Determination of Total Nitrogen in Proteins and Their Hy drolyzates Improved M e t h o d and Apparatus RAYMOND JONNARD The Warner Institute for Therapeutic Research, 113 West Eighteenth St., N e w York,
A reliable semimicro modified Kjeldahl method for the rapid serial routine determination of total nitrogen, principally in proteins and their hydrolyrates, is described. A convenient distillation head used in connection with this method is presented. Reproducible, maximum figures for the total nitrogen content of certain amino acid, protein, and polypeptide mixtures often require as long as 1 P to 16 hours of continuous digestion under the conditions of the method. The method gives satisfactory figures, and is recommended for the analysis of biological fluids which have been submitted to conventional fractionation procedures and which may contain disturbing mineral precipitating agents such as sodium tungstate and phosphotungstic acid.
N. Y.
digestion flask to the all-glass distillation apparatus when such a design was utilized. These sources of error were eliminated by designing the distillation head shown in Figure 1 (manufactured by Ace Glas:, Inc., Vineland, N. J.).
A pinhole a t the lower level of the bent tube in the trap allows the liquid condensed therein to drain back into the flask,wheyeI t !e stripped of its ammonia when, a t the end of the distillation, it reaches the hottest parts of the apparatus. The long tube extending from the alkali-measuring ampoule inside the trap was found necessary to prevent mist entrainment of some adhering alkali solution with the distillate. KO rinsing is necessary after delivering the alkali. The condenser, which is to be attached to the 19/38 standard-taper joint, is rinsed in the usual way, at the end of a run, through the lateral stopcock connection. The tapered joint is preferably lubricated with molybdenite, although ordinary stopcock grease wag satisfactory if applied with care. Transfer of the digestion mixture is avoided by carrying out the digestion in standard 100-ml. Kjeldahl.flask.3 and then directly attaching these flasks to the distillation apparatus by means of frequently renewed rubber stoppers; this kind of connection was found satisfactory by Redemann (as). The volume of the trap is reduced to a minimum, as a large volume of air present at the beginning of the ammonia distillation may result in losses. With this apparatus, a delivery tube 2 mm. in diameter,
THE
determination of total nitrogen in proteins by the Kjeldahl method and its numerous modifications presents well-known difficulties, mostly due t o the slowness of the sulfuric acid digestion in the absence of the proper catalyst. Additional difficulties are met in protein hydrolyxates, owing t o the very unequal sensibility of their various components to the digestion procedure, the ease with which some polypeptides form free nitrogen in presence of certain oxidation catalysts, and the frequent presence of inhibiting mineral substances (in analytical precipitation mixtures. deproteinated clinical blood and urine specimens, etc.). Many of these difficulties are eliminated in the microprocedures described in the literature, such as that of Clark (11). However, these procedures do not easily lend themselves to routine analysis of a very large number of specimens, as often required in industrial research and production control. A timely warning was recently sounded by Chibnall et al. ( 9 ) , who were obliged to revise all the Kjeldahl nitrogen determinations of egg albumin and other proteins made during the previous ten years in their laboratory, when i t was discovered that the technique followed resulted in a probably incomplete digestion. In the course of preliminary work on the production of casein hydrolycates ($1), considerable discrepancies were recorded between duplicate analyses in solutions which were treated with either sodium tungstate or phosphotungstic acid for fractionation purpose. This observation motivated a reinvestigation of the influence of the duration of the digestion required for the analysis of this material in the presence of various catalysts and precipitating agents. Finally, additional sources of error having been found in the ammonia distillation procedure when a conventional trap was used, this part of the apparatus was redesigned with a view to perfecting a reliable semimicromethod.
8 OD,
c-
50
MODIFIED SEMIMICRO-KJELDAHL DISTILLATION HEAD
Although steam-distillation of ammonia has been adequately described in connection with microanalytical methods, the difficulties inherent in quantitative distillation without steam entrainment on a semimicro scale have not yet been satisfactorily solved. Various arrangements of distillation apparatus, particularly that of Dewey and Witt (IQ), were tried with mixed success. Ultimately it was found that errors arose mainly from two sources: (1) retention of a significant quantity of ammonia in the few drops of liquid contained in the conventional trap, and (2) losses encountered during the transfer of the reaction mixture from the
________
Figure 1.
246
U
---
Distillation H e a d
ANALYTICAL EDITION
April, 1945
Table
I.
Nitrogen Determination on Various Compounds Nitrogen Found Procedure A Procedure C’C
Teat Substances
%
Lot 2751
13.33
13.80
Melting Points Reported Found 0
c.
163-6 S+fanilamide, U.8.P. 187.6-90 Hip una acid, C.P. 176-88 2 4-binitroaniline. reagent 4lMethyl-Z-hydroxyquinoline 2 2 3 . 7 0
... ... ... ...
21.26 21.19 21.13 10.32 10.89 10.36 10.40
...
Aoetanilide (theoretical, 10.37%) a
Miscellaneous Substancee
%
... ...
Ammonium sulfate (theoretical, 21.19%1°
Nitrogen Nitrogen Found b y Reported Procedure C’
c. 162 187.5 178.0 219.0
%
%
16.20 7.82 22.93 8.80
16.29-16.33 7.76- 7 . 7 4 20.46-20.74 8.77- 8 . 7 2
A.C.S. specification reagent.
b Highly purded material. C
Commercial specimers of various origin.
provided with a 10-ml. capacity safety bulb, and dipping about 10 to 15 mm. into the standard acid solution, is recommended. It is advisable t o adjust, first, the rate of delivery of the alkali solution, and then the rate of heating, so that no gas bubble will escape from the acid solution. After the first 3 minutes of the distillation, when all air has been purged, full heat can be applied. With the quantities of reagents and test solution indicated below, a complete distillation takes 15 to 20 minutes. No bumping is experienced even if gas heating is employed, if a few pieces of washed Carborundum are put in the flask before the apparatus is connected. Stainless-steel gauze around the flask was found best to ensure safe uniform heating, so that the distillation requires no attention until the end. With a battery of twelve apparatus, a single operator can run 36 semimicroanalyses per day, and still have time for the required cleaning and steaming out operations. Reliable, reproducible results are obtained if the apparatus is steamed out aft>erevery three or four rum. EXPERIMENTAL PROCEDURES
KJELDAHL DIGESTION. The various catalysts and digest ion methods tested were:
A. The ordinary Kjeldahl method, using 3 ml. of concentrated sulfuric acid, 10 mg. of copper sulfate, and 500 m potassium sulfate for every 5 mg. of nitrogen t o be titratef(Ef 1.5 to 3 ml. of a 5% protein solution or hydrolyzate), and adding 100 mg. of potassium persulfate after the first 2 hours of digestion. B. The method of Dupray ( l 7 ) , usin 1 ml. of perchloric acid a t the beginning of the digestion, for &e same proportions of nitrogen, sulfuric acid, and potassium sulfate as indicated above, keeping the temperature a t about 90’ C., adding after the first hour of digestion 50 mg. of selenious oxide plus 10 mg. of copper sulfate, and continuing the digestion for 5 t,o 8 hours, or longer if necessary. C. The Hotchkiss-Dubos method (go), in which 1 ml. of 57% hydriodic acid takes the place of the perchloric acid in procedure B. The hydriodic acid (“alkoxy1 determination purity”) was prepared according to Clark ( I O ) . C After some preliminary experiments, procedure C proved most satisfactory, and ultimately was combined with the simpler method of Pepkovitz, Prince, and Bear (85)-1 ml. of a 1.2oJ, solution of selenious oxide in concentrated sulfuric acid was added after the first hour of digestion in procedure C.
.
Heating was done by means of gas microburners, adjusted so that after decolorization the digestion mixtures were barely boiling. Preliminary experiments showed that the digestion was usually finished in 2 to 5 hours, b u t protein hydrolyzates and certsin amino acids required a much longer time.
247
DISTILLATION. The distillations were unifmmly carried out in the appamtus described above, after addition of 20 ml. of 30y0 sodium hydroxide solution. The precaution of saturating the alkali solution with Seignette salt in order to keep the copper oxide in solution and prevent bumping, as indicated by Cerbelaud and Bayard (8) was found unnecessary with this procedure, INHIBITING PRECIPITATING AGENTS. The interfering deproteinating agents studied in this preliminary work were sodium tungstate, according to the technique of and in the proportions indicated by F d i n and Wu (18); and 12-phosphotungstic acid, according to Drechsel(I5) and following the technique of Drummond (18). This reagent was purified according to Van Slyke et al. ($8). Commercial casein, purified casein, CASEINPREPARATIONS. and casein hydrolyzates were analyzed and purified by the following procedures:
Moisture. By dryin to constant weight a t 110’ C., after it was ascertained that t8e same value was obtained by prolonged drying over sulfuric acid in vacuum. Ash. By incineration a t 600’ to 6.50’ C., taking cognizance of St. John’s ($9) and Wichmann (36) observations. The value thus determined includes both the mineral impurities and the phosphoric acid and calcium constituents of the casein molecule minus perhaps a small fraction of the phosphorus lost by this procedure. Purijication. Specimen 136-IV was repeatedly precipitated at pH 4.6 from a solution alkalinized to pH 8.0 with ammoilia. following exactly the operational details outlined by Cohn anti Hendry ( I d ) and Blatt (4). Specimen 136-VI11 was purified in a similar way, but after each precipitation one partial fractionation was effected a t pH 6.3 to remove insoluble calcium caseinate, according to Loeb’s procedure (85). The final removal of calcium was by precipitation with oxalic acid a t p H 6.0, by the wellknown Van Slyke and Bosworth method. Hydrolyzates. The hydrolyzates reported were prepared by the method of Dakin ( I S ) , varying the duration of the process to buit the purposes. RESULTS
Repeated analyses were performed over a period of 1.5 years on various lots of ammonium sulfate, acetanilide (dried to constant weight a t 7 0 ° ,which requires some 15 days), and a number of other organic nitrogenous compounds (Table I). All weighing. were done on an Austrian-made Rueprecht balance, to 0.05 mp. Only the analyses on the A.C.S. specifications lot of amnioniuni sulfate, and purified acetanilide, were performed with calibrated glassware, with all due corrections; shelf reagents were analyzed in ordinary glassware of high commercial quality, since no attempt was made t o check the figures obtained against theoretical values,
Table II.
Nitro en Recovery in Presence and A b r e n c t of Sodium fuigrtatc and Phosphotungstic A c i d
Test Substance Ammonium sulfate’ (theoretical, 2 1 . 1 9 % )
Acetanilide e (theoretical, 10.37%)
Precipitating Agents
Nitrogen Found Procedure Procedure Procedurr A B C
%
%
%
...
... ...
21.30 21.10 21.05
17.80 18.20 19.20
Na tungstate
20.80 20.80
3.67 2.42
Phosphotungstic acid
26:02
l1:36
26:?5
...
10.25
l2:07 7.93
...
9.93 10.40 7.66 8.65
3.70 2.36
21.40 21.15 21.3nb 21-25) 21.40 21.30, 21.35) 20.95) 21.OOb 21.10 10.386 10.35) 10.20 10.22 10.14 10.25, 10.28) 10.10 10.28) 10.22b
...
...
Na tungstate
...
...
... ... ...
...
Phospho9.75 7.40 10.48 tungstio 7.51 acid ... ... * Different lots of commercial product of reagent purity. a Duplicate analyses made at different dates on same lot of reagent. 5 Three lots of acetanigde purified as for experimenta reported in Table 1.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
248 Table
Ill. Nitrogen Content of Casein Determined b y Proposed
Casein Lot 136
136-IV
hloisture, % 9.40 9.35 9.35 7.70 7.68
...
136-VI11
6.54 5.53 5.57
Method Ash, 5% of Dry Protein 5.40 5.30 5.40 1.31 1.30 1.32 0.12 0.20 0.11
Nitrogen, % of Dry. Ash-Free Protein 14.80 14.90 14.84 15.20 15.20 15.22 15.90 15.85 15.87
Table IV. Comparative Nitrogen Recovery [In protein (casein) hydrolyzates in presence of the precipitating reagent, phosphotungstic acid 1 Nitrogen Content of Nitrogen Recovered with Duration of Original Solution, Phosphotungstic Acida Hydrolysis Procedure C Procedure A Procedure C‘ Hour8 Mg./100 mi. A i o . / l O O ml. Mo./lOO ml. 6 59 1 430 590 525’ 52% 2.16 442 505 1.15 480 508 618 7.30 547 616 583 7.45 ... 588 17.00 476 428 473 a
Sum of nitrogen in precipitate and in supernatant fluid.
but only to ascertain the reliability and reproducibility inherent in the method. The recovery of nitrogen from ammonium sulfate and acetanilide reported in Table I agrees satisfactorily with the values reported in the literature. Van Slyke, Hiller, and Dillon (Jb) reported that all the catalysts recommended in the literature for the Kjeldahl procedure failed in their hands to yield more than 90% of the total nitrogen of tryptophane and lysine. The results shown in Table I, obtained by procedure C’, are somewhat more reproducible and always higher than those obtained by the usual procedure, A, and seem close enough to the expected values for such commercial preparations of unknown purity. Of the other few organic compounds (of unknown purity) tested to date, only 2,Pdinitroaniline appears not analyzable. and this should be expected. On the other hand, 4-methyl-2hydroxyquinoline is now directly analyzable by the modified Kjeldahl procedure, and the figures obtained on duplicates are close enough to each another, within the limits of p%rmissible error. Table I1 contains the comparative results obtained with the three methods investigated, in presence and absence of the inhibiting precipitating agents, with several lots of ammonium sulfate and acetanilide not specially purified. The use of perchloric acid results in losses of nitrogen even with ammonium sulfate (column 3) as reported by Wicks and Firminger ( S T ) , and this effect is increased in presence of the precipitating agents mentioned. These agents interfere much less in the determination by method C’. The results obtained by the proposed procedure are reproducible and agree closely on the same lot of reagent. Table I11 contains the results of repeated analyses on several casein preparatims. Following Chibnall’s observation ( 9 ) , it waa necessary to investigate first the duration of digestion required to obtain maximum nitrogen values; 12 to 16 hours proved necessary. The maximum values recorded are higher than those reported by Block (5)who found 14.7% ih casein (uncorrected for moisture and ash, 6), or by Ramsdall and Whittier (27) who found 14.6% in casein and 15.5y0after correction for calcium and phosphorus. They agree closely (for specimen 136-VIII) with the value reported by Chibnall ( 9 ) , who found 15.7%, also after correction for calcium and phosphorus. The effect of the interfering precipitating agents is mostly felt in the analysis of protein hydrolvzntes where they are used for
Vol. 17, No. 4
fractionation purposes. When these reagents are present in solution a specific precipitate forms whose nitrogen content added to the nitrogen content of the supernatant usually fails to add up to the nitrogen content of the original solution, when method A or B is used for the analysis. Table IV contains comparative results obtained on casein hydrolyzates, containing various proportions of polypeptides, diketopiperazines, and by-products of hydrolysis. Column 2‘ shows the nitrogen content of the original solutions determined by method C’. Column 3 shows the sum of the nitrogen content of the precipitates and supernatants (separated by centrifugation), obtained in presence of phosphotungstic acid, and determined by method A, and column 4 shows the sum of the nitrogen content of the same fractions determined by method C‘. Nitrogen recovery by method C’ appears satisfactory. DISCUSSION
The unsatisfactory results obtained with perchloric acid are in agreement with the more extensive results of Wicks and Firminger (97). The discrepancies are attributed by Peters and Tan Slyke (26) to the formation of free nitrogen, and to undecomposed amines by Gortner and Hoffman ( 1 9 ) ,Villiers and MoreauTalon (33)), and more recently Mears and Hussey ( 2 4 ) . The satisfactory recovery reported by the proposed method seems to invalidate the conclusions of Belcher and Godbert (9) and Beet and Belcher (21, that the selenium catalyst proposed by Lauro (22) “does not justify the favor of so many chemists”. Their catalyst mixture (1 part of selenium for 5 parts of mercuric sulfate and 32 parts of potassium sulfate) is but a modification of Wagner’s (34) digestion mixture which was found satisfactory by Acree (1) and by Taylor and Smith (SI). Thiscatalyst, however, results in the added complication that the mercury-ammonium complexes must be destroyed before distillation. The selenium concentration in the recommended procedure outlined above remains within the limits indicated by Bradstreet (‘7). Hydriodic acid was used for the preliminary reduction of difficultly digestible compounds in the so-called Friedrich modification of the Kieldahl method, as described by Scarrow and Allen (90)and by Clark (11). The original procedure involves a 45minute digestion n-ith 57% hydriodic acid (1 ml. per 10 mg. of test material. plus a trace of red phosphorus) in absence of sulfuric acid, followed by a distillation of the iodine with dilute sulfuric acid. The procedure was found time-consuming by Belcher et al. ( 2 , 3). The ahove reported results indicate that, a t least in the cases considered, hydriodic acid in sulfuric acid solution is a sufficiently strong reducing agent and acts long enough for a satisfactory and reproducible nitrogen recovery, provided that the subsequent digestion period is continued for a sufficient time. Clark (11) insisted that many compounds, although requiring a very long digestion period (no figure given) will nevertheless yield their nitrogen quantitatively to one or another of the modified Kjeldahl procedures. The author’s experience with casein and its hydrolyxates confirms the conclusions of Chibnall et al. (9) that “the digestion time required for complete nitrogen recovery is unusually long (12 to 16 hours) with some amino acid (lysine, histidine, etc.) and polypeptide mixtures, even though the preparation is rapidly decolorized during the early phases of the reaction”. [Since completion of this work, there appeared a publication by Warner (%), pointing to the possible loss of some phosphorus a t 600’ C., and a method to prevent it was described. Therefore it is possible that the true nitrogen content of the author’s casein specimen is still higher than reported here.] ACKNOWLEDGMENT
Very valuable technical assistance from H. Crowley is gratefully acknowledged.
ANALYTICAL EDITION
April, 1945 LITERATURE CITED
Acree, F., Jr., J . Assoc. Oficial A&. Chem., 3, 648-51 (1941). Beet, A . E., and Belcher, R., Mikrochemie, 24, 145-8 (1938). Belcher, R., and Godbert, A. L., J . SOC.Chem. I d . , 60, 196-8 (1941).
Blatt, A. H., “Organic Syntheses”, Collective Vol. 11, p. 120, New York, John Wiley & Sons, 1944. Block, R. J., “Determination of Amino Acids”, Burgess Publishing Co., Minneapolis, Minn., 1942. Block, R. J., personal communication. Bradstreet, R. B., IND.ESG. CHEX..,ANAL.ED.,12, 657 (1940). Cerbelaud, R., and Bayard, C., “Manuel Clinique d’iinalyses Bacteriologiques”, p. 189, Paris, Bayard, 1907. Chibnall, A. C., Rees, hl. W., and Williams, E. F. Biochem. J., 37, 354-9 (1943).
Clark, E. P., IND.ENG.CHEM., .4NAL. ED., 10,677 (1938). Clark, E. P., J . Assoc. Oficial Agr. Chem., 24, 641-7 (1941). Cohn, E. J., and Hendry, J. L., J . Gen. Phyaiol., 5 , 521 (1923). Dakin, H. D., J . B i d . Chem., 44, 499-529 (1920). Dewey, B. T., and Witt, N. F., J . A m . Pharm. Assoc., 32, 65 (1943).
Drechsel, J . , J . prakt. Chem. ( 2 ) ,39, 425 (1889). Drummond, J. C., Biochem. J . , 12, 5-24 (1918). Dupray, h i . , J . Lab. Clin. &Wed.,12, 387 (1926). Folin, O., and Wu, H., J . Bid. Chem., 38, 81 (1919). Gortner, R. A . , and Hoffman, W. F., Ibid., 70,457 (1926). Hotchkiss, R. D., and Dubos, R., Ibid., 141, 155-62 (1941). Jonnard, R., and Fischer, L. O., Abstracts of Papers, AM. CHEM. SOC. Meeting, Pittsburgh, p . 21-B (Sept., 1943). LRUrO. M .F., IND.ENQ.C H E M . , .4NiL. ED.,3,401-2 (1931).
249
(23) Loeb, J., J . Gen. Phusiol., 3, 547 (192C-21). (24) Xlears. B., and Hussey, R. E., IXD.ENG.CHEM.,A N A L .ED., 13, 1054 (1942). ( 2 6 ) Pepkovitz, L. P., Prince, A . L., and Bear, F. E., I b i d . , 14, 856-7 (1942). (26) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemical Methods”, p . 519, Baltimore, Williams 8: Kilkins, 1932. (Pi) Ramsdall, G. A . , and Whittier, E. C., J . Bid. Chem., 151, 413-19 ( 1944). (28) Redemann, C. E., IND.Esq. CHEM.,Aix.4~. Eo., 11, 635-7 (1939). (29) St. John, J. L., J . Assoc. Oficial Agr. Chem., 24, 932-5 ~ 1 9 4 1 ~ . (30) Scarrow, J. A., and Allen, C. F. H., Can. J . Research. 10, 73-6 (1934). (31) Taylor, W. H., and Smith, G. F., ISD. ENQ.CHEX.,.\NL. En., 14, 437-9 (1942). (32) Van Slyke, D. D., Hiller, .1.,and Dillon, R . T., J . B d Chem., 146, 137-57 (1942). (33) Villiers, A., and Moreau-Talon, -I,, Bull. S O C . chirn., 23, 308 (1918). (34) Wagner, E. C., IND. ENG.CHEM.,ANAL.ED.,12, i i l - 2 (1940). (35) Warner, R., J . Am. C h m . SOC.,66, 1725-31 (1944). (36) Wichmann, H. J., J . Assoc. Oficial Agr. Chem., 26, 522-59 (1943). (37) Wicks: L. E., and Firminger, H. I., IND. ENG.CHEsr.. Ax.9~. ED.,14, 760-2 (1942). PREBESTED in part before t h e Division of Biological Chemistry a t the 108tll Meeting of t h e AMERICAN CHE\IICAL SOCIETY, N e w York, N . 1.
Determination of Copper in Copper Proteins Using the Dropping M e r c u r y Electr0.de STANLEY R. AMES’
AND
CHARLES R. DAWSON
Department of Chemistry, Columbia University, N e w York, N. Y.
A specific method for the determination of copper in copper proteins involves use of the dropping mercury electrode after an acid extraction of the copper. The base solution for analysis is an acid sodium citrate buffer containing 0.005% fuchsin as a maximum suppressor. The copper can b e quantitatively extracted and the presence of native protein and protein breakdown products has been shown to b e permissible within prescribed limits. The method therefore elimi-
D
URIKG the past decade, interest in the role of minute amounts of copper in various biological processes has been revived as a result of the isolation of a number of copper proteins. These conjugated proteins, containing, in so far as is known at present, only copper as the nonprotein constituent, have been isolated from both plant and animal sources (3, 4,12, 16, f7,19, 21, 22, 29). As we gain knowledge of the importance of these copper proteins in plant and animal tissues, the need for a specific method for the rapid and precise microdetermination of copper in copper protein solutions becomes apparent. Several methods (2, 6 23 34) are available for the anaylsis of copper in plant and antma! tissue extracts, but most of these involve an ashing procedure which is time-consuming and productive of errors through contamination. For laboratories havin re$irometer equipment (6),the manometric method of d r b u r g (33) as modified b y Warburg and Krebs (34) has met with approval, since i t does not involve a long ashing procedure. This method has the advantage of speed, and has been extensively used in these laboratories (3,4, 16, f7,21, 2 2 ) . This experience has revealed, however, t h a t the precision of the method is not all that could be desired. T o obtain reliable results, the operator must be highly skilled in the technique. Even then a series of 1 Present address, Department of Madison, Wis.
Biochemistry, University of Wisconsin,
nates the necessity of a tedious arhing procedure. The half-wave potential at 25.0’ C. for cupric ion in the above base solution i s -0.18 volt vs. the saturated calomel electrode. The diffusion coefFicient at 25.0’ C. OF the cupric citrate complex in the above medium is equal to 0.43 X 10-6 cm.2 sec.-l. Practical limits of the method are from 1 to 75 to 100 micrograms per ml. of copper in the base solution with an average deviation of + 3%.
determinations on the same sample shows n mean deviation of about *1070. Recently, Reed and Cummings (23) have reported that copper in plant materials can be satisfactorily determined by use of the dropping mercury electrode. The earlier investigations of the rlarographic analysis for copper in the ash of hioloqical material y Kamegai (a), Mandai ( l a ) , Shoji (N),and Roncato and Bassani (24) were of a preliminary nature. Thanheiser and Maassen ( 3 2 ) , Such9 (31), and Stout, Levy, and,Williams (SO) have suggested procedures for the polarographic analysis of co er in steel, brass, and other nonbiological materials. prepare the sample for analysis according to the method of Reed and Cummings ( 6 3 ) , the lant material is subjected to a rather long and tedious wet-asfiing procedure, involving concentrated nitric, sulfuric, and perchloric acids. Interfering iron is then removed by precipitation with ammonium hydroxide. An acid sodium citrate buffer, containing acid fuchsin m a maximum suppressor, is used as the regulating solution for the polarographic analysis. The limits of the method, using a 1-grani sample of plant material, were reported as from 200 to 0 . 2 microgram of copper. T h e precision was not estimated and the temperature was not given.
E
It has been well established that copper proteins lose their copper in acid solutions-Le., below a p H of 3.0 (3,4,‘7, 1 2 ) . Copper proteins contain copper in both the cupric and cuprous form, but the copper in solution after an acid extraction would t.end to be in the cupric form, since cuprous copper is readily
