968
ANALYTICAL CHEMISTRY
cal results; the choice of procedure will depend on individual preference. The dry ashing procedure is the most time consuming of the three. Wet ashing is rapid, but the anion exchange columns require regeneration more frequently when this teohnique is used because the resin becomes saturated with perchlorate ions. I n fact, only one 10-ml. aliquot of wet digest can be passed through a 3-gram column while as many as 15 aliquots of dry ash solution (prepared as described above) can be passed through before regeneration is necessary. Acid precipitation of casein affords a quick and simple means of preparing a clear solution containing all of the milk calcium and magnesium. One slight drawback of this method is the fact that the somewhat uncertain vdume of the casein precipitate must be taken into account in calculating results. However, this point is of minor consideration in most work.
Biedermann, W., and Schwarzenbach, G., Chimia Prague, 2, 56-9 (1948).
Bushill, J. H., Lampitt, L. H., and Filmer, D. F., J . SOC.Chem. Ind. (London),56, 411T413T (1937).
Cheng, K. L., and Bray, R. H., Soil Sei., 72,449-58 (1951). Cheng, K. L., Kurtz, T., and Bray, R. H., AXAL.CHEM.,24, 1640-1 (1952).
Connors, J. J., J . Am. W a t e r W o r k s Assoc., 42, 33-9 (1950). Diehl, H., Goetr, C. A., and Hach, C. C., I b i d . , pp. 40-8. Gastler, G. F., Proc. S. Dakota A c a d . Sci.,28, 77-81 (1949). Greenblatt, I. J . , and Hartman, S., AXAL.CHEM.,23, 1708-9 (1951).
Kibrick, A. C., Ross, M.,and Rogers, H. E., Proc. SOC.E x p . Bid. Med., 81, 353-5 (1952).
Morris, H. P., Nelson, J. W., and Palmer, L. S., IKD. EXG. CHEM.,ANAL.ED.,3, 164--7 (1931). Pyne, G . T., Analyst, 68, 330 (1943). Schwarzenbach, G., and Ackermann, H., Helu. Chim. A c t a , 30, 1798-1804 (1947).
Schwarrenbach, G., Biedermann, W., and Bangerter, F., Ibid., LITERATURE CITED (1) Banewicz, J. J., and Kenner, C. T., ANAL.CHEII., 24, 1186-7 (1952). (2) Betr, J. D., and Noll, C. 8.,J. Am. W a t e r W o r k s Assoc., 42,4956 (1950). (3) Ibid., pp. 749-54.
29,811-18 (1946).
Sobel, A. E., and Hanok, -4., Proc. SOC.E x p . Bid. X e d . , 77, 73740 (1951).
Willson, A. E., AXAL.CHEM.,22, 1571-2 (1950). RECEIVED for review Sovember 7 , 1952. Accepted February 11, 1953. Paper 2931, Scientific Journal Series, Minnesota dgricultural Experimental Station.
Rapid Modified Procedure for Determination of Kjeldahl Nitrogen C. H. PERRIN, Canada Packers, Ltd., Toronto, Ontario, Canada the spectacular success of Wilfarth and of Gunning in reS ducing the time required for Kjeldahl digestions, many chemists have attempted to increase the speed of the reactions still INCE
further. Typical efforts are described by Shedd ( 6 ) , Gerritz and S t . John (2), Lauro ( d ) , and Stubblefield and DeTurk ( 7 ) . Digestion mixtures such as those cited are more rapid in their action than conventional mixtures when used to digest samples such as animal feeds. However, the author thought i t would be interesting to compare the reaction speeds of such forniulas on nicotinic acid, a very refractory heterocyclic nitrogen compound. Kicotinic acid has been included in a number of recent studies of Kjeldahl digestion mixtures (6, 8, 9). Table I gives a comparison of rates of the digestion of nicotinic acid when an extremely brief reaction period is used. Methods making use of oxidizing agents such as perchloric acid, selenium, persulfates, and hydrogen peroxide, which introduce uncertainties, were not included in this study, The “proposed” method referred to in Table I is the subject of this paper.
Table I.
Comparison Speeds of Nicotinic Acid Digestion Nicotinic Acid. 11.38% N (theoretical). 1!.26% N obtained by Kjeldahl-
Wilfarth-Gunning method with 3 hours’ digestion Digestion. 0.5-gram sample, 12 minutes using 550-watt heaters, starting hot. 4.33-minute boil test Method Nitrogen Found. % Kjeldahl-Filfarth-Gunning 1.66 With HgO 1.34 With CuSO4 ( I ) 4.63 Gerritz and St. John ( 8 ) 2 .00 Stubblefield and DeTurk ( 7 ) 11.29 Proposed
believed to be the most rapid macrotechnique yet described. Although no use is made of selenium, phosphates, and other controversial additives, or of unusually intense heat, a complete nitrogen macrodetermination can be made in about 50 minutes. I n the case of all materials investigated to date, the point at which the digestion is complete can be determined visually. The small quantity of acid and alkali required reduces the violence of the reactions, contributes to accuracy, safety, speed of cooling, and makes possible the use of caustic pellets in the distillation step. (The preparation and handling of large quantities of liquid caustic are, perhaps, the most hazardous and unpleasant features of the Kjeldahl determination.) Bumping and foaming in the distillation are virtually eliminated. This permits a more rapid boiling. The chief disadvantage of the proposed method is that it mill stand less abuse than the method of the Association of Official Agricultural Chemists ( I ) . It requires more careful control of heat during digestion and more care in measuring chemicals. With some samples gentle heating during the first few minutes is particularly important because of the high boiling point of the digestion mixture. RE4GENTS
Silica, smooth boiling granules, not selenized (Hengar Co., Philadelphia, Pa.) Mercuric oxide, red, X.F. grade or Ion- in nitrogen. Sodium hydroxide pellets or flakes, low in nitrogen. Boric acid solution. Dissolve 4 grams of C . P . crystalline H3B03 in 100 ml. of distilled nater. Methyl red-bromorresol green indicator. Mix 5 parts of O.2Y0 bromocresol green solution with 1 part of 0.2% methyl red solution, both in alcohol. PROCEDURE
An investigation of a wide variety of catalysts and digestion mixtures led to the conclusion that the efficiency of the KjeldahlWilfarth-Gunning acid-salt-mercury combination ( I ) can be increased to the point where additional oxidizing agents become superfluous. This new combination permitted the development of a rapid Kjeldahl method which offers the following advantages. Except for methods making use of added oxidizing agents, it is
Place a 0.5- or 1.0-gram sample in a dry 500-ml. Kjeldahl digestion flask and add approximately six smooth boiling silica granules, 1.3 to 1.5 grams of mercuric oxide, and 12 =t0.5 grams of potassium sulfate. (Add no filter paper or other matter.) Swirl flask to mix, add 15 f 0.5 ml. of concentrated sulfuric acid, and mix again. Digest for 5 minutes (or until frothing ceases) a t low heat, then boil a t “full heat” until digestion is complete. (By full heat is meant a heating intensity sufficient to bring to a rolling boil 250 ml. of water in a 500-ml. Kjeldahl flask in from 4 to 5 minutes. This is called the boil test.)
V O L U M E 2 5 , N O . 6, J U N E 1 9 5 3
969 Sodium sulfate is not a satisfactory substitute for potassium sulfate because of cake formation. Figure 1 illustrates the importance of carefully measuring the heat input when a highly refractory form of nitrogen is encountered. Although it is possible to recover nitrogen completely from nicotinic acid using heaters that are below specification in their heat input, additional time must be allowed. The reaction times in Figure 1 were particularly short, because nicotinic acid can be digested without the initial period of low heat.
Heating conditions are correct for most samples if the following schedule is obtained: Low heat Full heat T o t a l digestion time
5 minutes 8 t o 12 minutes 13 t o 20 minutes
The total time required for complete clearing should be from 10 to 11 minutes. Avoid intense heating of flask above level of liquid. Khen digestion is complete. cool and promptly add about 200 ml. of cold water. Swirl if necessary to dissolve the cake and add in quick succession (and in the following order) approximately 25 grams of sodium hydroxide pellets, 5 grams of sodium thiosulfate pentahydrate, and 0.5 gram of 20-mesh zinc. Connect to the Iijeldahl distillation apparatus and receive ammonia in about 7 5 nil. of the boric acid solution. Titrate with standard acid using the mixed indicator.
Protein analyses were performed on 60 samples of feeding concentrates (31 dry-rendered tankage, 11 wet-rendered tankage, 5 lung meal, and 13 blood meal) received from 7 different laboratories. These samples varied in protein content (42 to 89%) and in fineness of grinding. A 1-gram portion was analyzed once by the proposed method and once by the official method ( I ) , except that the ammonia was received in boric acid as specified by the proposed method.
h-OTES O S DETERMINATION
The digestion is stopped when the appearance of the mixture has remained unchanged for 3 to 5 minutes using heaters with a 4- to 5-minute boil test. Nearly all part.icles of carbon will have disappeared and the dome of fine bubbles arising from the silica granules will be replaced by the evolution of relatively large and few t)ubbles. If many determinations are carried out simultaneously, the analyst may prefer an empirical control of the digestion by using a standard time interval rather than depending on the visual end point. Periodic check of the heaters (using the boil test) and careful control of other conditions will then be particularly necessary (Figure 1). Under no circumstances should digestion periods be required in excess of 30 minutes. If gas heat is used for digest,ions, care must be taken to avoid overheating the flasks above the surface of the liquid. After a little practice it is easy to adjust the flame to meet specified heat requirements. If single-heat, electric units are used, i t usually is safe t,o place the flask on the cool heater and then mitch on. This procedure automatically provides the initial low heat. requirement. Soiiie samples can be digested a t full heat immediately and do not require initial low temperature treatment. I n such cases fonming does no harni, as long as none of the organic matter rises into the neck of the flask. Where this trmtnient can be applied, it frequently is possible to carry out an entire digestion in less than 10 minutes. If desired, a solution of sodium hydroxide arid sodium thiosulfate may be used in the distillation step in place of the solid cheniicxls. (The solution should contain 23 grsnis of sodium hydroxide and 5 grams of sodium thiosulfate pentahydrate.) Samples which are higher than about 15% nitrogen or which consume an unusually large weight of sulfuric acid-for example, certain organic compounds and substances of higher than 20y0 fat content-should not exceed 0.5 gram. Substances showing an unusually great tendency to foam may also require this higher ratio of acid to sample.
Table 11. Determination of Nitrogen in a Yariety of Substances (Comparison of proposed method with Kjeldahl-Wilfarth-Gunning method) Nitrogen, Per Cent AOAC method Proposed method Sample Casein Hoof and horn mea Fish meal Wheat flour Soluble dried blood Cottonseed meal Groying mash k eed Hi3tidine hydrochloride monohydrate Theoretical 20.06 4 0.6-gram sample.
The average result for the 60 samples was 59.61% by the official method and 59.63% by the proposed method. Therefore, the rapid method has no positive or negative trend on these materials. The average difference between the two methods was 0.16 percentage unit of protein. I n 57 out of the 60 comparisons the agreement was within 0.3 percentage unit. Table I1 illustrates the performance of the proposrd rapid method on a variety of other substances. I n all comparisons, the hOAC Kjeldahl-IKlfarth-Gunning method was used M ith the mercuric oxide catalyst. Tables I11 and IV compare the precision of the proposed method with that of the A0.4C ( 1 ) method on two compounds containing nitrogen in a refractory form. The standard deviations and Studmt's t values 11 ere calculated for these results, which were obtained by one analyst using the same equipment and degree of care for all determinations. On both samples the proposed method was significantly more precise than the AOAC method a t the 99% level and its averages were closer to theoretical. DISCUSSION
401
6
'
I
8
IO
I I2
DIGESTION TIME I N MINUTES
1
I
14
16
(STARTING
I
18 W I T H FULL HEAT)
Figure 1. Digestion of 0.5-Gram Samples of Nicotinic Acid Proposed rapid method
20
I n the conventional Kjeldahl digestion of most substances, i t is considered necessary to continue heating until all the carbon has been oxidized and all the nitrogen converted into ammonium sulfate. The latter reaction, sometimes called the mineralization of the nitrogen, is not considered complete when the mixture clears. An afterboil period of from 10 minutes to 16 hours is used, depending on the type of material analyzed, the composition of the digestion mixture, and the source of heat. Much time is wasted through the desire to be certain that the reaction is complete. A simple and rapid means of determining the end point of the reaction is badly needed.
970
ANALYTICAL CHEMISTRY
upper limit of 410" C. for a 60-minute digestion presented by Lake, McCutchan, Van Meter, and Nee1 ( 3 ) is not exceeded in the proposed method. (Nicotinic acid, theoretical, 11.38% N. 0.5-gram samples. Electric heater. SILICAGRAXULES.The use of silica granules 4.3-minute boil test) causes a small but consistent increase in the diDigestion Av. % S o . of Standard Student's Method Time N Detns. Deviation t Value t 0.05 1 0.01 gestion speed of tankage samples. It was obOfficial 3 hr. 11.26 16 0.026 served that 6 granules are superior to 1 or to 6 3.40 2.042 2.750 Proposed 14 min. 11.29 16 0.024 granules crushed to a powder. However, when nicotinic acid was analyzed, no speed advantage Table IV. Relative Precision of Proposed and Kjeldahl-Wilfarthin favor of the granules could be observed. Gunning Methods in Analysis of Tryptophan Their use is justified because they assist in the (Tryptophan, theoretical, 13.72% N. 0.5-gram samples. Electric heater. visual detection of the completion of the reaction 4.3-minute boil test) and cause smoother boiling. Digestion -417. % No. of Standard Student's N Detns. Deviation t Value t 0.05 t 0.01 Method Time The reagent ratios (acid-salt-catalyst) in the Official 3 hr. 13.54 10 0.064 proposed rapid digestion procedure may be repre5.80 2 . 1 2 0 2.921 Proposed 14 min. 13.68 8 0.025 sented as 15/12/1.5/6 granules. Figure 2 shows that some of the benefits of high reaction speed may be enjoyed using greater amounts of reagents in the flask. The first curve represents ratios In the proposed method the combustion of the carbon and the similar to those in the proposed method, while the last curve mineralization of the nitrogen proceed swiftly and apparently shows the performance of the AOAC (1) method. approach completion almost simultaneously. As even samples Considerable favorable experience was gained with the 25/20/ containing nitrogen in a highly refractory form require only a 2.0/6 granules combination because i t was felt that some few minutes' afterboil, this method makes it possible t o deterchemists may prefer to continue using 25 ml. of sulfuric acid and mine, with considerable assurance, just when the digestion is comstill enjoy a substantial increase in digestion speed. plete. The cost of the reagents in the proposed method is approxiThe appearance of this digestion reaction is interesting. Clearmately the same as that of the AOAC Kjeldahl-Wilfarth-Gunning ing is sudden and frequently a black ring of carbon is seen around method using potassium sulfate and mercuric oxide. The extra the clear central mass. Before the reaction is complete, a dome cost of mercuric oxide and sodium thiosulfate is balanced by the of fine bubbles rises from the silica granules. The termination of saving in potassium sulfate, sulfuric acid, and sodium hydroxide. the digestion is marked by a relatively calm surface of the mixThe silica granules are easily recovered. ture and a noticeable reduction in the rate of production of bubbles. When this condition prevails for 3 to 5 minutes, the heat is removed. Effect of Modifications of Method. Superficially the digestion mixture of the proposed method is much like that of the usual Kjeldahl-Wilfarth-Gunning method. It seems strange that a few small modifications should increase the reaction speed several times. Probably the three most important factors explaining this are a high concentration of mercury, intense heating, and the use of silica granules. MERCURY CONCE~YTRATIOX. The concentration of mercury is about four times as high as that used in the 8 0 9 C method ( 1 ) . That an increase in the concentration of mercury is effective is illustrated in Table V. 10 12 14 16 I8 2 0 2 2 24 26 28 30
Table 111. Relative Precision of Proposed and Kjeldahl-WilfarthGunning Methods in Analysis of Nicotinic Acid
DIGESTION TIME IN MlNUTfS (STARTING WITH FULL HEAT)
Table V.
Effect of Mercury Concentration
(0.5 gram of nicotinic acid, 1 5 ml. of HzSOd, 12 grams of KzSOd, 6 granules Digestion 12 minutes. Starting hot) HgO, Grams Nitrogen, 7% 0.5 6.78 1.0 8.95 1.5 10.58 As heaters were adjusted to only 450 watts, recovery of nitrogen was incomplete in all cases.
-4number of similar experiments performed on casein and tankage confirmed the effectiveness of high mercury concentrations when proteins are analyzed. TEMPERATURE. After 15 minutes' digesti,on, the temperature of the mixture was approximately 370' C. Under the same conditions the AOAC method (1)showed a digestion temperature of approximately 345" C. This temperature advantrsge enjoyed by the proposed method in the early stages of the digestion is probably enhanced by the smaller weight of chemicals in the flask. Under these conditions considerable superheating of the glass surface beneath the digestion mixture would be expected. The safe
Figure 2.
Digestion of 0.5-Gram Samples of Nicotinic Acid
4.5-minute boil test, electric heater
The work which led to the development of the proposed rapid method included a study of various catalysts and combinations of catalysts. Selenium proved treacherous in these high temperature digestions, causing unpredictable nitrogen losses. An interesting objection to a common method of using copper sulfate was also noticed. When 1-gram samples of tankage were analyzed using l gram of cupric sulfate with 25 ml. of sulfuric acid and 16 grams of potassium sulfate as in the AOAC method ( I ) , values approximately one percentage unit of protein higher than those obtained with the alternative mercuric oxide modification ( I ) were obtained. However, when either of the following two precautions was taken, no difference between the copper and mercury values was observed. 1. The copper was precipitated in the presence of thiosulfate before distillation (exactly as in the mercuric oxide alternative procedure).
971
V O L U M E 2 5 , NO. 6, J U N E 1 9 5 3 2. The contents of the receiver were again distilled into boric acid or standard acid solution. The Eastern Regional Research Laboratory, United States Department of Agriculture, as well as a number of other laboratories, repeated the above experiment and obtained comparable errors. C . L. Ogg, Eastern Regional Research Laboratory, concluded that the errors were caused by alkali carried over during the first distillation. Shedd (6) also reported trouble with copper sulfate and shomd that the error could be reduced by precipitating the copper with polysulfide. Several simple experiments proved that as little as 0.01 gram of copper sulfate in the distillation flask greatly increased the volume of hydrogen generated by the zinc during a normal distillation. That sodium hydroxide caused the high titrations was proved by receiving the ammonia in distilled water, boiling to dryness, and titrating the residue. Solutions of the residue were also compared with respect to sodium content using a flame photometer. As even the most efficient type of scrubber connecting bulbs permitted large errors in some cases, certain precautions should be taken when copper sulfate is employed as a catalyst. A suitable eubstitute for zinc should be used to prevent bumping during distillation, or the minimum weight of zinc should be used, with a thiosulfate or sulfide precipitation of the copper. By replacing potassium sulfate with dipotassium phosphate, it n as possible to obtain complete nitrogen recovery from nicotinic acid in only 6 minutes’ total digestion time. However, comparisons of the two salts in the digestion of feeds and feeding concentrates brought out practical objections to the use of the phosphate. The flasks were etched and low results were occasionally obtained. The nitrogen losses probably were caused by the temperature, which frequently exceeded 400” C. 4 s the digestion in the proposed method is extremely rapid, some concern was felt over the possibility of nitrogen losses caused by accidental continuation of the afterboil. Evidence that a considerable safety factor exists is furnished by the following study, carried out on samples of tankage digested by both gas and electric heat following the heating conditions recommended by
the procedure. The nitrogen recovery was still 100% after a total digestion time of 60 minutes. However, after 90- and 120-minute digestion times the nitrogen recovery fell to 99 and 98%, respectively. In laboratories that do not possess regular Kjeldahl digestion apparatus the digestion may be conducted in narrow-necked 500ml. Erlenmeyer flasks placed in a fume cupboard. Each flask is fitted with a 3-inch funnel which returns condensed acid and prevents mechanical loss. The heating schedule of the proposed procedure is followed approximately. Somewhat greater care is used to avoid overheating during the early stages of the reaction, The proposed method has already replaced conventional Kjeldah1 procedures in several large feed analysis laboratories. ACKNOWLEDGMENT
Very helpful criticisms and suggestions were received from
C. 0. Willits and C. L. Ogg, Eastern Regional Research Laboratory, U. S. Department of Agriculture, and from H. -4.Davis, University of New Hampshire. The author also wifihes to thank Ralph Gibson and Donald Sinclair of Canada Packers’ research division for their assistance in testing the method on a variety of materials. LITERATURE CITED (1) Assoc. of Offic. Agr. Chemists, “Methods of Analysis,” p. 2.24. 1950. (2) Gerritz, H. W., and St. John, J. L., IND.ENG.CHEM.,ANAL.ED., 7, 380 (1935). (3) Lake, G. R., McCutchan, Philip, Van Meter, Robin, and Keel, J. C., ANAL.CHEM.,23, 1634 (1951). (4) Lauro, M. F., IND. ENG.CHEM.,ANAL.ED.,3, 401 (1931). (5) Ogg, C. L., Brand, R. W., and Willits, C. O., J . Assoc. Ofic.A g t . Chemists, 31, 663 (1948). (6) Shedd, 0. M., Ibid., 10,507 (1927).
(7) Stubblefield, F. M., and DeTurk, E. E., IND.ERG. CHEM..
ANAL.ED., 12, 396 (1940). (8) White, L. M., and Long, M. C., ANAL.CHEM.,23, 363 (1951). (9) Willits, C. O., Coe, hl. R., and Ogg, C. L., J . Assoc. O f i c . Agr. Chemists, 32, 118 (1949). RECEIVED for review .4prll 7,
1952. Bccepted December 10,1952.
Ultraviolet Spectrophotometric Determination of Cerium GEORGE TELEP’ A N D D. F. BOLTZ Wayne University, Detroit 1 , Mich.
vv
a dilute solution of cerium(II1) is treated with a concentrated solution of potassium carbonate, a white precipitate is formed which dissolves in an excess of the reagent. The resulting cerous complex is presumably oxidized by the oxygen in the air to give a yellow ceric complex. Plank ( 4 ) found that a turbidity developed if hydrogen peroxide was used to facilitate the oxidation. Therefore, Plank preferred to oxidize with a stream of pure oxygen for 10 to 15 minutes. Although his work showed the applicability of this colored complex to the colorimetric determination of cerium, the method has found little use. This study mas undertaken to determine the ultraviolet absorption spectrum of this cerium complex and to develop a spectrophotometric method for determining small amounts of cerium. HEN
APPARATUS AND SOLUTIONS
A Beckman Model DU spectrophotometer with 1.00-em. silica cells was used for all the absorbancy measurements. A hydrogen discharge tube was used for measurements taken from 220 to 400 mfi and a tungsten filament lamp for the region from 400 to 500 m/r. A glass electrode was used for all pH measurements. A standard cerium(II1) solution was prepared by dissolving Present address, E. I. du P o n t de Nemours & Co., Inc., Dacron Division, Kmston, K. C. 1
0.3130 gram of the hydrated cerous perchlorate in redistilled water and diluting to 1 liter. This solution was standardized using the gravimetric procedure of Brinton and James (I), according to which the cerium was initially precipitated as the iodate, converted to the oxalate, and then ignited to the oxide in a platinum crucible. One milliliter of this solution contained 0.086 mg. of cerium(II1). The hydrogen peroxide used was 3% and 28% analytical reagent grade. The potassium carbonate (100 grams/100 ml.) solution was prepared from the reagent grade salt dissolved in redistilled water. FUNDAMENTAL REACTION
9 dilute solution of cerium(III), when made basic with a concentrated solution of potassium carbonate and treated with hydrogen peroxide, gives an intense yellow complex. This complex exhibits maximum absorbancy in the ultraviolet region of the spectrum. The formula of the yellow complex is unknown, and an investigation is in progress to determine the exact constitution of this complex. EXPERIMENTAL
Cerium Concentration. The absorption spectra for several concentrations of cerium were determined under two sets of conditions with the position of the absorbancy maximum being
