Estimation of Phytin Phosphorus ROBERTS. HARRISAND L. MALCOLM MOSHER Department of Biology and Public Health, Massachusetts Institute of Technology, Cambridge, Mass.
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H E nutritional value of phosphorus varies with the type of compound to which it is bound. Because of these differences, in working with plant materials, especially grains, an accurate method is needed for the determination of the peculiar type of phosphorus which prevails to the extent of 70 to 90 per cent in grains and seeds. In nature this so-called phytin phosphorus presumably occurs as sodium and magnesium salt of inositol hexaphosphoric acid, though Posternak (6) has suggested that the sodium-calcium salt occurs in nature, with the formula C6H60~4PsCa~Nas.3H~0. Both Anderson (1) and Posternak have prepared phytic acid, C&&P6, from various plant materials. It seems likely, therefore, that phytin phosphorus occurs as some salt of inositol hexaphosphoric acid (phytic acid).
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PROCEDURE Extract an 8-gram sample with 200 CG. of 2.0 per cent hydrochloric acid for 3 hours with occasional shaking and filter through a double layer of hardened filter paper. To a 50-cc. aliquot of this filtrate in a 400-cc. beaker add 10 cc. of a 0.3 per cent ammonium thiocyanate solution and 107 cc. of distilled water (resultant acidity is therefore 0.6 per cent). Add standardized ferric chloride solution (0.001 gram of iron per cc.) from a buret until the typical brown color of ferric thiocyanate no longer appears to be fading, then add 0.25 to 0.50 cc. more. Allow to stand until the precipitate flocculates (10 to 20 minutes) and filter through two layers of hardened filter paper. The ffocculation may be induced by occasional stirring. Wash the precipitate with three 11-cc. portions of distilled water. , Make up a blank by adding 10 cc. of 0.3 per cent ammonium thiocyanate and 3.16 cc. of hydrochloric acid (sp. gr. 1.19) to 157 cc. of distilled water. Add ferric chloride solution to the blank until the color matches that of the unknown. Calculate the percentage of phytin phosphorus by the following formula:
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Per cent of phytin phosphorus =
XARDY COLOR ANALYZER (ABSORPTION
CURVES)
50( CURVE A- TESTSAMPLE. CURVLB- BLANK
;MATCHI;A;PL~.
DFEPC RE BLANK CURVEC- EXCESSFeClj
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The authors have tested the suggestion of Andrew5 and Bailey (2) that this difficulty can be removed by titrating the unknown short of the end point and then completing the titration on the filtrate. Unless one titrates very close to the end point the ferric phytate still interferes somewhat. This necessitates a preliminary determination of the end point by the Heubner (4) method. Even so, the determinations give a value of phytin phosphorus which is lower than that obtained with the Heubner method. The authors have modified this Heubner procedure in such a way that this difficulty is entirely removed.
460h
480h
Thq authors are not yet convinced that this 1.19 factor is correct.. Tests have shown that the gravity filtration, taking perhaps 1 hour, causes no perceptible diminution of the color. The advantage of the proposed modification in terms of reduced variability as compared with the original Heubner method is shown by the data in Table I.
WAVE LENGTH
The best method which has been offered is that of Heubner and Stadler (4). Phytin phosphorus is extracted from pulverized plant material with 2 per cent hydrochloric acid for 3 hours, Cltered by gravity through hardened filter paper, and precipitated from the filtrate with ferric chloride in acid sohtion with the formation of insoluble ferric phytate. Ammonium thiocyanate is used as indicator of the ferric thiocyanate which forms after all of the ferric phytate has been precipitated. This method has been modified several times. Rather (7) has used a 1.2 per cent acid solution for the extraction of the grain. Averill and King (3)have used a strong ferric chloride solution (the upper limit suggested by Heubner) for titration. Knowles and Watkin (5) have used sodium salicylate rather than ammonium thiocyanate as indicator, on the basis that the pink end point is easier to read than the yellow-brown end point given by the ammonium thiocyanate. In the authors’ hands these modifications have been inconsequential and have not removed the chief difficulty of the Heubner determination: titration of a solution containing a slow-settling colloidal precipitate. Check titrations are extremely difficult because the end point is uncertain.
cc. of ferric ohloride (sample blank) X grams of iron per cc. X 1.19 X 100 weight of sample
TABLEI. COMPARISON OF METHODS (Corn extract) FFJRRIC CBLORIDEI~ INVBSTIQATOR Heubner Harris-Mosher cc. c c. . . 3.89 B. P. 3.59 3.88 3.85 3.92 P . K. B . 3.95 3.91 3.55 3.93 8. J. 4.03 3.91 3.17 J. M. B . 4.24 3.92 . 3.61 3.92 G. B. A. 3.82 3.87 3.59 3.83 3.90 3.94 3.89 3.91 J. F. 3.88 3.81 3.87 3.70 Average Average deviation Maximum deviation 0
PHYTINPHOSPHORUS Heubner Harris-Moeher Gram aer 100 mama 0.209 0.227 0.225 0.227 0.230 0.229 0.207 0.228 0.236 0.229 0.185 0.228 0.248 0.229 0.211 0.229 0.223 0.226 0.227 0.224 0.230 0.227 0.229 0.227 0.227 0.222 0.217 0.226 0.221 0.228 . 0.011 0.001 0.036 0.002
0.000965 gram of iron per cc.
Table I1 demonstrates that this modification is equally applicable to determinations of phytin phosphorus in grains other than corn. The phytin phosphorus content of pure calcium phytate is 19.7 grams per 100 grams (calculated). The crude sample 320
September 15,1934
INDUSTRIAL AND ENGINEERING CHEMISTRY
included in the determinations in Table I1 is about 50 per cent pure. TABLE11. HARRIS-MOSHER METHOD FERRIC CHLORIDE^
cc .
3.94 3.95 3.73 3.75 3.80 Corn 3.79 Wheat gluten 3.24 3.23 Calcium phytate (crude) 4.38 4.40 4 0.000965 gram of iron per cc.
Oats
Barley
PHYTIN PHOSPHORUS Grams/100 grams 0.228 0.229 0.216 0.217 0.222 0.222 0.186 ' 0.185 10.07 10.11
SAMPLE USED
Grams 2.0000 2.0000 2.0000 2.0000 0.05
There is always a possibility, in colorimetric titrations of plant extracts, that a substance may be present which contributes a color which either masks the true end point or produces a false one. To check this possibility the authors subjected three solutions'to the Hardy color analyzer.' The results are shown in the figure. Curve A was made by an extract of corn which had been matched with a blank (curve B). The two curves show that the matching of the color with the eye is quite accurate. They also show that the only color which was visible (and therefore used in matching the solutions) was that produced by the ferric thiocyanate. Therefore false end points are absent from the titration. 1 Description of an early model, J . O p t i d Soc. Am., 18, 98 (1928). Description of instrument used for these determinations will appear in the same journal.
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A third solution containing a large excess of ferric chloride was also tested (curve C). This curve indicates that a t about 420 X the absorption produced by ferric thiocyanate reached a maximum. CONCLUSIONS The Heubner-Stadler method for the determination of phytin phosphorus has been modified by titrating the unknown solution with ferric chloride beyond the end point, filtering, and matching the color of the filtrate with a blank. Determinations are much more accurate because the insoluble colloidal ferric phytate which clouds the end point has been removed. Color analyses have shown that there is no color of plant origin which interferes with the end point in the titration of corn extracts with this method. The color produced by ferric thiocyanate reaches a maximum absorption a t 420 A. LITERATURE CITED (1) Anderson, R.J., N. Y. Agr. Expt. Sta., Bull. 19, 1912. (2) Andrews, J. S.,and Bailey, C. H., IND. ENO.CHEM.,24,80 (1932). (3) Averill, H.P.,and King, C. G . ,J.Am. Chem. Soc., 48,724(1926). and Stadler, H., Biochem. Z., 64,422 (1914). (4) Heubner, W., (5) Knowles, F.,and Watkin, J. E., J. Agr. Sci., 22, 755 (1932). (6) Posternak, S.,Compt. rend., 169,138 (1919). (7) Rather, J. B., J.Am. Chem. Soc., 39,2506 (1917). RNOEWEDMay 25, 1934. Presented before the Division of Biological chemistry at the 87th Meeting of the American Chemical Society, St. Petersburg, Fla., March 25 to 30, 1934. This paper is Contribution 31 of the Department of Biology and Public Health, Massachusetts Institute of Technology.
Improved Method for Determination of Percentage Acetyl in Organic Compounds MAX PHILLIPS, Bureau of Chemistry and Soils, Washington, D. C. NUMBER of methods have been described in the always reliable, particularly with small quantities of subliterature for the quantitative estimation of the per- stance. Perkin hydrolyzes the substance with alcohol and centage acetyl in organic compounds. All these meth- concentrated sulfuric acid, distills off the ethyl acetate ods consist essentially in formed i n t o an excess of first splitting off the acetyl 0.5 N alkali, saponifies the group and then determinethyl acetate, and then deing quantitatively the acetic termines the excess of alkali. acid produced. The variThe Perkin m e t h o d , a l ous methods described in though relatively simple to t h e l i t e r a t u r e have been carry out, suffers from the adequately r e v i e w e d b y disadvantage that charring Meyer (a). Of the m o r e of the sample by the sulfuric commonly employed methacid may take place with , ods, mention need only be the p r o d u c t i o n of sulfur made in this connection of dioxide which, of course, inthe method of Wenzel (6) terferes with the determinaand of Perkin (3). Wenzel tion. With the t e c h n i c hydrolyzes the s u b s t a n c e recommended by Perkin it with slightly diluted sulis difficult to obtain quantifuric acid, primary sodium tative results, particularly phosphate and metaphoswith smaller quantities of p h o r i c acid being t h e n material. Sudborough and added and the acetic acid T h o m a s (6) h a v e used distilled off under reduced aromatic sulfonic acids such pressure. Such a procedure, as benzene sulfonic a c i d however, involves as a rule or a- and @-naphthalene long and tedious operations, sulfonic acids. Freudenand the r e s u l t s a r e n o t FIGURE 1. DIAGRAM OF APPARATUS berg and Harder ( I ) have
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