ANALYTICAL EDITION
January 15, 1942
77
Literature Cited
Kolthoff, I. M., and Lingane, J. J., “Polarography”, New York, Interscience Publishers, 1941. Mackey, E., and Butler, A. M., in Peters and Van Siyke’s “Quantitative Clinical Chemistry”, Vol. 11, p. 859, Baltimore, Williams and Wilkins Co., 1932. Stern, Adolph, and Macy, I. G., “Application of Polarographic Microdetermination t o Foods, Urine, and Feces”, presented before Division of Biological Chemistry at 100th Meeting of AMERICAN CH~MICAL SOCIETY, Detroit, Mich. Thanheiser, G., and Willems, T., Arch. Eisenh.tlttenw., 13, 73 (1939). (6) Uhl, F. A., 2.anal. Chem., 110,102 (1937).
(1) Hohn, H., “Chemische Analysen mit dem Polarographen”,p. 18, Berlin, Julius Springer, 1937.
PREBENTED before the Division of Micro Chemistry a t the 100th Meeting of the AKERICANCHEMICAL SOCIETY,Detroit, Mich.
monium phosphomolybdate. The method requires small amounts of material, effects a great saving of time in routine analyses, and eliminates some of the sources of errors involved in the chemical method. The method may be applied to the determination of phosphorus in organic material as well as plant ash. Illustrative results of determinations upon foods, urine, and feces by both polarographic and chemical methods are presented.
Spectrophotometric Determination
of Phosphorus T. D. FONTAINE’, Mellon Institute, Pittsburgh, Penna.
A
Calibration data are set forth graphically in Figure 2. Beer’s law applies for concentrations up to 1 microgram of phosphorus per ml. of the colored solution. The percentage transmittance was measured against blanks and the extrapolation to zero concentration of phosphorus gave a value of 98 per cent. The results obtained when the final concentration of sulfuric acid was varied from 1.7 to 2.4 AT,using a constant quantity of the other reagents, are given in Table I. Blanks in this acid range developed a faint yellow color which did not interfere with the determination. I n acid concentrations less than 1.7 AT, the blue color appeared in the blank and erratic results were obtained. It is evident that the error involved in determinations in which the acid concentration might vary from 1.7 to 2.1 N would be less than 2 per cent. The amount of stannous chloride reagent may be doubled without any change in the transmittance of the standard at 820 mp, although there is a slight increase in the yellow color of the blank. A study of the stability of the color developed under the conditions finally adopted showed no variation in intensity over a period of 24 hours if the colored solutions were kept
XUhlBER of investigators have described colorimetric
methods for the determination of phosphorus based upon the reduction of molybdic acid. As far as can be determined, however, no complete spectral data on the characteristics of the blue color are available. I n the course of investigations on cottonseed phospholipids and proteins, information of this nature has been obtained and an improved method for the determination of phosphorus has been devised. In a study of the reaction mechanisms involved in the quantitative determination of phosphorus, Berenblum and Chain ( 2 ) demonstrated that the phosphate ion acted as a catalyst for the reduction of molybdic acid by stannous chloride; they concluded that optimal results for phosphorus could be obtained only within a very narrow range of acid concentration (1.1 N ) . An examination of their graph reveals that a fairly constant intensity of color is recorded also in the acid concentration region of 1.8 to 2.0 N , thereby suggesting a method wherein final concentration of acid does not require careful adjustment. Inasmuch as high acid concentrations inhibit the rate of the reduction of molybdic acid, it was found necessary to develop the color by heating, as recommended by Benedict and Theis (1) for the reduction by hydroquinone, and by Horecker, Ma, and Haas (4) for the reduction by 1,2,4-aminonaahtholsulfonic acid.
Experimental A Coleman spectrophotometer (model 10sDM, 7.5 mp slit) was used to measure the transmittance of the solutions. The spectral curves, from 400 t o 950 m9, for three concentrations of phosphorus in 2 N sulfuric acid are shown in Figure 1. It is apparent that a definite minimum transmittance occurs at 820 mp. Dyer and Wrenshall (3) recorded a minimum at 660 mp (photoelectric colorimeter with filters), although it was later reported that the stannous chloride which had been used was very impure ( 7 ) . McCune and Weech (6) showed that up t o 750 mp a minimum was not reached (Hardy recording spectrophotometer), which is in line with the results presented in the figure. The nature of the spectral curve indicates that, if filters are used, they should transmit light in the red region of the spectrum, so that maximum sensitivity and minimum interference are obtained. 1 Present address, Southern Regional Research Laboratory, Kew Orleans, La.
?S!O
4dO
4b
5bO
550
6bO 660 7 k O 7:O 8bO WAVE L E N G T H - M I L L I M I C R O N S
8:O
9bO
9bO
IJOO
FIGURE1. TRANSMITTANCE-WAVE LENGTHCURVESFOR THREECONCENTRATIONS OF PHOSPHORUS
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 1
heat the mixture slowly, with shaking, in order to get maximum clarification. Repeat such treatment until the solution becomes clear, after which add water to the cooled digest and again heat the solution to boiling in order to convert the pyrophosphate to orthophosphate. Dilute the digest and measure an aliquot into a 25-ml. volumetric flask. Add sulfuric acid (10 N) in quantity sufEcient to make the final concentration approximately 2 N . The remainder of the procedure has been described above. Compare the final color against a blank treated in exactly the same manner, but without added phosphorus. It is evident that, depending upon the size of sample and amount of phosphorus contained, the quantity of sulfuric acid used for digestion may be varied, because the adjustment to the desired final concentration is made later.
Advantages The method described appears t o have the following advantages: The reducing agent, stannous chloride, is cheaper and more readily available than the organic reducing agents that have been suggested. The amount of stannous chloride used can be varied without affecting the intensity of the developed color. It is not necessary t o make an accurate adjustment of the final concentration of acid, which may vary from 1.7 to 2.1 N . The developed color is stable for at least 24 hours if the solution is kept in a closed flask. Greatest accuracy is attained by measuring the percentage transmittance at 820 mp. The difficulties observed by Willard and Center (8) in the determination of phosphorus-in steels by the stannous chloride method might possibly be eliminated by the modifications here described. TABLEI. P H O S P H O R U S , pQ.
POI
ACID CONCENTRATION COLORINTENSITY
INFLUENCE OF VARYINQ
ml
FIGURE 2. CONCENTRATION OF PHOSPHORUS us. TRANSMITTANCI
Concentration of
Reagents Sodium molybdate (phosphate-free), Dissolve 75 grams in 1 liter of distilled water. Stannous chloride, Dissolve 10 grams of the c. P. salt in 25 ml. of concentrated hydrochloric acid and store in a glassmtoppered brown bottle. Kuttner and Lichtenstein (6) suggest that the solution should not be used longer than 4 weeks. Dilute 1 ml. of the stock solution to 200 ml. with water for each set of determinations. Standard phosphate solution. Dissolve 0.4389 gram of anhydrous potassium dihydrogen phosphate in 1 liter of distilled water (6). This solution (0.1 mg. of phosphorus per ml.) may be diluted to give the required concentrations.
Recommended Procedure STANDARDIZATION. To known quantities of phosphate solution (2 to 24 micrograms of phosphorus) in a 25-ml. glass-stop pered volumetric flask add 5.0 ml. of 10 N sulfuric acid, 2.5 ml. of 7.5 per cent sodium molybdate solution, and sueicient water to bring the volume to approximately 22 ml. It is necessary to shake the mixture a t this point in order to avoid the appearance of a premature blue color upon the addition of the reducing agent. Then add 2.5 ml. of stannous chloride and again mix the contents of the flask. Place the loosely stoppered flask in boiling water for 20 minutes to develop the color, cool to room temperature, and adjust the volume to 25 ml. Transfer approximately 5 ml. of this solution to the cuvette of the spectrophotometer and measure the percentage transmittance a t 820 mp, against a blank receiving identical treatment. The calibration curve needs to be determined only once. UNKNOWN.To 15 to 20 m . of phospholipid or 30 to 40 mg. of protein, in a micro-Kjeldahf flask, add 10 ml. of 10 N sulfuric acid. Heat the contents of the flask over an open flame until the water is removed and the sample is charred. After cooling, add a drop of 30 per cent hydrogen peroxide (c. P.) and again
Transmittance at 820 m p
Sulfuric Acid
N
%
1.7
49.8 49.6 49.5 49.4 49.a 48.8 47.6
i.8 1.9 2.0 2.1
in closed flasks. A maximum decrease in color of about 2 per cent occurred if the solution was allowed to stand in the cuvette of the spectrophotometer for 1 hour.
ON
2.2 2.4
' 0.32 microgram of phosphorua per ml. Summary
The spectral curve for the blue color which is obtained from molybdic acid by reducing agents in the presence of phosphorus, and which is used for the quantitative determination of phosphorus, is described. Minimum transmittance occurs at 820 mp. An improved micromethod, utilizing several procedures developed by previous investigators, is presented.
Acknowledgment The author is indebted t o H. S. Olcott for proposing the problem and for guidance in preparing the manuscript.
Literature Cited (I) Benedict, S. R., and Theis, R. C., J . Biol. C h m . , 61,63 (1924). (2) Berenblum, I.,and Chain, E., Biochem. J.,32,286,295 (1938). (3) Dyer, W. J., and Wrenshall, C. L., Can. J . Reaulrch, 16B, 97 (1938). (4) Horecker, B. L., Ma, T. S., and Haas, E., J. Biol. Chem., 136,
775 (1940). (6) Kuttner, T., and Liohtenstein, L., I W . , 86,671 (1930). (13) . . McCune, D.J., and Weech, A. A., Proc. SOC.Ezpptl. Biol. Med., 45, 659 (1940). (7) Smith, G. R., Dyer, W. J., Wrenshall, C. L., and De Long, W. A., Can. J. Rcsearch, 17B, 178 (1939). (.8,) Willard. IS. H.. and Center, E. J., IND.ENG.Cxnx., ANAL.ED., 13,81 (1941). CONTRIBUTION from the Multiple Fellowship of the Cotton Research Foundstion at hlellon Institute.
