AACC International Method 46-12.01

Nitrogen AACC International Method 46-12.01

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Crude Protein—Kjeldahl Method, Boric Acid Modification

Final approval October 8, 1976; Reapproval November 3, 1999

Objective

This method is a modification of the Kjeldahl method, using boric acid in the indicator solution. The method determines total nitrogen by digestion of sample in H2SO4. It is applicable to wheat and flour mill products only.

Apparatus

1. Kjeldahl flasks, Pyrex, 650- to 800-ml capacity; used for both digestion and distillation.

2. Digestion heaters, 500–600 W (depending on voltage and on how close bulb of flask is to electric heating elements). Heater unit should boil 250 ml water starting at 25° in 5 min with hot burners.

3. Digestion unit; consists of electric heaters, large lead tube, and plastic fume stack with suction fan capable of exhausting toxic fumes to outside air.

4. Distillation unit; to consist of Iowa State-type connecting bulbs (traps) 36 × 100 mm, Pyrex glass condenser tubes, pure gum-rubber stoppers and tubing, electric heating units (600 W), and condenser tubes capable of being kept cool with adequate amounts of cool water during distillation and with thermo-water control on stills. Upper ends of bulbs or traps connect with high-quality rubber tubing to condenser tubes and lower ends connect with rubber stoppers to 650- to 800-ml distillation flask. Lower ends of condenser tubes have rubber-connected glass or polyethylene tubes that lead to:

5. Receiving bottles or flasks, 300-ml capacity.

6. Proper burets for dispensing a) concentrated H2SO4, b) concentrated NaOH, c) boric acid-indicator solution, and d) 0.1000N H2SO4. See Note 1.

Reagents

1. H2SO4, concentrated, 95–98%, nitrogen-free.

2. Catalyst. Polyethylene packets containing 15 g potassium sulfate, 0.7 g mercuric oxide, and approximately 0.10 g pumice stone. See Note 2.

3. Antibumping agent. Either zinc metal, 20-mesh, or pumice stone (if pumice is not already in catalyst mixture).

4. NaOH, pellets or solution, nitrate-free. For solution, dissolve approximately 450 g solid NaOH in 1 liter water. (Specific gravity of solution should be 1.48 or more.) Since mercury is used as catalyst, add 80 g sodium thiosulfate per liter to NaOH solution to precipitate mercury.

5. Methyl red-methylene blue indicator. Mix 2 parts 0.2% alcohol methyl red solution with 1 part 0.2% alcohol methylene blue solution. Other indicators may be used satisfactorily. See Note 3.

http://dx.doi.org/10.1094/AACCIntMethod-46-12.01

Nitrogen AACC International Method 46-12.01

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Crude Protein—Kjeldahl Method, Boric Acid Modification (continued)

6. Standard H2SO4, approximately 0.1N but accurately standardized by Method 70-80.01. Other recognized standardization methods may be used. See Note 4.

7. Boric acid-methyl red-methylene blue receiver solution. Add 360 g boric acid (crystals) and 48 ml methyl red-methylene blue indicator (reagent 5) to 18 liter water. See Note 5.

Procedure

1. Weigh quickly and accurately 1.0 g finely ground sample. Place in digestion flask. (Sample may be placed in nitrogen-free paper to prevent clinging to sides of flask.) Add polyethylene packet of catalyst, or equivalent, and 25 ml concentrated H2SO4 (reagent 1) to flask. Digest till solution is clear and then 30 min longer; remove and cool but do not allow to crystallize.

2. Place 300-ml bottle or flask containing 50 ml boric acid-methyl red-methylene blue indicator solution (reagent 7) under condenser tube with tip of condenser tube immersed under surface of solution. Add to original flask that is cooling 250–300 ml tap water and antibumping agent, if not previously added. Gently add 50 ml concentrated NaOH (reagent 4), connect to condenser with tight-fitting rubber stopper, and swirl. Boil until all ammonia has distilled (at least 150 ml of distillate), and then set receiving bottle down so that condenser tube is completely drained.

3. Titrate distillate to neutrality with standard 0.1N H2SO4,

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