Perchloric Acid in Micro-Kjeldahl Digestions L. F. WICKS
AND H.
I. FIRRIINGER, Research Department, The Barnard Free Skin and Cancer Hospital, St. Louis, Rlo.
Data are presented in an effort to discourage the use of perchloric acid as an aid in micro-Kjeldahl digestions, as the authors are convinced that this oxidizer can cause a serious loss of nitrogen.
I
nitrogen in the digestion period”. Myers (16) doubts that it is as satisfactory as hydrogen peroxide for urine and blood analyses. The most emphatic pronouncement is in the text of Peters and Van Slyke (IY), who maintain, “if, however, the organic material present does not reduce all the perchloric acid the latter destroys part of the ammonia formed. For this reason perchloric acid cannot be used as a general reagent in Kjeldahl digestions.”
S 1921, Mears and Hussey (1.4) suggested that the powerful oxidizer, perchloric acid, might be of value to shorten the time of Kjeldahl digestions, but warned that “an excess” should be avoided. Their figures and curves illustrate strikingly that low results will follow if such surplus be present. Shortly before this publication, Willard and Cake ( 2 4 had tried 60 to 70 per cent perchloric acid, with and without sulfuric acid, and concluded that “it is practically impossible to avoid loss of nitrogen if the organic matter is completely oxidized”. Not long after, Parker and Terrell (16) used perchloric acid to aid in the difficult digestion of leather, but it was clear to them also that low results could easily follow. Nevertheless, perchloric acid so readily destroys otherwise resistant organic matter that many workers soon attempted to apply it to all sorts of Kjeldahl digestions.
It is to such opinion that the authors hold, but this is iiot a general conviction. Nitrogen determinations, both macro and micro, that employ the aid of perchloric acid are still being done and still being proposed. Some of the better known digestion mixtures containing perchloric acid which hare appeared in the literature are shown in Table I. TABLE
I. IsIT1.4L CONCEiYTRATIOsS O F PERCHLORIC ACID DIGESTION MIXTURES
Lematte, Boinot, and Kahane (12) employed a very strong mixture for digesting animal organs, and Frey, Jenkins, and J o s h (6) claimed agreement with +her methods when they accelerated the digestion of leather with potassium perchlorate. However, Fiszerman and Fiszerman-Garber (4) reported that in their Kjeldahl digestions (of liver and kidney meals), those aided by perchlorates revealed loss of nitrogen, and Yoe (26) had cautioned earlier that in the case of fluids such as milk and urine, low results would always follow unless a certain minimum of perchloric acid was used. Kearly all the experimenters named above had carried through their digestions with mixed perchloric and sulfuric acids. LeTourneur-Hugon and Chambionnot ( I S ) recommended that the perchloric acid be cautiously added dropwise to the boiling sulfuric acid after some preliminary destruction. They also stated that not all methods of using it are suitable and that it should not be applied to milk or flour. Gauduchon-Truchot (8), in a doctorate thesis on the Kjeldahl technique, cited some analysts who distrusted perchloric arid, along with those who favored it, but on the whole this author considered it to give generally satisfactory results.
(Percentages on weightlweight basis) Digestion Mixture HClOh HzSOa HaPo, NanSOa Meam and Hussey 1.46 87..5 ... ... Parker and Terrell 1.59 78.2 ,.. ... Rose 21.8 27.2 ... , . . Dupray 1.31 57.0 2.90 ... Lematte, Boinot, and Kahane 39.3 35.4 ... ... Chiles 2.06 51.6 ... 6.89” Zakrzewski and Fuchs 0.126 61, a ... ... a Xot all will remain dissolved.
CuSOa 1.99 2.83 .,. ,..
...
0.46
...
Experimental Work The authors had often suspected that micro-Kjeldahl determinations gave lower results when done with the aid of perchloric acid. The literature reveals differences of opinion, but no good presentation of comparative analyses of various substances by both perchloric acid and other digestion procedures. No modification of the Kjeldahl method gives nitrogen quantitatively on all compounds, but the technique is suited for a fair approximation of this element as found in such very heterogeneous mixtures as biological materials. One does not generally employ the Kjeldahl method for pure organic compounds. The authors present here comparable nitrogen analyses of materials digested with perchloric acid techniques and with other methods, and have concerned themselves with micro quantities only, for i t is there that the use of this terrific oxidizer is most objectionable.
When perchloric acid was applied to microquantities as of biological fluids, etc., one might expect that investigators here and there would mention lower results. Rose (18), however, employing a powerful sulfuric-perchloric acid mixture (supplemented by hydrogen peroxide) for micro quantities, claimed good agreement with the calculated nitrogen of various organic compounds and with a ‘‘macromethod” otherwise not identified. Dupray (3)suggested a much milder digestion mixture for nonprotein nitrogen determinations on blood filtrate but gave no figures at all. Chiles (1)used a digestion mixture containing some perchloric acid but preferred to omit this oxidizer when dealing with urine. mentioning “somewhat low results”. He did, nevertheless, employ it for other biological fluids. Kit0 (IO)adopted Chiles’ formula for flour. milk, etc., but accelerated the digestion by the addition of selenium. Walters ( 2 1 ) . interested in the nitrogen of brewery products, rlearly stated that the digestion methods of Mears and Hussey and of Rose seemed to cause a loss of nitrogen and that the findings were always less than those of regular Kjeldahl digestions. Doneen ( 2 ) attempted to determine the nitrogen of plant material with what must have been a large excess of perchloric acid. [The micro digestion mixtures of Fuchs and Falkenhausen ( 7 ) ,and Zakrzewski and Fuchs (279, are so low in perchloric acid that it is scarcely significant.]
For the major experiment, they chose samples of four typical biological fluids: serum, cowJs milk, human urine, and proteinfree blood filtrates. These last were prepared from serum and trichloroacetic arid, or from whole blood and tungstic acid. Determinations were done in triplicate on each sample of suitably diluted fluid, taking aliquots containing 0.2 to 0.5 mg. of nitrogen. Digestions were carried out in Folin-Wu tubes or in 25 X 200 mm. Pyrex test tubes and heated over microburners. The hydrogen peroxide digestion of Koch and McMeekin (11) and the selenium-sulfate mixture of West and Brandon (22) were selected as reference procedures to be compared with various perchloric acid techniques. All analyses were by direct nesslerization of the diluted digest and reading in a Klett-Bio colorimeter. The Nessler’s reagent used was that of Koch and McMeekin but prepared with mercuric oxide as described elsewhere (25). Every ordinary precaution was taken, The distilled water was all redistilled from dilute sulfuric acid-permanganate solution to remove traces of ammonia, Blanks were run on every reagent.
One encounters scattered general statements in the literature questioning the advisability of utilizing perchloric acid in Kjeldahl digestions. Smith (19) remarks in passing that “unfortunately the process in certain applications gives low results, presumably due to loss of
760
ANALYTICAL EDITION
September 15, 1942 T.4BLE
11. MICRO-KJELDAHL DIGESTIONS COMPARIXG PERCHLORIC ACID METHODS WITH OTHERPROCEDERES
Biological Fluid
Approximate Difference
c/o
%
Total Protein
Koch and Mchleekin 1 drop 60% HC104 Koch and SlcMeekin Rose West a n d Brandon 2 drops 7270 HClOi West a n d Brandon Rose West and Brandon Chiles
7.2, 7.7, 7 . 8 5.4,5.1,5.2 7.5, 7.6, 7.8 6.9, 5.6, 6 . 3 8.1.7.9,8.2 4,5,3.4,4.2 7.6, 8.4, 7.6 6.2,7.1, 6.1 7.4, 7.8, 7 . 5 1.7, 5 , 3 ,2 . 8 , 6 . 4
7.6 5.2 7.6 6.3 8.1 4.0 7.9 6.5 7.6 4.0'
1 Koch and h1cMeekin 2 d r o p s 7 2 % HClOa 2 Koch and McMeekin Rose 3 West and Brandon 1 drop 72% HClOi 4 West a n d Brandon Rose 5 Koch and h l c l f e e k i n Chiles
3.4,3.5,3.3 2.1, 1.9, 2 . 4 3.7, 3.7, 3 . 8 3.0, 2.7, 2.8, 3 0 4.4,4.7, 3.9 3.0,3.0, 2.9 3.4.3.4. 3.7 3.0, 2.6, 3.2 3.3, 3.3, 3 . 2 1.5, 1.2, 1.7. 1 . 3
3.3 2.1 3.7 2.9 4.3 3.0 3.6 2.9 3.3 1.4
L-'0
Serum
l\Iilk
hverage
Digestion Method
32 17 51
761
Five-cc. portions of an ammonium sulfate solution (= 0.5 mg. of nitrogen) containing 0.05 per cent dextrose were pipetted into ignition tubes together with 1 cc. of 1 to 1 sulfuric acid and two small quartz pebbles. (The dextrose served to mark the charring point a t which some workers drip in the perchloric acid.) The tubes were heated (electrically, for uniformity) until the dextrose charred and sulfur trioxide fumes escaped. They were then lifted from the heater coils and let cool slightly to avoid splatter when the 72 per cent perchloric acid was introduced. This was added in varying numbers of small drops, and the tubes were covered with 30-mm. watch glasses, returned to the coils, and heated for an additional 5 minutes. They were then diluted, nesslerized, and read in the usual manner.
I8
47 38 22
OF MICRO-KJELDAHL DIGESTIOM os TABLE 111. COMPARISON HEXOLYZED BLOOD
Digestion Method
30 19 57
Average
Nitrogen Found
Significant
-Mo. p e r 100 cc. Koch and XlcMeekin West and Brandon Wong
33.6, 35.7, 34.3 34.4, 33.4, 34.1 33.8, 34.0, 3 3 . 6
34.5 34.0 33.8
34 34 34
Rose Dupray 1 t o 2 drops of 72Y0 HClOx added
26.7, 22.6. 24.9, 24.3 29.6, 27.9. 28.4, 26.5
24.6 28.1
25 28
26 18
2 7 . 2 . 2 4 . 2 , 26.8. 2 5 . 6
26.0
26
24
Approximate Difference from Above,
Total Nitrogen
% Koch and 11cMeekin 1 drop 60% HClOa Koch and McMeekin Rose Koch a n d McMeekin Rose West a n d Brandon 1 drop 7 2 7 HClOi West and firandon Rose
Crine
2 . 0 , 2 .O, 1 . 9 1 . 7 , 1 . 8 ,1 , 8 2,3,2 . 6 , 2 . 4 2.2.2.2,2.2 0.71, 0.71, 0.71 0.64, 0.64, 0.67, 0,62 1.4, 1.4, 1 . 3 1.1, 1.2. 1 . 2 2.1, 2 , 5 , 2 . 3 1.9,1 . 8
2.0 1.8 2.4 2.2 0.71 0.64 1.4 1.2 2.3 1.8
8
10 14 22
Sonprotein S i t r o g e n
Filtrate 1
2 3 4
5
Koch and Dupray West and Dupray Koch and Dupray West and Dupray Folin a n d Dupray
Mc\leekin Brandon McRleekin Brandon
W u (6)
.Mg./IOO cc. 54, 52 41,47,44 43, 45, 48 39, 41 36, 37, 36 22, 28, 27 44, 47, 43 26, 24, 27 31, 29, 28 25. 20, 27
.Mg./
100 cc.
53 44 45 40 36 26 45 26 29 24
5
10
li 11
28 42
17
The results are illustrated in Figure 1, each point representing the average of three to four runs. The curve is necessarily a ragged one, but i t does show progressive loss of nitrogen with increased perchloric acid. Heating time was a little longer and perchloric acid concentration somewhat higher than would have been required by most microdigestions. The curve illustrates simply that perchloric acid can remove nitrogen from a heated ammonium sulfate-sulfuric acid solution.
I t , is, of course,, almost meaningless t o average such scattered results. Chiles' mixture can give very varying figures, depending partly upon t h e critical heating time. 0
The standard solutions for the colorimetric comparison were always of similar acidity as the samples, and standard and sample were brought to the same temperature before matching. Six careful readings of the colorimeter were averaged for each single determination. Typical findings are given in Table 11. In every instance, the results from the perchloric acid digests were lower and usually more scattered than those of the other procedures. As a further experiment, a number of microdigestion techniques were all compared on a single sample of complex nitrogenous content. This was a solution of hemolyzed blood, consisting of oxalated whole blood which had been diluted 1 t o 100 with water containing a trace of saponin. The results are shown in Table 111. There is good agreement between the reference methods, and lower results with greater scattering where perchloric acid was present. (The authors have found in other work also that the digestion procedures of Koch and Mchleekin and West and Brandon, and the persulfate oxidation of Wong, 26, always give essentially the same figures.) Whatever the reasons for low results with perchloric acid, at least part of the loss can occur in the final digest after charring has quite cleared. If an ammonium sulfate-sulfuric acid solution be heated in the presence of perchloric acid, a disappearance of nitrogen can result. To illustrate this, the following experiment was performed :
0.4
.
1 0.3 .
2
i? 1
0.2
'
0.2
*
-
a
O
~
Z
9
-DROPS FIGURE1. DESTRUCTION OF
4
5
7
72% ffC/04-NITROGEN IN -4MZMONIEM
ACID SOLUTION PERCHLORIC ACID
SULF.4TE-DEXTROSE-SULFERIC
6
BY
ADDED
8
762
INDUSTRIAL AND ENGINEERING CHEMISTRY
Discussion The mechanism of the apparent loss of nitrogen is not clear. Mears and Hussey suggested that possibly an excess of perchloric acid formed ammonium perchlorate which then decomposed. Walters, Smith, Willard and Cake, and Peters and Van Slyke evidently believed that part of the ammonia (as ammonium sulfate) could be oxidized to free nitrogen. Myers regarded the lower results in colorimetric micro determinations as probably due to the formation of amines which, he stated, would not give full color development with Nessler’s reagent. This does not seem to be sufficient explanation, although Gortner and Hoffman (9) pointed out that about 7 per cent of amine nitrogen results with ordinary Kjeldahl digestion, and Villiers and Moreau-Talon (20)earlier claimed that the presence of strong oxidizers promotes amine formation. (Their paper was prior to the introduction of perchloric acid.) Certain analysts may protest that, granting all the above, if an excess of perchloric acid be avoided, the error will still be negligible. This may be true for sizable samples, and where just enough perchloric acid is added to clear the solution after preliminary digestion by sulfuric acid. But for microquantities, even a small excess may release a considerable portion of the nitrogen-for example, 2 small drops may be inadequate and 3 drops excessive.
Temperature Apparently temperature has an important influence on the oxidizing power of perchloric acid. The acid does not seem to have much effect on the organic matter until sufficient water has been driven off from the digest to elevate the boiling point considerably, after which the combustion is vigorous and rapid. A little perchloric acid added then is much more potent than a great deal present in the original digestion mixture. Certain experiments suggest that, with very careful temperature regulation, a zone might be found where one could complete the oxidation of the carbon without loss of nitrogen. The authors wonder, however, whether such procedure would prove either practical or trustworthy.
Vol. 14, No. 9
Acknowledgment The authors are grateful to G. F. Smith of the University of Illinois for the loan of a monograph (8) otherwise difficult to obtain. Literature Cited (1) Chiles, H. M., J . Am. Chem. Soc., 50, 217 (1928). (2) Doneen, L. D., Piant Physiol., 7, 717 (1932). (3) Dupray, M., J . Lab. Clin. Med., 12, 386 (1926).
(4) Fisaerman, J. H., and Fiszerman-Garber, D., Bull. SOC.Pharmacol., 40, 210 (1933). (5) Folin, O., and Wu, H., J . B i d . Chem.. 38, 81 (1919). (6) Frey, R. W., Jenkins, L. J., and Joslin, A. ~ .J . ,Am. Leather Chem. Assoc., 23, 397 (1928). (7) Fuchs, H. J., and Falkenhausen, hl. Y., Biochem. Z., 245, 304 (1932). (8) Gauduchon-Truchot, H., “Contribution 1 l’Etude de la MMBthode de Kjeldahl”, Paris, Imprimerie Henry, 1936. (9) Gortner, R. A., and Hoffman, IT. F., J . B i d . Chem., 70, 457 (1926). (10) Kito, W. H., A n a l y s t , 59, 733 (1934). (11) Koch, F. C., and McMeekin, T. L., J . Am. Chem. Soc., 46, 2066 (1924). (12) Lematte, L., Boinot, G., and Kahane, E., J . pharm. chim., 5, 325, 361 (1927). (13) LeTourneur-Hugon and Chambionnot, Ann. f a l s . , 29, 227 (1936). (14) Mears, B., and Hussey, R., J. ISD. ESG. C H m r . , 13,1054 (1921). (15) Myers, V. C., J . Lab. Clin. Med., 17, 227 (1931). (16) Parker, J. G., and Terrell, J. T., J . SOC.Leather Trades Chem., 5, 380 (1921). (17) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry. Methods”, p. 519, Baltimore, \Tilliams & Wilkins Co., 1932. (18) Rose, A. R., J . B i d . Chem., 64, 253 (1925). (19) Smith, G. F., IND.ENG.CHEY.,ANAL.ED.,6, 229 (1934). (20) Villiers, A., and Moreau-Talon, A , , Bull. soc. chim., 23, 308 ( 1918). (21) Walters, L. C., -4ustralian J . Erpcl. BioZ. M e d . Sci., 7 , 113 (1930). i i ~ aED., ~ . 4, (22) West, E. S., and Brandon, A . L., IND.E N G .CHEM., 314 (1932). (23) Wicks, L. F., J . Lab. Clin. M e d . , 27, 118 (1941). (24) Willard, H . H., and Cake, W. E., J . Am. Chem. Soc.. 42, 2646 (1920). (25) Wong, S. Y., J . Bid. Chem., 55, 431 (1923). (26) Yoe, J. H., Ann. chim. anal., 7, 193 (1925). (27) Zakrzewski, Z., and Fuchs, H. J., Biochem. Z., 285, 391 (1936).
Improvements in the Colorimetric Microdetermination of Phosphorus C. P. SIDERIS, Pineapple Research Institute of Hawaii, Honolulu, T. H.
T
W O fundamentally distinct methods have been in use for
almost two decades for the colorimetric determination of phosphorus-the Bell-Doisy (1) and Copaux ( 4 ) methods. The former, depending on the estimation of a blue color formed by the yellow phosphomolybdate compound reacting with some reducing agent, has undergone many modifications in the hands of various workers. A very excellent account of this method and modifications has been presented by Snell (6) and Peters and Van Slyke ( 5 ) . Zinzadze’s studies (7, 8 ) pertaining to amounts of reducing reagent, time, temperature, etc., are interesting. A great improvement in the colorimetric determination of phosphorus was made with the introduction of the Berenblum-Chain (2, 3) method. Incorporating the features of both the Bell-Doisy and Copaux methods, this proved more successful than either. In the Berenblum-Chain method the yellow phosphomolybdate compound is extracted with isobutyl alcohol instead of ether as in the method of Copaux, and then changed into the blue phosphomolybdate compound by
means of a reducing agent (stannous chloride in 37 per cent hydrochloric acid). Also, the ether phosphomolybdate extract obtained by the method of Copaux can be easily converted into a blue color by addition of the tin-hydrochloric acid reagent used by the writer. However, this procedure is not recommended, owing to the high degree of volatility of the ether and rapid changes in the volume of the extract. nButyl alcohol is preferred to ether as a solvent. The main points of divergence of the writer’s technique from that of Berenblum-Chain are in the use of metallic tin instead of stannous chloride in 37 per cent hydrochloric acid, elimination of ethyl alcohol for washing the separatory funnel and dilution of the blue phosphomolybdate compound, heating the mixture containing the unknown, 2 N sulfuric acid, and ammonium molybdate, and the use of normal instead of isobutyl alcohol. A reducing agent prepared by mixing stannous chloride and 3 i per cent hydrochloric acid sometimes loses its reducing power and fails to give an intense indigo blue color during an
