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.
321
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.
A
'
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 f o r m e d i n t o an excess of H 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 phoric 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 s u l f o n i c a c i d however, involves as a rule or a- and @-naphthalene long and tedious operations, s u l f o n i c 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
322
ANALYTICAL E D I T I O N
employed p-toluene sulfonic acid for breaking u p the acetylated compound. The method described in this paper is a modification of the Perkin method. It has none of the disadvantages of the original method, and is an improvement over the modified procedure employed by Freudenberg and Harder. Using the technic described i t has been possible to obtain quantitative results not only with -0-acetyl but also with -N-acetyl compounds. APPARATU~ The apparatus used is illustrated in Figure 1. I t is constructed of Pyrex glass and consists of a reaction flask D, which is provided with an inlet tube, B, and stopcock, d‘. D is placed in water bath M , which is heated with microburner Q. Connection between D and condenser F is made through interchangeable ground-glass joint E (No. 11) and is held fast by means of springs 0. Condenser F is connected through glass tube H to condenser G. K is a 300-cc. Erlenmeyer flask upon which two marks have been placed indicating a volume of 100 ahd 150 cc., respectively. During the distillation L serves as an ice bath, and during the hydrolysis of the ethyl acetate, as a water bath. J is a rubber stopper, the opening of which has been lubricated with glycerol so that it can readily slide up and down tube I . N is a two-wa stopcock through which cold water for condenser F is p a s s e l By turning N and opening pinchcock P it is possible to drain the water immediately from F. A is a 500-cc. Erlenmeyer flask, partly filled with aldehyde-free 95 per cent ethanol and used or generating alcohol va or. It is heated by means of an electric hot plate, not shown on tEe drawing.
PROCEDURE FOR -O-ACETYL COMPOUNDS The weighed sample (0.3 to 0.4 gram) is laced in reaction flask D to which are also added 5 rams of togenesulfonic acid,’ a small piece of unglazed tile, a n t 2 5 cc. o f & per cent aldehydefree ethanol (prepared by the method of Stout and6Schuette, 4). Into receiver K , 25 cc. (accurately measured with a pipet) of an alcoholic potassium hydroxide solution (approximately 0.2 N ) and a small piece of unglazed porcelain are put. J is moved up close to the inner seal of G, leaving K open to the atmosphere. K is surrounded with crushed ice contained in L,which is supported by means of a ring and stand. The height of the ring is so adjusted that I reaches close to the bottom of K. E is lubricated with stopcock grease and is attached to F with springs, 0. Cold water is circulated through both condensers, and D is placed in water bath M , and the reaction mixture is refluxed for 15 minutes. The water in condenser F is then drained, stopcock C is opened, and a slow stream of alcohol vapor is allowed to pass into D from generator A . This operation is continued until the total volume in K measures 150 cc. During the distillation, M is heated with microburner Q at such a rate that a t the end of the distillation the volume in D is reduced ap roximately one-half. K is then lowered so that the end of P i s above the level of the liquid. Stopcock C is shut off, and the rubber tube attached to generator A is disconnected from B. D is detached from F , and J is lowered until it is approximately 2 inches (5 cm.) from the lower end of I and is securely attached to K. The ice in L is re laced with hot water, and the distillate is refluxed for one-half tour. During this period L is heated with a microburner. K is then disconnected, diluted with distilled water, and the unused potassium hydroxide determined by titration with 0.1 N acid, phenolphthalein being used as the indicator. A somewhat sharper end point is obtained by running in a known excess of 0.1 N acid and ,determining this excess by titration with 0.1 N alkali. A blank determination is made following the procedure above described, except that no sample is added to D. From the blank determination the normality of the alcoholic potassium hydroxide solution is accurately determined. The number of cubic centimeters of 0.2 N potassium hydroxide solution used when multiplied by 0.86 and divided by the weight of the sample gives the percentage acetyl in the compound. PROCEDURE FOR -N-ACETYLCOMPOUNDS The analytical procedure described above is modified as follows: 1 Thia is purified as follows: A strong aqueous solution of p-toluenesulfonic acid is placed in a distilling flask which is attached to a condenser, and a current of steam passed through the solution until the distillate coming over no longer reacts acid. The residual solution of the sulfonic acid is concentrated on the steam bath and allowed to crystalliee. The crystals are filtered off and dried in a vacuum desiccator over sulfuric acid.
Vol. 6 , No. 5
Instead of refluxing the solution in D for 15 minutes, it is refluxed for 2 hours. The water in condenser F is then drained, and alcohol vapor from generator A is allowed to pass through the solution contained in reaction flask D. This operation is continued until the volume of the solution in K measures 100 cc. During this distillation the heating of M is so regulated that the volume of the reaction mixture in D is reduced one-half. C is then shut off, and cold water is again circulated through F. The solution in D is then refluxed for 30 minutes, after which the cold water circulating through F is shut off, the jacket of the condenser F is drained, and a current of alcohol vapor from A again passed through the solution in D. This operation is continued until the volume of the li uid in K measures 150 cc. The refluxing of the solution in K a n j the determination of the excess of potassium hydroxide sdution are carried out exactly as described above for -0-acetyl compounds. The results obtained with several -0-acetyl compounds
and with acetanilide are given in Table I. TABLE I. DETERMINATION OF ACETYL WEIQHT OF
COMPOUND
SAMPLE
Acetyl dehydrotoxicarol Cellobiose octaacetate Acetanilide Acetanilide
0.2 N XOH
U
~
ACETYL D Found Calculated
Cram
cc.
%
%
0.4000 0.4000 0.2000 0.3000 0.3500 0.2000 0.3500 0.3500
10.87 10.88 5.35 6.95 3.90 23.9 13.1 13.08
23.3 23.3 23.0 17.05 9.58 51.3 32.1 32.1
23.1 23.1 23.1 17.4 9.66 60.74 31.8 31.8
LITERATURE CITED (1) Freudenberg and Harder, Ann., 433,230 (1923). (2) Meyer, H., “Analyse und Konstitutionsermittlung organisoher Verbindung,” 4th ed., pp. 670-83,J. Springer, Berlin, 1922. (3) Perkin, Proc. Chem. SOC.,20, 171 (1904);J. Chem. Soc., 87, 107 (1905). ENQ.CHEM.,Anal, Ed., 5, 100 (1933). (4) Stout and Sohuette, IND. ( 5 ) Sudborough and Thomas, Proc. Chem. Soc., 21,88 (1905). (6) Wensel, ref. 2,pp. 678-81. REOEIYEDMay 8, 1934. 238th Contribution from the Color and3Farm Waste Division, Bureau of ,Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C.
Suction Device LOUISCOHEN College of the City of New York, New York, N. Y.
I
N laboratory filtrations employing suction, i t is fre-
quently desired to introduce the filtrate directly into the Erlenmeyer or any other flask. For such a purpose, the piece of apparatus shown in the illustration as used with a Walter crucible holder has proved convenient. This device is particularly applicable in the analysis of reducing sugars.’ T h e a p p a r a t u s may b e r e a d i l y constructed from a Pyrex test tube, by attaching a side arm at a convenient distance from its mouth, expanding the mouth to fit a Walter crucible holder, and cutting the test tube at a convenient distance from the bottom, to allow a portion of the holder stem to extend beyond the so-called s u c t i o n funnel stem. RECEIVBD June 9, 1934. 1 ASEOC. O5cial Agr. Chem., Official and Tentative Methods, pp. 379-80 (1930).
