INEC Carob Manual 1 2 12.

Chemical Methods

12. Chemical Methods

Crude protein – Kjeldahl method, boric acid modification

1 Scope methyl red-methylene blue indicator (reagent 3.5) to

18 L water. (See 5.5).
Applicable to wheat and flour mill products only.
4 Procedure
2 Apparatus
4.1 Weigh quickly and accurately 1 g finely
2.1 Kjeldahl flasks, Pyrex, 800 ml capacity; used ground sample. Place in digestion flask. (Sample may
for both digestion and distillation (distn). be placed in nitrogen-free paper to prevent clinging to
2.2 Digestion heaters, 600 W (more or less, de- sides of flask.) Add polyethylene packet of catalyst, or
pending on voltage). Heater unit should boil 250 ml equiv., and 25 ml conc. H2SO4 to flask (reagent 3.1).
water starting at 25°C in 5 min with hot burners. Digest till soln is clear and then 30 min longer; remove
2.3 Digestion unit; consists of electric heaters, and cool but do not allow to crystallise.
large lead tube, fume stack (plastic), and suction fan 4.2 Place 300 ml bottle or flask contg 50ml boric
capable of exhausting toxic fumes to outside air. acid-methyl red-methylene blue indicator soln (reagent
2.4 Distn unit (see footnote); to consist of Iowa 3.7) under condenser tube with tip of condenser tube
State type connecting bulbs (traps) 36 x 100 mm, Py- immersed under surface of soln. Add to original flask
rex glass condenser tubes, pure gum-rubber stoppers that is cooling 250-30 ml tap water and anti bumping
and tubing, electric heating units (600 W), condenser agent, if not previously added. Gently add 50 ml conc.
tubes capable of being kept cool with adequate amts of NaOH (reagent 3.4), connect to condenser with tight
cool water during distn and with thermo-water control fitting rubber stopper, and swirl. Boil until all ammo-
on stills. Upper ends of bulbs or traps connect with nia has dist (at least 150 ml of distillate), and then set
high quality rubber tubing to condenser tubes and receiving bottle down so that condenser tube is com-
lower ends with rubber stoppers to 800 ml distn flask. pletely drained.
Lower ends of condenser tubes have rubber-connected 4.3 Titrate distillate to neutrality with std 0.1 N
glass or polyethylene tubes that lead to: H2SO4, using burette graduated in 0.1 ml. Read ml of
2.5 Receiving bottles or flasks, 300 ml capacity. acid used, directly from burette.
2.6 Proper burettes for dispensing (a) conc. 4.4 Run blank detn periodically, using all ingredi-
H2SO4, (b) conc. NaOH, (c) boric acid-indicator soln, ents except sample. Corr burette reading for nitrogen in
and (d) class A burette for dispensing 0.1000 N H2SO4. reagents as shown by blank.
(See 5.1).
5 Notes
3 Reagents
5.1 In routine testing of large no. of samples, use
3.1 H2SO4, conc., 93-98 %, nitrogen-free. large dispensing burettes for conc. acid and alk and
Schellbach automatic zero burets at titration table.
3.2 Catalyst. Polyethylene packets contg 15 g
potassium sulphate, 0.7 g mercuric oxide, and approx. 5.2 Precaution: Copper sulphate is recommended
0.10 g pumice stone. (See 5.2). as a less hazardous catalyst than either mercury or
selenium, or their compounds (see Ref. 4). Adequate
3.3 Anti bumping agent. Either zinc metal, 20 exhaust ventilation must be provided in digestion-distn
mesh, or pumice stone (if pumice is not already com-
area. With mercury as catalyst and 40 min digestion
bined in catalyst mixt).
time, use polyethylene packets contg 9.9 g potassium
3.4 NaOH, pellets or soln, nitrate-free. For soln, sulphate, 0.41 g mercuric oxide, 0.08 g copper sul-
dissolve approx. 450 g solid NaOH in 1 L water. (Sp gr phate, and approx. 0.10 g pumice stone.
of soln should be 1.36 or more.) Since mercury is used
5.3 Mixed indicator consisting of 0.75 g methyl
as catalyst, add 80 g sodium thiosulphate per L to
red and 0.625 g Guinea green per L or 0.75 g methyl
NaOH soln to ppt mercury.
red and 0.5 g methylene blue (Ref. 8) dissolved in 300
3.5 Methyl red-methylene blue indicator. Mix 2 ml alc may be used. Any indicator used should have
parts 0.2 % alc methyl red soln with 1 part 0.2 % alc sharp end point and distinct colour change. Use 35 ml
methylene blue soln. Other indicators may be used to 18 L bottle.
satisfactorily. (See 5.3).
5.4 Rodkey (Ref. 8) has successfully applied tris
3.6 Std H2SO4, approx. 0.1 N but accurately stdze (hydroxymethyl) aminomethane as a convenient pri-
(See 5.4). mary std for direct stdn of acid solns.
3.7 Boric acid-methyl red-methylene blue receiv-
er soln. Add 360 g boric acid (crystals) and 48 ml
INEC Carob Manual 2 12. Chemical Methods

5.5 For proteins higher than 22 %, use 720 g (4 2. Corn Industries Research Foundation. 1957.
%) boric acid crystals and 48 ml methyl red-methylene Standard Analytical Methods. Method A18. The Foun-
blue indicator to 18 L bottle of water. dation, Washington, DC.
5.6 To check entire protein method, digest 0.1 g 3. Johnson, A. H., and Green, J. R. 1930. Modi-
pure ammonium oxalate (monohydrate) with 2 g pure fied methyl red and sodium alizarin sulphonate indica-
sugar using regular procedure. Resulting protein tors. Ind. Eng. Chem., Anal. Ed. 2:2.
should be 11.24 % calcd as follows: (NH4)2C2O4 • 4. Kane, P. F. 1984. Comparison of HgO and
H2O with mol wt of 142.12 contains 28.016 g nitrogen CuSO4 as digestion catalysts in manual Kjeldahl de-
or 19.713 %; 19.713 % x 5.7 : 10(0.1g sample) = 11.25 terminations of crude protein in animal feeds. Collabo-
% protein. rative study. J. Assoc. Off. Anal. Chem. 67:869.
5.7 It is best that Boric Acid modification be used 5. Kent-Jones, D. W., and Amos, A. J. 1957.
in air-conditioned laboratory ( Ref. 5). Ammonia may Nitrogen and crude protein. Page 548 in: Modern Ce-
be lost if contents of receiver flasks exceed 40 °C. real Chemistry, 5th ed. Northern Pub. Co., Liverpool.
5.8 Use of 0.1253 N H2SO4 simplifies calculation 6. Neill, C. D. 1962. The Kjeldahl protein test.
by making % protein equal to the ml of 0.1253 N Cereal Sci. Today 7:6. also 4:310 (1959).
H2SO4 used for titrating sample, minus the ml used for
7. Reeder, W., and Patton, J. 1957. Standard
titrating blank.
acid for Kjeldahl. Cereal Sci. Today 2:176.
6 Calculation 8. Rodkey, F. L. 1964. Tris (hydroxymethyl)
aminomethane as a standard for Kjeldahl nitrogen
(ml std H2SO4 x N of H2SO4) x 1.4007 x 5.7 analysis. Clin. Chem. 10:606.
% Protein
sample wt (g) 9. Wagner, E. C. 1940. Titration of ammonia in
presence of boric acid in the macro-, semimicro-, and
where: factor for wheat, flour, and bread = 5.7.
micro-Kjeldahl procedures, using methyl red indicator
and the colour matching end point . Ind. Eng. Chem.,
7 References
Anal. Ed. 12:771.
1. Coleman, D. A., Fellows, H. C., and Dixon, 10 Willard, H. H., Furman, N. H., and Bricker,
H. B. 1925. A study of methods for making protein C. E. 1960. Elements of Quantitative Analysis. D. Van
tests on wheat. Cereal Chem. 2:132. Nostrand, Princeton, NJ. p. 164.