Colorimetric Oetermination of Inorganic Phosphate in Microgram Quantities MORRIS ROCKSTEIN AND P.IUL W.H E K K O S State College of Washington, Pcdlman, Wash. H E determination of phosphate ion is an important and widely Tused procedure in analytical and biological chemical studies. However, most commonly employed methods have distinct Iiniitations, especially as regards the reducing agent employed. In a n attempt to find a technique for determining yuantitatively the release of phosphate from organic phosphate, the authors found that a little mentioned and rarely used modification of the Bell and Doisy ( 1 ) method, originated by Sumner ( 7 ) ,\T:E adaptable to measurement of phosphorus as phosphate in mic.i,ogram quantities. Sumner’s use of ferrous sulfate as a reducaing agent is superior to the aminonaphtholsulfonic acid method of Fiske and Subbarow ( 3 ) and the rtannous chloride method of Kuttner, Cohen, and Lichtenstein ( 5 , 6) for several significant reasons. As Sumner suggested, a color end point is reachrd within a few seconds and the intensity of the color remains unchanged after more than 2 hours’ standing a t room temperaturt.. T h e reducing agent is easily and rapidly prepared, and problems of storage are eliminated by its fresh preparation for cacBh set of’ determinations. Sumner also reported that with this method. employing considerably less acid than the method of Kuttner el al., i,:ipid hydrolysis of labile phosphate esters does not occur, nor does trichloroacetic acid interfere with the color reaction (in rontrast to the stannous chloride method; cf. Bodansky, 9).
Table I. Absorbance at 720 nifi and Phosphorus Concentration (Mean room teniperature 28.0“ C . ) Standard Deviation, Mean ZDX Absorbance. Optical Density n-1 0,010 10.0028 0.024 10.0026 0.036 10.0061 0,047 f0.0024 0,072 i-0,0037 0,097 f0.0064 0.136 f0 ,0040 0.189 &O. 0163
Final Concentration, Y/ML 0.1 0.2 0.3 0.4 0.6 0.8 1.2 1.6
1.4036 grams of Baker’s C.P. potasium dihydrogen phosphate, previously dried over concentrated sulfuric acid in a desiccator for a t least 24 hours, were dissolved and made u p t o the 1000-ml. mark in volumetric flask with distilled water. One milliliter contains 0.320 mg. of phosphorus. COLORIVIETRIC PROCEDURE
To a 0.2-nil. sample of standard phosphate (or unknown) solution in a IO-ml. volumetric flask were added 9 ml. of acid molybdate followed by 0.8 ml. of freshly prepared ferrous sulfate solution. A similar “blank” preparation for 100% transmittance (zero optical density) setting on the spectrophotometer wa8 made, using 0.2 ml. of distilled water plus acid molybdate and feirous sulfate solutions as in the standard or unknown preparations, and not pure distilled water alone. The pH of the solution after color development is 0.75: final concentration of sulfuric acid in this solution is 0.76 S. H ESU LTS
are shown in Figure 1 for the several conrentrations of standard phosphate solutions, prepared by dilution of stock solution, espresetl in micrograms per milliliter of final reaction solution. From thew data, i t is obvious that the optimum wave length for this colorimetric determination for the spectrophotometer employed lies in the vicinity of 720 niw, the region of the maximum absorbance \vith the Irast devintion in absorhm c e for slight variations in wave length. Table I and Figure 2 demonst,rate the reproducibility and sensitivity of this reaction as determined with si.; replications for each point. The curve in Figure 2, drawn by the method of least squares, indicates that Beer’s Ian. is valid a t 720 mp for the rolor
This study was undertaken to determine the agreement witli Beer’s law by this method a t microgram lwrls of phosphorus :i$ inorganic phosphate.
.I 5
MATERIALS AND METHODS W 0
Spectrophotometer. T h e Model B Beckman instruinerit \vas used with sets of four matched 10-mm. square plastics cells, having transmittance characteristics similar to those of Corex cells. Determinations were made a t room temperatuw (28’) :it a wave length of 720 mp. Reagents (7). Stock sulfuric acid, 7.5 N . Stork niol?t)date solution, 6.6%, prepared from Baker’s C . P . fine crystal reagent ammonium molybdate tetrahydrate ( phosphute content 0.000370). This is stable for at least G monthr at i’ooni teniperature. Acid Molybdate Solution. To 200 nil. of solution contaiiiiiig 25 nil. of 6.670 stock molybdate solution were s l o ~ ~ added ly 25 ml. of 7.5 N sulfuric arid. Thip is also stable indefinitely at i’ooni temperature. Ferrous sulfate solution, pIep:irc~d irnmedi:~trl>.bcfo mination. T o a 50-nil. volumcltric flask were a d d d 5 Baker’s C.P. special ferrous ~ u l f a t e heptahydrate ( phosphate), followed by 1 ml. of 7.5 S sulfuric acid, and mark with distilled water. Because of the tendency to m i d y oxidation, ferrous sulfate solutions should be prepared fresh every 2 hours, according to Sumner ( 7 ) . For determination of sets of as many as 16 different Pamples, each set taking 20 minutes, the solution, with ferrous sulfate in great excess, was found to lose no effectiveness as a reducing agent after a n hour a t roo111 temperature, for the concentrations of phosphorus studied. St:iii(lard Phosphate Stock Solution. I n distilled witer
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550
600
650
700
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W A V E L E N G T H mp, Figure 1. Absorption Spectra for Several Concentrations of Phosphorus
1500
V O L U M E 2 3 , N O . 10, O C T O B E R 1 9 5 1
1501
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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 e m p l o ~ . 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.,
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.05
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0.50
0.75
MICROGRAMS
Figure 2.
1.00
1.25
IS0
PER ML.
Phosphorus C o n c e n t r a t i o n 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 s o n ~ 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 solutio11 of 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
