TOTAL KJELDAHL NITROGEN DETERMINATION

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1 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 1 of 9 Crop and Environmental Sciences Division Standard Operating Procedure TOTAL KJELDAHL NITROGEN DETERMINATION -AP04 This document is issued under the authority of MS. LILIA R. MOLINA Assistant Manager II THIS IS A CONTROLLED DOCUMENT

2 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 2 of 9 SIGNATURE PAGE The signatures below signify approval and concurrence of the signatories with the contents of this SOP coupled with the commitment to provide the resources necessary to ensure proper quality operation of the ASL laboratory. Issuing Authority: Name & Title Signature Date (yyyy-mm-dd) Lilia R. Molina Manager/QM Christy S. Daniel Deputy Quality Manager

3 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 3 of 9 -AP04 TOTAL KJELDAHL NITROGEN DETERMINATION 1.0 Scope and application This SOP describes the procedure for the determination of total Kjeldahl nitrogen concentration in test samples (plant or soil) via a two-step process. Firstly, the sample is digested using sulfuric acid and digestion catalyst which converts the organic nitrogen content to the ammonium form. The sample digest is then analyzed for ammonium-n colorimetrically in an auto-analyzer utilizing the Berthelot reaction. The procedure does not quantitatively digest nitrogen from heterocyclic compounds (bound in a carbon ring), oxidized forms such as nitrate and nitrite, or ammonium from within mineral lattice structures. 2.0 Summary Total Kjeldahl Nitrogen is determined by converting the various organic nitrogen forms to NH + 4 and then measuring the NH + 4 -N concentration. To accomplish this, a measured weight of dry, oven-dried ground sample material is digested in a mixture of conc. H 2 SO 4, K 2 SO 4 and Selenium at about 380 C for a certain period of time. The ammonium formed is determined colorimetrically in a Technicon AutoAnalyzer II (TAA) by reacting it with sodium salicylate in the presence of sodium hypochlorite and nitroprusside to form an emerald green complex (the Berthelot reaction) whose intensity is measured at 660 nm. This method only partially converts nitrate, thus samples with high nitrate levels must be pretreated with salicylic acid-thiosulfate to ensure complete conversion (Bremner and Mulvaney, 1982; Bremner, 1996). 3.0 Apparatus and materials 3.1 Technicon AutoAnalyzer II unit consisting of sampler, manifold, proportioning pump, heating bath, colorimeter (with 650 nm filter, 1.5 mm x 15 mm), and recorder (BranLubbe 310), computer software attached to the colorimeter for data acquisition (FASPRO 1 v.2.01) and data processing (FASPRO 2) or any other equivalent continuous flow, segmented stream autoanalyzer system 3.2 Analytical balance, readability of 0.01g and 0.001g 3.3 Aluminum block digestor with automatic temperature control suitable for digestion x 20 cm calibrated digestion tubes

4 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 4 of Dispenser, Repipet 10 ml or equivalent 3.6 Vortex Genie 2 cyclone mixer or equivalent 3.7 Calibrated scoops (for catalyst mixture) 3.8 Polyethylene bottles 3.9 Volumetric flasks 3.10 Funnel 3.11 Autosampler sample cups 4.0 Reagents and standards 4.1 Concentrated sulfuric acid (Analytical Grade). 4.2 Potassium sulfate/selenium mixture. Grind selenium powder to pass a 100 mesh sieve, and add 2 g of this mixture to 100 g of anhydrous potassium sulfate. Mix well each time before use as the mixture tends to separate. 4.3 Sodium hydroxide solution, 20% (Stock). To 600 ml deionized water, add 200 g of sodium hydroxide pellets. Cool to room temperature and dilute to one liter with deionized water. 4.4 Sodium potassium tartrate solution, 20% (Stock). Dissolve 200 g of sodium potassium tartrate tetrahydrate in about 600 ml of deionized water, let it cool to room temp. Dilute to one liter with deionized water and mix thoroughly M Buffer solution (Stock). Dissolve 134 g of sodium phosphate, dibasic, crystal in about 800 ml of distilled water. Add 20 g sodium hydroxide pellets, dilute to one liter with deionized water and mix thoroughly. 4.6 Working buffer solution. Combine the reagents in the stated order: add 250 ml of stock 20% sodium potassium tartrate solution, to 200 ml of 0.5 M stock buffer solution with swirling. Slowly, with swirling, add 250 ml of 20% sodium hydroxide solution. Dilute to one liter with deionized water, add 1.0 ml of 30% Brij-35 solution and mix thoroughly. 4.7 Sulfuric acid/sodium chloride solution (as carrier or diluent). Dissolve 100 g of sodium chloride in about 600 ml of deionized water. Add 7.5 ml of sulfuric acid and dilute to one liter with deionized water. Add about 1.0 ml of 30% Brij-35 (about 20 drops) and mix thoroughly. 4.8 Sodium salicylate/sodium nitroprusside solution. Dissolve 150 g of sodium salicylate (NaC 7 H 5 O 3 ) and add 0.3g of sodium nitroprusside (disodium pentacyanonitrosylferrate, Na 2 Fe(CN) 5 NO 5H 2 O) in about 600 ml of deionized

5 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 5 of 9 water. Filter through fast filter paper into 1L volumetric flask and dilute to volume. Add about 1 ml of 30% Brij-35 and mix thoroughly. Store in amber bottle. 4.9 Sodium hypochlorite solution, 0.525%. Dilute10.0 ml of any commercial bleach solution (containing 5.25% available chlorine is satisfactory) to 100 ml with deionized water. Add 0.1 ml (2 drops) of 30% Brij-35 and mix thoroughly. Prepare fresh daily Internal reference standards In-house Rice Plant- 99S In-house Rice Plant- 99 G CRM WEPAL IPE CRM- NIST Tomato Leaves Soil In-house Reference Material SIRM 2007 Control no CRM- WEPAL ISE 987 Control no EDTA Calibration standard Solution A (Stock ppm N). Weigh g ammonium sulfate (99.4%purity, previously dried at 105 C), dissolve in deionized water, add a few drops of concentrated sulfuric acid and make to final mass ( g) with deionized water. Shake well to mix Standard solution B (100 ppm N): Dilute g of stock solution A with deionized water to g Working calibration standards. All standards are made up in 10% v/w sulfuric acid and 5% w/w potassium sulfate. Prepare the working standards as follows (Refer to Table 1): Weigh the required amount of Std soln A or B; Add 5.00 g. of potassium sulfate; Add deionized water until the volume is about 70 ml; Add 10 ml of concentrated sulfuric acid; When cool make up to the final mass by adding deionized water.

6 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 6 of 9 Table 1. Working Calibration Standards from Standard Solution A and B. Concn. of Working Std (ppm N) Standard Sol n, (g) Conc. H 2 SO 4 (ml) K 2 SO 4, (g) Final Mass, (g) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) (Std Sol n B ) * 9 (Std Sol n A) * 10 (Std Sol n A) * 12 (Std Sol n A) * 15 (Std Sol n A) *applicable only for high N samples Ammonium chloride stock solution (1000 ppm N). Weigh 3.84 g of ammonium chloride (99.5%purity, previously dried at 105 C), dissolve in deionized water, add a few drops of concentrated sulfuric acid and make to final mass (1 000 g) with deionized water. Shake well to mix Ammonium chloride standard solution (100 ppm N). Weigh g of ammonium chloride stock solution (1000 ppm N) and dilute to g with deionized water Initial Calibration Verification Standard (50 ppm N). Weigh 50g of ammonium chloride standard solution (100 ppm N), add 5.00 g. of potassium sulfate, add deionized water until the volume is about 70 ml and add 10 ml of concentrated sulfuric acid. When cool, make up to the final mass of g with deionized water. 5.0 Sample preparation, preservation and storage See procedures in QM-AD036-MP24- Plant and soil sample preparation procedures and QM-AD038-MP25/SFT03- Sample receipt and Chain of custody for the appropriate sample preparation, handling, and storage.

7 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 7 of Procedure 6.1 Sample Digestion Weigh 50.0 ± 0.1 mg of oven dried sample in a 2.5 x 20 cm precalibrated digestion tube. In the case of soil samples, weigh ± 0.1 mg of finely ground air dried soil (100 mesh size) Add 1 g of the potassium sulfate/selenium mixture into the digestion tube Add 2.0 ml concentrated sulfuric acid and mix using a "Vortex" mixer Place the digestion tubes on the Aluminum block digestor and turn on the temperature controller which is preset at a temperature of 370 ± 10 o C Shake the tube if sample particles are clinging to the side of the tube The digestion is complete when the solution has been clear for 15 min Remove the digestion tubes from the digestor, cool. When the digestion tubes are still warm to touch add, very carefully, a few ml of deionized water to mix. This prevents solidification of the mixture in the digestion tube When cooled, add deionized water up to the calibration mark. Shake well to mix Transfer the diluted digest into a 20 ml test tube and stand overnight to allow for sedimentation before analysis Ammonium-Nitrogen is analyzed using the automated Technicon Autoanalyzer II described below Internal standards are digested also together with the sample. 6.2 Colorimetric Determination of Nitrogen using the Technicon AutoAnalyzer II Set up autoanalyzer and analyze the samples and standards using the flow diagram shown in Figure Follow the operating procedure for the Technicon AutoAnalyzer II.

8 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 8 of 9 Figure 1. Flow diagram for the determination of NH 4 -N in sample digests. 7.0 Quality Control 7.1 Each analytical batch of samples for digestion and colorimetric analysis should contain at least two method blanks, one chemical standard (5 mg EDTA standard), certified and in-house reference materials, sample repeats, initial calibration verification standard and QC check standard. 7.2 Unusual situations occurring during sample preparation and analysis must be documented on the work sheet, and non-conformity/corrective action report must be issued if necessary.

9 SOP Code: AP04 Issue Date: Revision No.: 03 Page: 9 of Calculations The concentration of NH 4 + in the digest is determined using the equation obtained by linear regression of the concentrations of the standards on the corresponding absorbance measurements. Calculate total Kjeldahl nitrogen as: % N = (mg l -1 NH 4 -N in digest mg l -1 NH 4 -N in method blank) V s (100) (sample weight, mg) or % N = (mg kg -1 NH 4 -N in digest mg kg -1 NH 4 -N in method blank) w s (100) (sample weight, mg) where w s is the final mass of digest in kg. where V s is the final volume of digest in liter. 9.0 References 9.1 Anonymous Manual Technicon AutoAnalyzer II Industrial Method No W/B. Release January 1976/Revised March Individual/simultaneous determination of nitrogen and/or phosphorus in BD Acid Digest. 9.2 Bremner, J. M Chapter 37 Nitrogen Total: In Methods of Soil Analysis. Part 3. Chemical Methods. SSSA Book Series no Bremner, J. M., and C. S. Mulvaney Nitrogen-Total. P , In: A.L. Page et. al. (eds). Methods of soil analysis. Part 2. 2 nd ed. Agron. Monograph Kalra YP, Maynard DG Methods manual for forest soil and plant analysis. Information Report NOR-X-319. Edmonton, Alberta (Canada): Forestry of Canada, Northwest Region, North Forestry Centre. 9.5 Mulvaney, R. L Chapter 38 Nitrogen Inorganic Forms: In Methods of Soil Analysis. Part 3. Chemical Methods. SSSA Book Series no Varley, J. A. (1966). The determination of nitrogen, phosphorus and potassium ion plant materials. Analyst. 91: (END OF DOCUMENT)

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