V O L U M E 2 3 , N O . 10, O C T O B E R 1 9 5 1
1501
.m
Ita sensitivity, permittiiig deterniiriatioii from 0.2-nil. saiiiplw. of as little as 0.1 microgram of phosphorus per ml. of color react-
ing solution, compares favorably with other methods emplo~.c~l for inorganic phosphate deterniination. As the writers have riot cheeked the stability of the color produced beyond 2.5 hours, this method cannot be favorably compared with other methods in this respect. I n a report which appeared after the present, manuwript was submitted for publication, Griswold el a2. ( 4 ) reported the color produced as being stable for a t least 24 hours, for a new modification of the Fiske and Subharow method, involving arninonaphtholsulfonic acid as a reducing agent and heating in a 1 .Y sulfuric acid solution. Their method is described as h:tviiig greater sensitivity, convenience, alid specificity for the determination of inorganic phosphates arid phosphate esters in hiologicnl nratei~ial. However, the present writers believe that, for the determination of inorganic phosphatases as such, the adapted Suniner method described in this paper is considerably more corivenient. This is true from the standpoint of both simplicity and ease of preparation of a relatively cheap arid easily obtainable reducing agent-Le., ferrous sulfate-as well as in the actual procedure involved in the production of color (cf. met,hod of Griswnld et nl.,
.I5
u W 2
u.10
P
sm
a
.05
4 1. 0.25
0.50
0.75
MICROGRAMS
Figure 2.
1.00
1.25
IS0
PER ML.
Phosphorus Concentration a n d Absorbance a t 720 mw
reaction obtained by this procedu1.e for thc range of concelitrations of phosphorus employed.
Because the filial acid concentration of 0.76 AV is considered more than adequate to prevent formation of silicomolybdic acid for silicon in amounts over 200 times that of phosphorus (Fiske mid Subbarow, 3 ) ,as well as sufficient to prevent color production through reduction of molybdic acid itself (Sumner, 7), unspecific c*olor production by this method is minimized in this respect. The method is recommended for analy,tical and biochemical studiw involving phosphate determination, as a relatively simple and reproducible colorimetric procedure.
UISCVSSION
LITERATURE CITED
I3ecnuse of‘ the extreme sensitivity of the test, the presence of vcry small quaiititiw of phosphate in the reagents employed (in this case the molybdate) gives a barely discernible bluish coloration to the blank, whirh gives a definite reading on the spectrophotometer. Ji-ith pure distilled \mter in the blank cell, a standard curve parallel to that of Figure 2, but which does not pass through the origin, is obtained.. This method is currently being employed in a htudy on the chrmistry of phosphatases in the honeyhee with satisfactory resulta.
(1) Bell, R. D., and Doisy, E. A., J . B i d . ChenL., 44, 55 (1920). (2) Bodansky, A,, Ibid.,99, 197 (1932). 1,3) Fiske, C . H., and Subbarow, Y., Ibid.,66, 375 (1925).
Griswold, B. L., Humoller, E’. L., and McIntyre, A . 13.. - 1 x . k ~ . (?HEM., 23,192 (1951). ( 3 ) Kuttncr, T., and Cohen, H. It., J . B i d . Chem., 75, 517 (1927). (6) Kuttner, T., and Lichtenstein, L., Ibid.,86, 671 (1930). ( 7 ) Gumner, J. B., Science, 100,413 (1944). (4)
RECEIVED December 11, 1950. This inXTestigation was supported in part by funds proliided for biological a n d medical research h y the State of Washington Initiative Measure Yo. 171.
Photometric Analysis of Furfural Dissolved in Xylene I‘se of p - B r o r n o a d i n e EUGEYE W.RICE‘ The Helen L. a n d ,%furyE . II.’artt*ickMenzorial, T h e George Washington University School of .Iledicine, Washington, I). C . Y CONIiECTIOS I\ ith son~ebiochemical investigatioile, the -author has determined the furfural-yielding capacities of nuniorous biological compounds by the conrcnient technique of Ito(\vcsand bIunro ( Z ) .
In this method, 5.00 nil. of an aqueous solutio11of the furfural prerursor are gently refluxed for 150 minutes in :in all-glass apparatus in the presence of 3.00 ml. of concentrated hydrochlqric acid and 25.00 ml. of xylene. After cooling, the acid layer 1s separated from the xylene and the latter is dried wit,h anhydrous sodium acetate. Finally, the furfural content of an aliquot of the xylene solution is determined hy the familiar aniline acetate met hod. the color formed as found t o bc uiistuble and not well suited for photometric measurements, thr p-bromoanilinc rcay:nt pro1 Present address. Departriicnt of Riologirnl Chemistry and Siitrition, Creighton University School of Medirine, Oiiialia, S e b .
posed by Roe and Rice for the dctcrinination of pentoses ( 3 , 5) was tried. Although the method is well suited for the analysis of neutral aqueous furfural solutions, the original rvagmt cannot Ix. used in the presence of xylene. The present, communication dewribes an improved method for the determination of furfural dissolved in xylene. ‘The modified reagent produces a color with excellent photometric qualities and, moreover, does not darken upon standing. I t consists of 2.00 grams of p-bromoaniline (Eastman Kodak Co.) dissolved in a solvent composed of 95.0 ml. of thiourea-saturated glacial acetic acid plus 5.0 ml. of distilled water. The reagent is stored in a glass-stoppered brown bottle. To analyze a solution of furfural in xylene, 1.00 rnl. of the un1 2
E g
- 70 - 10 - 90
=ANILINE ACETATE METHOD 20-
,bo 2b0 CONCENTRATION OF FURFURAL, y PER ALIQUOT ANALYZED 210
410
$0
-
2
’
100
8‘0
Figure 1. Standard Curves for Furfural Solutions Measured against Reagent Blank with Evelyn Colorimeter at 520 m,u gent are added to each tube and immediately mixed by lateral shaking. After 30 minutes’ standing a t room temperature in the dark, the optical densities are determined with the Evelyn colorimeter using a 520 filter and setting the instrument to zero density with the blank tube. The color reaches a maximum in approximately 30 minutes and remains constant for about 20 minutes at room temperature, then fades slowly. The total concentration of furfural is calculated by means of the customary formula of spectrophotometry and the original data of Reeves and Munro (2).
The range and the accuracy of both methods were established by plotting the absorptancy against logarithm of concentration of furfural, according to the proposal of Ringbom ( 4 ) and Ayres(1). These data are shown in Figure 1. Inspection of the two curves shows the p-bromoaniline method to have an optimum range (0.9 to 6 micrograms) almost twice that of the original aniline acetate method (4 to 14 micrograms). The sensitivity, in turn, is three times greater than the original method. A relative analysis error of 3.2% per 1%absolute photometric error for both methods is established by dividing 230 by the slopes of the respective curves, which is the absorptancy change, in per cent, Qorresponding to a tenfold change in concentration (1). The p-bromoaniline method has been extensively employed in this laboratory for over a year for the analysis of furfural derived from numerous sources. It has proved superior to any other method tried by the author. LITERATURE CITED
(1) Syres, G.H., $NAL. CHEW., 21,652 (1949). (2) Reeves, R. E., and Munro, J., IND.ENQ.CHEM.,ANAL.ED.,12, 551 (1940). (3) Rice, E.W., and Roe, J. H., J . Bid. Chem.. 188,463 (1951). (4) Ringbom, A., Z.anal. Chem., 115,332(1939). (5) Roe, J. H., and Rice, E. W., J . Biol. Chem., 173,507 (1948). January 16, 1951. Work aided, in part, by an Institutional Grant. American Cancer Society, Inc.
RECEIVED
Semimicroanalysis of Organoselenium Compounds EDWIN S. GOULD Brooklyn Polytechnic Institute, Brooklyn, N . Y .
the flame combustion method for analysis of orALTHOUGH ganoselenium compounds described by McCullough, Camp-
cases, the sudden formation of a relatively volatile selenide often resulted in too rapid combustion: In attempts to devise a method for the analysis of organoselenium compounds which was rapid, required only small samples, was applicable to compounds containing mercury or bromine or both, and required no extra precautions in analyzing compounds
bell, and Krilanovich ( 3 ) gives highly accurate results when applied to compounds of a number of different types, it is inapplicable to compounds containing bromine, mercury, or sulfur in addition to selenium. For best results with the combustion method, a sample containing 20 to 80 mg. of selenium is desirable, a complete analysis requiring from 60 to 90 minutes in the hands of a skilled operator. It was found that compounds containing a seleTable I. Analysis of Organoselenium Compounds by the niuni-oxygen linkage often burn less smoothly Digestion Method than selenides, diselenides, and selenocyanates. Selenium, % Deviation, Most benzeneseleninic acids and their derivatives Compound Formula, Theory Analysis % decomposed, leaving a nonvolatile residue containDi-p-tolylselenium CidHirSe 30.3 30.1 -0.7 p,p’-Dimethoxydiphenylselenium ClrHirOtSe 26.9 26.9 0.0 ing selenium in t’he burner. Such residues re24.6 24.5 -0.4 ~,p’-Diethoxydiphenylselenium CieHisOzSe mained, even at the hottest telnperature obtainDibenzoselenophene ClzHsSe 34.1 34.1 0.0 CsH7NSe 40.6 40.3 -0.7 able by the heater, and combustion could be com~;$$~~,~~~~enzene ClHdNClSe 36.4 36.5 0.3 p,p’-Dimethoxydiphenylselenoxide ClaHiaOsSe 25.5 25.5 0.0 pleted only by heating the outside of the burner 23.4 23.4 0 0 P p’-Diethoxydiphenylselenoxide CieHisOaSe with the flame from a nlicroburner until ignition Dibensoselenophene oxide CiaHsOSe 31.9 31.7 -0.6 p p’-Dimethoxydiphenylselenone CiaHidOaSe 24.3 24.1 -0.8 of the residue in the stream of oxygen occurred. p:p-Diethoxydiphenylselenone C16HlsOPSe 22.5 22.4 -0.4 p-Tolueneseleninio acid CiHsOzSe 38.9 38.7 -0.5 It was essential that the latter operation be carried m-Bromobenzeneseleninic acid CeHaOzBrSe 29.4 29.2 -0.7 out with great care, for too rapid heating resulted m-Nitrobeneeneseleninic acid CeHsOaNSe 33.7 33.5 -0.6 ClrHloSez 50.6 50.3 -0.6 in a semiexplosive cornbustion or decomposition ~-’,,;$$~~$d;iselenide CzrHisSez 34.3 34.0 -0.9 p,p-Diethoxydiphenylselenium of the residue and the escape of some of the seleClsHiaO~ClzSe 20.1 20.0 -0.5 dichloride niuin dioride. If the residue exploded without Diphenylselenium &bromide CnHioBrzSe 20.1 20.0 -0.6 complete combustion, elementary selenium and ~-(o-BiphenylylsBleno) acid propionic Cd’hcOtSe 25.9 25.5 -1.6 sizable amounts of soot were carried into the abp,p’-Dimethoxydiphenyl selenoxide-mercury(I1) chloride CiaHlaOsSeHgClz 1 3 . 6 13.5 -0.7 sorption tube, necessitating a thorough rinsing of p,p,-Diethoxydiphenyl selenoxidemercury(I1) bromide Ci6HisOsSeHgBrz 1 1 . 3 11.3 0.0 the apparatus with chromic acid-sulfuric acid Dibenzoselenophene oxidesolution. Many selenoxides decompose to the mercury(I1) bromide CuHeOSeHgBrz 13.1 13.0 -0.8 parent selenides at, their melting points; in such
