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.