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15

N Analysis Service Department of NRES University of Illinois at U-C

C-227 Turner Hall 1102 South Goodwin Avenue Urbana, IL 61801

tel.: (217) 333-9467 (217) 244-7592 fax: (217) 244-2755

Technical Note 97-03

Total-N and 15N Analysis by the Kjeldahl Method Apparati Aluminum block digestion stand.

digester

or

micro-Kjeldahl

Pyrex® digestion tubes (50-250 mL)1 and/or Kjeldahl flasks (100 mL, with 24/40 inner joint). Semimicrodistillation apparatus (Fig. 1). The unit illustrated is a modified version of an apparatus originally designed for inorganic-N analyses (Bremner, 1965). It must be fabricated by a glassblower2. An adapter allows attachment of a 100-mL Kjeldahl flask equipped with a ground-glass 3 joint , which serves as the distillation chamber. Steam for distillation is generated by boiling deionized water in a 5-L flask containing a few Teflon boiling chips and equipped with an electric heating mantle controlled by a variable transformer. Before use, the apparatus should be steamed out for about 10 min, during which the transformer is adjusted so that distillate is collected at the rate of 7 to 8 mL min-1. A glass or plastic trap is installed on the outlet end of the condenser to prevent condensate that collects on the outside surface of the condenser from contaminating the distillate. Microburette (5 mL, graduated at 0.01-mL intervals) or automatic titrator. Electric hot plate. A commercial griddle is satisfactory, such as the West Bend Model 76220.

Fig. 1. Steam distillation apparatus.

Microplate. If isotopic analyses are to be performed by the 15N Analysis Service, a Microtiter® plate will be required that is manufactured by Dynatech Laboratories, Chantilly, VA (cat. no. 001-010-2205). This plate is available from Fisher Scientific (cat. no. 14-245-71). Reagents Sulfuric acid, concentrated (18 M).

1

If a 40-place Al block digester is used, digestions are conveniently carried out in Pyrex® 50-mL nonprotein-N Folin-Wu tubes. 2 A commercial source of the distillation apparatus described is O’Brien’s Scientific Glass Blowing, 750 West Railroad Street, Monticello, IL 61856 (phone: 217762-3636). 3 The joint should not be greased.

Potassium permanganate solution. Dissolve 25 g of KMnO4 in 500 mL of deionized water. Store in an amber bottle. Sulfuric acid (9 M). To 500 mL of deionized water in a 2-L Pyrex® flask, slowly add 500 mL of concentrated H2SO4. Mix carefully and cool. Reduced iron powder. The certified reagent available from Fisher Scientific is acceptable. Technical Note 97-03 (rev. b) Page 1 of 3


15

N Analysis Service Department of NRES University of Illinois at U-C

C-227 Turner Hall 1102 South Goodwin Avenue Urbana, IL 61801

Potassium sulfate-catalyst mixture. Powder 20 g of CuSO4·5H2O by grinding in a mortar, and mix intimately with 2 g of Se and 200 g of K2SO4 (powder). Sodium hydroxide solution (approximately 10 M). To 12 L of deionized water in a heavy-walled 20-L Pyrex® bottle, add 6.0 kg of NaOH pellets. Dissolve the pellets with vigorous stirring from a motorized stirrer. Cool the solution, bring the volume to 15 L by adding deionized water, and mix thoroughly with the motorized stirrer. Stopper the bottle during storage. Boric-acid indicator solution. Add 400 g of reagentgrade H3BO3 to 18 L of deionized water in a 20-L Pyrex® bottle marked to indicate a volume of 20 L, and stir vigorously with a motorized stirrer to dissolve the H3BO3. Then add 0.396 g of bromocresol green and 0.264 g of methyl red (as water-soluble sodium salts), and bring the volume to 20 L with deionized water. With continuous stirring, adjust the pH to approximately 5.0, or until the solution assumes a reddish-purple tint, by cautiously adding 1 M NaOH4. Dispense the solution through Teflon tubing. Dilute sulfuric acid, 0.005 M standard (for titration) and 1 M (for acidulation of distillates). Formic acid (approximately 1 M). Dilute 10.5 mL of concentrated formic acid to 250 mL with deionized water in a volumetric flask. Ethanol (95% v/v). Procedures Digestion. Place a sample of soil, plant material, or solution (≤ 20 mL of natural water or 0.5 M K2SO4 soil extract) containing about 1 mg of N in a Kjeldahl flask or digestion tube, add 1 mL of KMnO4 solution (by dispenser or pipette), and swirl to mix completely. Allow the sample to stand for about 30 s; then pipette 2 mL of 9 M H2SO4 down the side of the flask or tube, taking care that no 4

If excess NaOH is added, the pH can be reduced by adding dilute HCl.

tel.: (217) 333-9467 (217) 244-7592 fax: (217) 244-2755

sample material is left on the wall. After 5 min, add 0.50 ± 0.01 g of reduced Fe powder through a funnel that reaches to within 1-2 cm of the mixture at the bottom. Swirl the flask or tube to bring all of the Fe into contact with the acid, allow it to stand for about 15 min (preferably in a fume hood), and apply gentle heat (approx. 90°C) for 45 min using the digestion stand or block in a fume hood. Allow a few minutes for cooling; then add 1.5 g of K2SO4-catalyst mixture (through a long-stemmed funnel) and 4 mL of concentrated H2SO4, and heat at about 150°C until the water is removed and white fumes appear (30 min to 1 h for soil or plant samples; up to 4 h for solution samples). Increase the heat (to about 250°C) until the color of the mixture becomes yellowish green, and then boil the mixture gently (340-360°C) for 5 h. Distillation. After digestion is complete, allow time for cooling, and then add about 10 mL of deionized water to the digest from a wash bottle. If digestion was performed using an Al block, mix the diluted digest using a vortex mixer, and transfer the mixture to a 100-mL Kjeldahl flask equipped with a 24/40 inner joint5. Repeat the transfer three times with a total of 15 to 20 mL of deionized water. Prepare the steam distillation apparatus for use by opening the lower stopcock on the steam-bypass assembly and closing the upper stopcock, which is connected to the distillation head (Fig. 1). Add 2 mL of H3BO3indicator solution to a 100- or 150-mL beaker marked to indicate a volume of 35 mL, and position the beaker under the condenser of the distillation apparatus. Then add 20 mL of 10 M NaOH to the funnel affixed to the top of the distillation head, attach the Kjeldahl flask to the distillation apparatus, and lift the peg stopper in the funnel to allow the NaOH to flow slowly into the flask. Then seal the funnel with the peg stopper, and immediately commence distillation by opening the upper stopcock on the steam-bypass assembly and closing the lower stopcock. When the volume of distillate reaches 35 mL, rinse the tip of the condenser, and stop the distillation by opening the lower stopcock 5

Transfer is aided if the digest has not cooled below 100°C; transfer is unnecessary if the tubes are equipped with ground-glass joints for connection to the distillation apparatus. Technical Note 97-03 (rev. b) Page 2 of 3


15

N Analysis Service Department of NRES University of Illinois at U-C

C-227 Turner Hall 1102 South Goodwin Avenue Urbana, IL 61801

tel.: (217) 333-9467 (217) 244-7592 fax: (217) 244-2755

on the steam-bypass assembly. Determine NH4+-N in the distillate by titration with 0.005 M H2SO4. At the end-point, the color changes from green to a faint pink. Calculate the amount of N in the digest using the expression, (S − C) × T, where S is the volume of H2SO4 used in titration of the sample digest, C is the volume used in titration of a control digest (obtained by carrying out digestion in the absence of any 6 sample), and T is the titer of the titrant . After removing the Kjeldahl flask from the distillation apparatus, attach a flask containing 1 mL of 1 M formic acid, and carry out steam distillation for 1 min. Then detach the flask, and replace it with one containing 25 mL of ethanol. Steam-distill the ethanol for 3 to 4 min. For 15N analysis of N in the digest, add 1 M H2SO4 to the H3BO3 solution after titration (0.1 µL µg N-1), and evaporate the acidified solution to dryness on a hot plate (approx. 90°C). Dissolve the (NH4)2SO4 in the beaker in a sufficient volume of deionized water that a 0.05- to 0.3-mL aliquot containing 50 to 200 µg of N can be transferred to a plastic microplate. Carry out final drying of samples by placing the microplate in a low-temperature oven (≤ 70°C). References Bremner, J. M. 1965. Total nitrogen. p. 11491178. In C. A. Black et al. (ed.) Methods of soil analysis. Part 2. Agron. Monogr. 9. American Society of Agronomy, Madison, WI. Bremner, J. M. 1996. Nitrogen−Total. p. 10851121. In D. L. Sparks et al. (ed.) Methods of soil analysis. Part 3. SSSA Book Ser. 5. Soil Science Society of America, Madison, WI. Mulvaney, R. L. 1993. Mass spectrometry. p. 1157. In R. Knowles and T. H. Blackburn (ed.) Nitrogen isotope techniques. Academic Press, San Diego, CA.

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The titer is obtained by multiplying the molarity of the titrant by 28000. For 0.005 M H2SO4, T = 140 µg of N mL-1. Technical Note 97-03 (rev. b) Page 3 of 3


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