cautiously and rapidly pipet a 20-ml sample (or smaller 
aliquot diluted to 20 ml) to a 75-ml digestion tube. Rinse the 
pipet with a small amount of ammonia-free water to remove adhering 
particles and combine with sample (NOTES 1 and 2).

Note 1. Do not allow tip of pipet to touch the blades of the 
blender when withdrawing a sample aliquot.

Note 2. The tip of the pipet must be cut off in order to withdraw 
and drain the water-sediment mixture.

6.2 Proceed as directed in "Nitrogen, dissolved organic and 
nitrogen, dissolved ammonia plus dissolved organic (Method 
I-2552)," beginning with paragraph 6.2.

7. Calculations 

7.1 Total ammonia plus organic (Kjeldahl) nigrogen as N is equal 
to NH3 as N as determined in paragraph 7.2, "Method 2552".

7.2 Total organic nitrogen as N is equal to the total ammonia plus 
organic (Kjeldahl) nitrogen as N minus total NH3 as N as 
determined in "Method I-3520 or I-4522".

8. Report

8.1 Report "Nitrogen, total organic (00605) or nitrogen, total 
ammonia plus total organic (total Kjeldahl) (00635)," 
concentrations as follows: Less than 1.0 mg/l, one decimal; 1.0 
mg/l and above, two significant figures.

9. Precision

9.1 No precision data are available.

      Nitrogen, total organic in bottom material and nitrogen,
        total ammonia plus total organic in bottom material

           Kjeldahl block digestion method (I-6552-76)

Parameters and codes: Nitrogen, organic, total in bottom
                        material (mg/kg as N):      none assigned
                      Nitrogen, ammonia plus organic,
                         total in bottom material
                         (mg/kg as N):              none assigned
                      Nitrogen, Kjeldahl, total in bottom
                        material (mg/kg as N):              00626

1. Application

1.1 This method may be used to analyze samples of bottom material 
containing at least 4 mg/kg as nitrogen. Concentration ranges for 
determining O to 120 and 80 to 400 mg/kg of nitrogen are used.

2. Summary of method

2.1 An accurately weighed portion of wet sample is mixed with 
ammonia-free water and digested with sulfuric acid, mercuric 
sulfate, and potassium sulfate in a block digestor. All of the 
ammonium ion subsequently present is determined by an automated 
salycylate-hypochlorite colorimetric reaction.

2. 2 See also "Nitrogen, dissolved organic and nitrogen, dissolved 
ammonia plus organic and (Method I-2552)."

3. Interferences

3.1 A comparison study of results obtained by this method with 
those from the digestion-distillation and indophenol methods 
(Methods I-1550 and I-2522) indicated the absence of 
interferences.

4. Apparatus

4.1 See "Nitrogen, dissolved organic and nitrogen, dissolved 
ammonia plus organic (Method I-2552)."

5. Reagents

5 .1 See "Nitrogen, dissolved organic and nitrogen, dissolved 
ammonia plus organic (Method I-2552)."

6. Procedure

All glassware should be rinsed with ammonia-free water.

6.1 Weigh, to the nearest milligram, an amount of wet sample (500 
mg maximum) containing less than 0.2 mg total organic or total 
ammonia plus total organic (total Kjeldahl) nitrogen.

6.2 Quantitatively transfer the weighed sample to a 75-ml 
digestion tube, rinsing the weighing container with ammonia-free 
water as needed. Add additional ammonia-free water as necessary to 
bring the liquid volume in the digestion tube to approx 20 ml.

6.3 Proceed as directed in "Nitrogen, dissolved organic and 
nitrogen, dissolved ammonia plus dissolved organic (Method I-
2552)," beginning with paragraph 6.2.

6.4 Determine the percent moisture for the wet sample by placing a 
representative portion (0.5 to 1.0 g) into a tared weighing 
bottle. Place the weighed bottle containing the accurately weighed 
sample in a drying oven and heat for 2 hr at 110 C. Cool in a 
desiccator for 30 min and immediately weigh.

6.5 Compute the loss in weight in grams, of the sample, on heating 
at 110!C.

6.6 Compute the percent moisture in the sample as follows:

                       Loss in wt, grams
Percent moisture =     __________________   X 100 
                       Wet sample wt, grams

                         Wet sample wt (100 - percent moisture)
Sample, dry wt, grams =  ______________________________________
                                        100

7. Calculations

7.1 Total ammonia plus total organic (total Kjeldahl) nitrogen as 
N is equal to NH3 as N as determined in paragraph 7.2, "Method 
I-2552."

7.2 Total organic nitrogen as N is equal to the total ammonia plus 
total organic nitrogen as N minus total NH3 as N as determined in 
"Method I-6522".

7.3 Convert to dry weight concentrations as follows:

Total organic or total ammonia plus total organic nitrogen in 
mg/kg =

                       mg NH3 as N in sample
                       _____________________    X 1000
                       Sample, dry wt, grams

Report

8.1 Report nitrogen, total organic or total ammonia plus total 
organic as N in mg/kg (milligrams per kilogram) of dry weight 
sample as follows: Less than 1,000 mg/kg, nearest 10 mg/kg; 1,000 
mg/kg and above, two significant figures.

Precision

9.1 No precision data are available.