Kjeldahl Method - Bradstreet

V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4

(44) Radt, F., Elseviers Encyclopaedia of Organic Chemistry,

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(47) Snell, F. D., and Snell, C. T., Colorimetric Methods of rlnaly-

(48) Stillman, J. W., and Dunlop, E. C., -4m. SOC. Testing Illaterials,

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Analytical Methods, 1953.

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(50) Vierordt, K., Die Anwendung des Spektralapparatus sur Photo- metric der Absorptions-spektren und sur quantitativen chemischen Analyse, Tubingen, H. Laupp, 1873.

(51) Welcher, F. J., Organic Analytical Reagents, New York, D. Van Nostrand Co., 194748.

(52) Yoe, J. H., Photometric Chemical Analysis, Vol. I, New York, John Wiley & Sons, 1928.

(53) Yoe, J. H., and Sarver, L. A., Organic Analytical Reagents, New York, John Wiley & Sons, 1941.

RECEIVED for review October 20, 1953. Accepted December 4, 1953. Presented before the Division of Chemical Literature at the 124th Meeting of the AMERICAN CHEMICAL SOCIETY, Chicago, Ill.

Kjeldahl Method for Organic Nitrogen R. B. BRADSTREET The Bradstreet Laboratories, he. , 1356 North Broad St., Hillside, N. 1.

H E extent to which the Kjeldahl method has been applied to T organic analysis has led to the publication of many data. From time to time, reviews (14 ,16 ,66 ) have been published cover- ing both macro- and micromethods. Comprehensive reports on microchemical methods have been made by Willits and Ogg (?I, 72). In a previous paper (9), the method was reviewed from its inception to 1939. This report covers the period from 1939 to the present date.

The Kjeldahl method, through the efforts of countless workers, has been modified to include many types of nitrogen compounds, and given a much wider scope than Kjeldahl could have antici- pated when he devised the method which was primarily to follow the course of protein changes in grain. The considerable use of the method arises mainly from the fact that it is a relatively un- complicated procedure. The ease with which the determination can be made has undoubtedly led workers to look upon it as a possible cure-all for problems in nitrogen analysis. It is obvious, however, from published data, that all forms of organic nitrogen cannot be converted quantitatively to ammonia.

In order to correlate the available data, the method has been broken down into its component parts-digestion, catalysts, distillation, titration, etc.

DIGESTION

The Kjeldahl method (27 ) is a wet oxidation using concentrated sulfuric acid. Originally, sulfuric acid alone was used, but because oxidation under such conditions was time-consuming, particularly if large samples were used, the method was soon modified by the addition of potassium sulfate (21). With the advantage of a higher boiling point, oxidation was possible in a much shorter time. Ogg and Willits ( 4 6 ) measured the tempera- ture of boiling digestion mixtures a t six different points, and having a potassium sulfate concentration from 0.25 to 0.875 gram per ml. of acid. Using nicotinic acid, they found that results varied from less than 1% to 11%, and attributed this dif- ference to the boiling temperatures or insufficient heating. Mil- bauer (44 ) also studied the acid digestion, using diphenylamine as a type compound with various additives, and found that a mixture of 25 ml. of sulfuric acid, 1 gram of mercury, 20 grams of (K2SZOB) and 25 ml. of hydrogen peroxide gave complete conversion in 4 minutes.

In the determination of nitrogen in foodstuffs (1 j , a digestion mixture of 40 grams of sodium sulfate and 1.6 grams of copper sulfate per 100 ml. of acid is recommended, with a digestion time of 6 hours. For the microdetermination of protein in 50% glycerol (51), bromine is used as an oxidizing agent, supplemented by 30% hydrogen peroxide. Rosenthaler (57 j , who studied the formation of ammonia by oxidation of nitrogen-containing compounds, stated that the final conversion to ammonia is caused by hydroly-

sis. Quartaroli (53) considered the digestion to take place in two steps: oxidation of the organic matter, and reduction of all nitrogen to the ammonium salt. From his experiments with pure compounds, he concluded that when oxidizing action is weak and slow, the accelerating action of the oxidizing phase is slight. Strong oxidizing agents are injurious in the reduction stage, resulting in the loss of nitrogen. Powdered pyrolusite is recommended as the most useful oxidizing agent.

At one time or another, there has been controversy as to the use of perchloric acid in the Kjeldahl digestion. By many workers its use is considered hazardous. However, shorter digestion times, with no loss of nitrogen, are reported. Pepkowitz and Shive (49) advocated the use of perchloric acid as a wet digestion micromethod, and claimed that for very resistant materials, the digestion is approximately four times as fast as the official method of the Association of Official Agricultural Chemists. Kaye and Weiner (26), however, stated that low results with heterocyclic compounds were obtained, although no loss of nitrogen from ammonium sulfate was observed. Koch ($88) reported its use in the digestion of grass, grain, and potato. In conjunction with selenium (68) for the digestion of soils and animal foods, i t is said to reduce the digestion time from 4 hours to 15 minutes. On the other hand ( 6 5 ) , it is reported that the method of Mall01 (39), using this combination, gave good results with the hydrochlorides of aniline and ephedrine, but poor results with such compounds as asparagine, aspartic acid, acetanilide, antipyrene, and caffeine. Wicks and Firminger (69) discouraged the use of perchloric acid, particularly in the micromethod, because large losses are developed by the use of a so-called excess which may not b? too ap- parent with a macromethod. It is an extremely powerful oxidia- ing agent and must be used with caution, particularly from the standpoint of loss of nitrogen either through formation and subsequent decomposition of ammonium perchlorate or oxidation of ammonium sulfate to nitrogen. Pepkowitz et a2. ( 4 8 ) obtained good results on addition of 10 drops of perchloric acid after strong heating of the sample in concentrated sulfuric acid, and cooling before the addition.

Hydrogen peroxide, also, is used frequently in both micro- and macro-Kjeldahl determinations as an oxidizing agent. Various workers (12, S4, 54, 69 ) report favorably on its use. Marquee and -4llista ( 4 2 ) found that the use of potassium pyrosulfate in conjunction with hydrogen peroxide gives rapid oxidations.

It is obvious that by increasing the severity of the reaction, oxidation will be accelerated. Disregarding for the moment the effect of catalysts, this is accomplished by the use of oxidizing agents and by increasing the salt content, with the consequent raising of the boiling point. The advantage of increasing the amount of potassium sulfate used, other than shortening of the digestion time, is that in many cases theoretical results are obtained which are not possible with the use of the conventional

186 A N A L Y T I C A L C H E M I S T R Y

10 grams of potassium sulfate. ous workers (8, S2, 47).

This has been confirmed by vari-

CATALYSTS

Xunierous elements and their salts have been used as catalysts in the Kjeldahl digestion. Mercury, as the metal, oxide, or sulfate, is probably universally accepted as the most efficient. Its only disadvantage, if it can be considered such, is that it is neces- sary to precipitate the mercury before distillations, since it forms complexes with ammonia. The use of other catalysts, such as copper sulfate or selenium, eliminates this difficulty.

A great amount of work has been done on the use of selenium as a catalyst since Lauro (33) first reported it. Sreenivasan and Sadasivan have explained the catalytic effect of selenium (64), and also reported that addition of mercuric oxide increased the efficiency of the catalyst. The kinetics of the Kjeldahl digestion using no catalyst, selenium, copper, mercury, and selenium- mercury, have been measured by Schwab and Schwab-Agallidis ( S I ) , who, working with aniline, showed this reaction to be of the first order. A review of the literature on the use of selenium as a catdyst has been published by Seebold (68).

In the determination of nitrogen in leather, Baker and Shuttle- worth (8) compared minimum digestion times and found that the catalyst efficiency was of the following decreasing order: mercury, selenium, copper, manganese. Lloyd (S6), reporting also on the nitrogen in leather, found slightly higher results with mercuric sulfate-selenium as compared with copper sulfate, and a digeation period which was shortened as much as 75% by the use of the mixed catalyst. A comparative study by Korek ( 1 9 ) on the effectiveness of various catalysts showed selenium-copper sulfate was more efficient in promoting complete conversion to ammonia. However, theoretical results were not obtained with aro- matic amines and heterocyclic compounds. Copper sulfate is a relatively EIOW catalyst, although the use of the anhydrous salt ( 4 ) increases the efficiency. A report of the catalytic activity of copper, cadmium, and calcium selenates by Dalrymple and King (15) on samples containing protein showed a shorter digestion time than with selenium alone. Losses occur on prolonged heating, and the authors do not recommend the use of selenates. Ac- cording to Hiller, Plazin, and Van Slyke (W), only digestion mixtures containing mercury as a catalyst gave nitrogen values for proteins comparable to those obtained by the Dumas method.

Willits, Coe, and Ogg ( 7 0 ) in their study of the determination of nitrogen in refractory heterocyclic compounds, did not recommend the use of selenium. A certain amount of caution must be exercised, as losses occur when more than 0.25 gram of selenium is used BS a catalyst (10) . Tellurium ( 2 4 ) has been proposed as a catalyst, replacing selenium. Comparative results ( 7 ) showed it to be unsatisfactory, both alone and in combination with copper sulfate or ferrous sulfate. An evaluation of copper sulfate and mercuric oxide by Potts, Parkham, and Schafer (52) in the determination of protein in fish meal showed that slightly higher results were obtained with mercuric oxide.

.

DISTILLATION AND DETERMINATION OF AhfMOXIA

The preparation of the Kjeldahl digest for distillation is largely mechanical, and the manipulative details are generally a t the operators discretion.

The recovery of ammonia, however, has been approached from various angles-e.g., direct distillation, steam distillation, aera- tion, and elimination of the distillation (40, 41) . Probably the most accepted macromethod .is direct distillation of the diluted and alkalinized digest. The recovery of ammonia is rapid, most of it coming over during the first few minutes boil. An hours continued boiling is apparently sufficient to remove all traces of ammonia. As an illustration, prepared mixtures simulating the Kjeldahl digest and containing ammonium sulfate were distilled (11) . The results in Table I indicate the recovery is complete

in 45 minutes. However, safe procedure nould dictate a 1-hour boil.

Alkali must be added carefully to the diluted and cooled digest to avoid loss of ammonia. Ynalvez (73) recommended an out- side boiler, and the introduction of the caustic through the steam tube. Fridman and Komissarova ( 1 9 ) transferred the digest to a distillation flask and neutralized with 20% sodium carbonate, using methyl orange as an indicator. Further dilution and addition of sodium bicarbonate are made prior to distillation. Distillation and determination of ammonia have also been made by the uee of a closed system (6, I S , 68) and subsequent direct titration. Minimum recovery has been reported to be 99.9%.

Table I. Recovery of Ammonia from Ammonium Sulfate

Sample, Gram

0.1000 0.1000 0.1000 0.1000 0.1000 0.1000

Distillation Time. hlin.

5 10 15 30 45 60

% Recovery

78.77 89.96, 91.97 98.63, 96.73 98.78, 98.38 99.97,100.16 99.98.100.09

The possibility of entrainment and subsequent carry-over of alkali is reduced by a regulated boil, with such added substances as boiling chips, or zinc and paraffin. Further reduction is obtainable by the use of an efficient trap.

The actual determination of ammonia may be accomplished in several ways, the most common of which is distillation of the ammonia into a measured excess of standard acid containing a suitable indicator. In place of the standard acid, there is frequently used a solution of boric acid (IS, 20, S5,37, 6 7 ) designated by investigators as saturated, 2%, 4%, etc. Taylor and Smith (65), using a micro-Kjeldahl method, distilled into exactly 10 mi. of 4% boric acid, made up to a final volume of 150 ml., and determined the pH. From a previously prepared calibration curve, the per cent nitrogen can be determined. -4 method which has found wide application is the so-called

formol titration (60) . This procedure, which eliminates distillation, is based on the following reaction with formaldehyde:

2(NH4)*S04 + GHCHO - (CHz)aN4 + 2HzS04 -I- 6Hz0 The digest is neutralized and treated with formaldehyde and

the resulting acid is titrated. The use of sodium hypobromite (35) has been adapted to micro-

Kjeldahl procedure, particularly in the determination of nitrogen in milk This is a very satisfactory method for small amounts of nitrogen and is represented by the following reactions:

2NHa + 2NaOBr -, 2NaBr + Nz + 3Hz0 SaOBr(excess) + 2KI + 2HC1 -P 2KC1 + NaCl + Iz + HzO

Iz + 2NazSz03 -, NazS40s + 2NaI The use of potassium biiodate ( 5 ) has also been suggested for

small amounts of nitrogen. In the determination of nitrogen in petroleum feed stocks (IS), a semimicro technique is used an ali- quot of the distillate nesslerized, and the nitrogen estimated colorimetrically.

APPLICATION TO MORE CO-MPLICATED FOR.MS O F NITROGEN

In order to extend the scope of the Kjeldahl method to include a larger variety of nitrogen-containing compounds, numerous modifications have been suggested. The use of pure cotton cellu- lose as a reducing agent for some nitro and azo compounds was proposed by Bhat (5 ) for estimating the dyestuffs on fiber. Azo compounds can also be reduced by treatment with 50 to

V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4 187

100% powdered copper (SO). Potassium iodide has been used frequently as a reducing agent. Rose and Ziliotto (66) applied this to the determination of nitrogen in nitriles. Preliminary reduction of nitrogen in side chains can be done by treatment with hydriodic acid and red phosphorus (43) . Fish (18) reduced hy- drazones, semicarbazones, and similar compounds by use of zinc dust and methanol in hydrochloric acid. I t has been claimed (63) that by the use of mercury or mercury-selenium oxychloride catalysts, and prolonged digestion, refractory compounds such as pyridine, quinoline, and nicotinic acid can be determined. This procedure haa been adapted to micro and semimicrodetermina- tions (46). Further work by Willits et al. (70) on refractory heter- ocycles showed that good results were obtainable with mercuric oxide, but that the results obtained by the addition of selenium were dependent upon the concentration of potassium sulfate and mercuric oxide, and digestion time. Perrin (60) found that the acid-salt-mercury ratio could be adjusted to the point where ad- ditional oxidizing agents were unnecessary. On the other hand, Dupuy (f7) determined the nitrogen in pyridine by the use of selenium oxychloride, 40 grams of potassium sulfate, and 1-hour digestion time.

For nitro compounds, the use of salicylic acid is generally es- tablished, followed by reduction with sodium thiosulfate. As this involves two separate operations, McCutchan and Roth (38) suggested the use of thiosalicylic acid in place of salicylic acid and sodium thiosulfate, and the increase of potassium sulfate to 20 grams. Their results indicate a marked improvement and increase the scope of the method. Lake (SI), in a report on cooper- ative work on nitrogen in petroleum and shale oil, also found that thiosalicylic acid gave results comparing favorably with the Dumas and ter Meulen methods.

Hydroxy compounds such as phenol and salicylic acid aid in the conversion of nitro groups and other nitrogen groupings. Ai study of various hydroxy compounds (8) showed that l-naphthol-pyro- gall01 in combination with sodium thiosulfate and 18 grams of potassium sulfate gave a marked increase in the amount of nitrogen recovered from refractory compounds, and in some cases reached theoretical values.

The importance of a general method for all forms of nitrogen need not be emphasized. Fundamentally, the physical operation of the method is simple, and has a further advantage in that it can be run in multiple. Whether or not a single modification em- bracing all forms of nitrogen can be developed will be in the hands of those workers to whom the Kjeldahl method is a valu- able tool.

LITERATURE CITED

(1) Alcock, R. S., Analyst, 71, 233-4 (1946). (2) Baker, W. F., and Shuttleworth, S. G., J . InteTn. SOC. Leathe7

(3) Ballentine, R., and Gregg, J. R., ANAL. CHEM., 19, 281-3

(4) Beatty, Croom, 111, IND. EWG. CHEM., ANAL. ED., 15, 476

(5) Bhat, R. V., Proc. Indian Acad. Sci., 13A, 269-72 (1941). (6) Bradley, J. A, , IND. EXG. CHEY., AXAL. ED., 14, 705-6 (1942). (7) Bradstreet, R. B., ANAL. CHEM., 21, 1012-13 (1949). (8) Ibid., in press. (9) Bradstreet, R. B., Chem. Reus., 27, 331-50 (1940):

Trades Chemists, 23, 488-91 (1939).

(1947).

(1943).

(10) Bradstreet, R. B., IND. ENG. CHEY., ANAL. ED., 12, 657 (1940). (11) Bradstreet, R. B., unpublished data. (12) Cartiaux, J., Ann . chim. anal. chim. appl , 22, 92 (1940). (13) Chand, Ram, J . Indian Chem. Soc., 24, 167-8 (1947). (14) Clark, E. P., J . Assoc. Ofic. Agr. Chemists, 24, 641-7 (1941). (15) Dalrymple, R. S., and King, G. B., IND. ENO. CHEM., ANAL.

(16) Drevon, B., and Roussin, J . pharm. chim., (9) 1, 18-24 (1940). (17) Dupuy, P., Compt. rend., 232, 836-8 (1951). (18) Fish, V. B., ANAL. CHEM., 24, 760 (1952). (19) Fridman, A. A., and Komissarova, Kh. F., Kozhevenno-Obus-

(20) Furukawa, T., and Nagase, O., J. Pharm. SOC. Japan, 70,690-1

(21) Gunning, J. W., 2. anal. Chem., 28, 188 (1899).

ED., 17, 403 (1945).

naya Prom. S.S.S.R., 19, No. 9-10, 27-30 (1940).

(1950).

(22) Hale, C. H., Hale, 21. N., and Jones, W. H., ANAL. CHEK, 21,

(23) Hiller, Alma, Plazin, J., and Van Slyke, D. D., J. Bwl. Chem.,

(24) Illarionov, V. V., and Solov'eva, N. A., 2. anal. Chem., 100,

(25) J h y , J., and Morvay, A., Ibid., 114, 120-5 (1938). (26) Kaye, I. A,, and Weiner, N., IND. ENG. CHEN., ANAL. ED., 17,

1549-51 (1949).

176, 1401-20 (1948).

328-43 (1935).

397-8 11945). (27) Kjeldahl,'J.,Medd Carlsberg Lab., 2, 1 (1883); 2. anal. Chem.,

(28) Koch, F. J., Ibid., 131, 426-7 (1950). (29) Korek, G. A, Trudy Leningrad Inst. Sovet. Torgoali, 1939, No. 2,

22, 366 (1883).

22-9; Khim. Referat. Zhur., 4, No 1, 89 (1941). (30) Kuznetsov, V. I., Zavodskaya Lab., 9, 1039 (1940); Khim.

(31) Lake, G. R., BNAL. CHEM., 24, 1806 (1952). (32) Lake, G. R., McCutohan, Philip, Van Meter, Robin, and Neel,

Referat. ZhlLT, 4, No. 3, 62-3 (1941).

J. C., Ibid., 23, 1634 (1951). (33) LaUrO, hf. F., ISD. ENG. CHEM., -4NAL. ED., 3, 401-2 (1931). (34) Leopold, H., 2. Lebensnt.-Unters~ccii. 7 ~ . -Forsch., 86, 220-3

(35) Ling, E. R., , I . SOC. Chem. Ind.$ 61, 194-6 (1942). (36) Lloyd, D. J., J . Intern. Roc Lmtiier Trades Chemists, 23, 275

(37) Ma, T. S., and Zuazaga, G., Ivn ENG. CHEV., -ANAL. ED , 14,

(38) McCutohan, P., and Roth, W. F., Ax.4~. CHEY., 24, 369 (1952). (39) .Mallol, Reu. real mad. cienc. Madrid, 39, 224-6 (1945). (40) Maroali, K., and Rieman, W., ANAL. CHEM., 20, 381-2 (1948). (41) MarCali, K., and Rieman, W., IND. ENG. C H E V . , zk- -%L. E D , 18,

(1943).

(1939).

280-2 (1 942).

709-10 (1946). (42) Maraues. M. C.. and Alista. G.. TBcnica. Y economh. (B6h/La,

Blanca, Arg.) , 1949, No. 2,'55-8. (43) Marsadro, Margherita, Mikrochernie ver. Mfcrociiim. Acta, 36/37,

(44) Milbauer. 6 . , C'hem. Ohzor., 16, 97-98 (1941) ; Chem. Zentr.,

(45) Ogg, C . L., Brand, R. W., and Willits, C ' . O., J . Assoc. O&.

(46) Ogg, C. L., and Willits, C. O., Ih id . , 33, 1 0 U M (1950).

(48) Pepkowitz, L. P., Prince, A. L., and Bear, F. E., IWD. ENG.

(49) Pepkowitz, L. P., and Shive, J . W., Ibid., 14, 914-16 (1942). (50) Perrin, C. 11.. . ~ N A L . CHEY., 25, 968 (1953). (51) Portner, P. E., Ibid,, 19, 502--3 (1947). (52) Potts, T. J . , I'arkham, 31. .i.. and Hchafer, I. M., 6. Assoc.

( 5 3 ) Quartaroli, rl., Ann. .fmaclltb ugrar. units. Pisa, 9, 9 6 9 (1948); 8

(54) Rautcrberg, E., and Benischke, II., and Schwab--\gallidis, Elly, J . A m . L'hen,. Soc.,

73, 803-9 (1'351). ( 6 2 ) Seebold, R . E , , J . A m . Leather C i t e m i s t s Assoe., 43, 362-i0

(63) Shirley, R. L., mid Becker, FV. \I-.. 1x11. Esc. ("HEK, -1x.4~

(64) Sreenivasan, A. and Sadasivaii, V., Ibid., 11, 314 (1939). (65) Taylor, W. H., and Smith, G. I?., Ibid., 14, 437-9 (1942). (66) Vickery, H. B., J . dssoc. Oflc. Ayr. Chemists, 29, 561-86 (1946). (67) Wagner, E. C., IND. ENG. CHmf., -1s.~~. ED., 12, 771-2 (1940). (68) Whitehead, E. I., and Olson, 0. E., J . Assoc. Ofic. Agr. Chem-

(69) Wicks, L. F., and Firminper, 1. I., IND. Esr:. CH

(70) Willits, C. O., r o e , AI. R., and Ogg, C . L.. J . dssoe. Ofic. A g r .

(71) Willits, C . O., and Ogg, C. L., Ibid., 32, 561-86 (1949). (72) Ibid., 33, 179-88 (1950). (73) Ynalvez, L. A, , Philippine Agr., 26, 823-6 (1938).

(1948).

ED., 17, 437-8 (1945).

ists, 25, 7G9-72 (1942).

ED., 14, 760-2 (1942).

Chentists, 32, 118-27 (1949).

RECEIVED for review Ootober 31, 1963. Bccepted December 4, 1953.

Kjeldahl Method - Bradstreet

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