ANALYTICAL EDITION
February 15, 1942
fore the next sample, and so on, without introducing an appreciable error. The benzaldehyde is dropped into a 75-ml. Erlenmeyer-shaped glass-stoppered flask containing 30 ml. of neutralized hydroxylammonium sulfate solution, and the flask is gently swirled. Samples lower than 90 per cent benzaldehyde usually go into solution with difficulty.
Acknowledgment The authors gratefully acknowledge the cooperation of the Commercial Solvents Corporation in furnishing samples of hydroxy~ammon~um sulfate,
Literature Cited
Titration Standard 0.1 N sodium hydroxide is run rapidly into the solution until within about 2 to 3 ml of the end point, then a few drops a t a time until the color is gre&-blue, and finally dropn7iseuntil the color is again the pale blue. The absence of direct sunlight is desirable. This part of the assay is almost exclusively responsible for any error. The results obtained will be more truly representative if the samples are kept in glass-stoppered Pyrex bottles whose lips can be thoroughly cleaned. Xl. of 0.1000 N SaOH X 0.0106 weight of >ample
155
loo = % benzaldehyde
OfficialAm. Chem., Official and Tentative Methods Analysis, 4th ed., p. 314, 1935.
(1) AssoC.
Of
(2) Cambier, and Brochet, A., Compt. rend.? l20, 449 (1895). (3) Donnab’, L. H., IBD.ENG. CHEM*?ANAL. ED., 5 ? 91-2 (l933). (4) Givaudanianl p‘
(5) Kolthoff, I. M., and Furman, N. H., “Volumetric rlnalysis”, 1st ed., Vol. 11, pp. 450-2, New York, John Wiley & Sons, 1929. (6) Kolthoff, I. M., and Rosenblum, C., “Acid-Base Indicators”, 1st English ed. (tr. from 4th German ed.). D. 123. New York. Macmalan Co., 1937. (7) Neubere. C.. and Gottschalk. A.. Biochem Z . . 146. 167 11924). (8) Ripper,YTfonatsh., 22. 1079 (1900): (9) U. S. Pharmacopoeia XI, p. 247 (1936).
Colorimetric Determination of Phosphorus in Biological Materials RUTH ADELE KOESIG AKD C. R. JOHNSON, University of Texas, Austin, Texas
ISSOS’S ( 3 ) method for the estimation of phosphorus has recently been adapt’ed to the determination of this element in iron, steel, and iron ores (4, 6 ) . During use of the method in food analyses in this laboratory, the fact became evident t’hat its range, sensitivity, and precision could be increased considerably in this application, where limiting interferences are rare. For this purpose the effect of time on the development of the yellow phosphovanadiomolybdate at various concentrations has been studied, the optimum acid concentration has been found, the range over which Beer’s law holds precisely has been determined at three wave lengths of light, and spectrophotometric calibration data have been obtained with nine series of standard solutions. The results of these experiments have been condensed in a convenient procedure for the rapid and precise determination of phosphorus in foods and other biological materials, and this procedure has been tested by comparison with official gravimetric methods.
Apparatus and Reagents
,
A Coleman Model 10-S spectrophotometer was used to measure transmittance values. The spectral band width was 30 mp. Matched square cuvettes were used to hold the reference liquids and test solutions. The dark current adjustment was frequently checked during the measurements, which were made a t temperatures between 25” and 30’ C. Two independently prepared sets of reagents were used. 15 N nitric acid, 6 N hydrochloric acid, 9 N perchloric acid, and 4 N aqua ammonia were purified by distillation methods. Standard potassium dihydrogen phosphate solution containing 0.1000 mg. of phosphorus per ml. was made by dissolving 0.4393 gram of twice recrystallized and vacuum-dried material in distilled water and making 1.0 1 liter. Another standard solution containing 0.1000 mg. of phosphorus per ml. was made by dissolving 1.351 grams of silver phosphate (8)in 5 ml. of 15 N nitric acid and diluting to 1 liter. Ammonium vanadate solutions were made by dissolving 2.346 grams of vacuum-desiccated ammonium metavanadate in 500 ml. of hot water, adding 10 ml. of 15 N nitric acid, and diluting to 1 liter. One sample of this salt was prepared by double recrystallization of reagent grade material with centrifugal draining; the other was a sample of atomic weight purity. One ammonium molybdate solution was prepared as described by Willard and Center ( 5 ) , using vacuum-dried molybdic acid purified by precipitation with 6 N hydrochloric acid, followed by two crystallizations from water, with centrifugal draining. A second solution of this reagent was a 10 per cent solution of ammonium molybdate shown to be free of phosphate
and chloride by blank tests; this was used with the systems containing silver. Fresh molybdate solutions were prepared frequently, as required.
Summary of Calibration Experiments I n obtaining calibration data, opportunity was taken to study the effect of various factors on the precision and accuracy of the method. Thus, while test solutions for the calibr$ions mere made by the same method described below for the analyses, standard phosphate solutions were used instead of digested samples, various concentrations and mixtures of nitric and perchloric acid were tried, and each system was kept under observation for a much longer time. Different combinations of solutions from the two sets of reagents were used, to eliminate constant errors. I n Table I are given the median transmittances from about 1200 calibration observations made approximately 30 minutes after color development. Mathematical treatment of the data as a whole showed that the best agreement with the medians in Table I for all phosphate concentrations studied was obtained with systems containing 5 ml. of 9 N perchloric or 3 ml. of 15 LV nitric acid per 100 ml. Delayed precipitation took place in all standards and blanks which contained only 2 ml. of 9 -Irperchloric acid per 100 ml., in the standards containing 10 ml. of 9 A‘ perchloric acid and 5 mg. of phosphorus per 100 ml., and in some corresponding solutions containing equivalent amounts of nitric acid, but not in systems with intermediate acid concentrations. I n such systems transmittance readings were sufficiently constant after 10 to 30 minutes for precise measurements: transmittances were almost invariably lower after 12 t o 24 hours, but rarely more than 1.0 unit lower. The minimum drop in transmittance occurred in systems containing 5 ml. of 9 N perchloric acid per 100 ml. On two counts, therefore, this is the optimum acid concentration. However, neither time nor acid concentration is extremely critical: transmittance readings taken from 10 minutes to many hours after making up the test solutions to 100 ml. with 2 ml. to 10 ml. of 9 N perchloric acid or equivalent amounts of nitric acid or nitric-perchloric acid mixtures give approximately correct results, even when precipitation of molybdic acid or the yellow complex is eventually to occur.
156
INDUSTRIAL AND ENGINEERING CHEMISTRY
N o significant differences due to interchange of reagents were observed during the calibrations. The order of addition of vanadate and molybdate should not be reversed. Readings taken with water as the reference liquid were a little more reproducible than those made with blanks, but water should be used as the reference liquid only when all the developing reagents are free from phosphate and the solutions to be analyzed are colorless.
TABLE I. Phosphorus Mg.li00 ml. 0.200 0.500
1.000
1.800 2.50 3.50 5.00
CONCENTRATION-TR.4NSMITTANCE DATAFOR PHORUS DETERMINATIONS
PXOS-
-Transmittance a t Various Wave Lengths3 Blank Reference Water Reference 430 mp 450 mp 470 mp 430 m p 450 mr 470 m p
%
%
%
%
%
%
77.1 53.0 28.6 10.7 [5.1]
84.0 66.0 44.2 23.5 13.5 [6.6]
88.6 76.0 58.7 38.8 27.2 16.4 [S.21
72.9 50.4 27.1 10.1 [4.8]
81.6 64.3 43.0 22.8 13.1 [6.3]
87.6 75.2 57.8 38.2 26.6 16.1
... ...
...
......
...
[8.l]
Graphical representation of the data in Table I shows that Beer’s law is followed very closely except for the bracketed values, which were not used in the final reduction of results. The 4 per cent deviation of these values is probably due to a n instrumental error; it is undesirable to use transmittances lower than 10 per cent in precise analyses.
Analytical Procedure The following procedure is designed for the determination of phosphorus in aliquot portions of the solution obtained by ashing a 10-gram sample of material by successive treatment with 60 ml. (and, if necessary, additional 30-ml. portions) of 15 N nitric acid and 24 ml. of 9 N perchloric acid, according to the method of Gerrite (1). When ashing is complete, evaporate the soluPROCEDURE. tion to about 50 ml., filter through a Munroe or Pyrex filtering crucible t o remove silica, dilute, boil for at least 30 minutes, cool, and make up to exactly 250 ml. Most of this solution may be reserved €or the determination of calcium and iron. To analyze a colorless solution for phosphate pipet a 10.00ml. or other suitable aliquot portion into a 100-ml. volumetric flask, add 5 ml. of 9 N perchloric acid or 3 ml. of 15 N nitric acid, and bring the volume t o 70 ml. Add 10.0 ml. of ammonium vanadate solution, gradually mix in 10.0 ml. of ammonium molybdate solution, and dilute to exactly 100 ml. After 30 minutes make duplicate transmittance readings with two portions of the solutions, at the optimum wave length. If the reagents are free from phosphate use water as a reference liquid; otherwise use a blank containing the reagents. Calculate the result of the analysis from the median transmittance by appropriate substitution in Equation l below, or by use of a graph derived from this equation. In analyzing colored solutions-e. g., the light yellow or yellowgreen solutions derived from many foods-use as a reference liquid a system containing the same aliquot portion of colored liquid as the test solution, with’all the reagents except the ammonium molybdate, which in this case must be free from phosphate. For convenience in calculating results with maximum precision the data in Table I have been reduced by the method of least squares to equations of the form: Mg. of P per 100 ml. of test solution
=
a
loglo T 1
+b
(1)
in which T is the percentage transmittance relative to the reference liquid, and I is the thickness of the solution in centimeters. Values of the constants a and b for the various conditions studied are given in Table 11. In the present calibrations I was 1.308 cm., and the data in Table I1 apply to cases in which matched square cuvettes of about this size are used ( 4 ) . The actual value of I should in any case be determined with a micrometer and calipers. Under the conditions specified, Equation 1 yields results correct to between
Vol. 14, No. 2
0.4 and 2 per cent over the range from 0.2 to 4.5 mg. of phosphorus per 100 ml. of final test solution.
Applications and Tests of Method The procedure described above has been used in the analysis of some 80 different materials, including vegetables, meats, flour, milk, eggs, fruits, grasses, berries, vinegar, yeast, gelatin, agar, condiments, urine, feces, baking powders, wine, beer, fertilizers, oils, pharmaceuticals, chemical reagents, and about 40 food concentrates. I n view of the small mineral content of most of these substances, few interferences were expected; the fact that results obtained a t all three wave lengths were generally in good agreement and independent of aliquot size may be taken as evidence of the absence of serious interferences. Of course, one wave length usually provides a result of maximum precision, depending on the size and phosphate content of the aliquot. The optimum conditions may be selected by the analyst, who may use the lowest wave length to obtain sensitivity, the highest to obtain range, or more than one for confirmatory purposes. TABLE11. CONST.4NTS
CONCENTRATION-TRANSXITTAXCE
FOR
EQUATIOXS
Wave Length MI 430 450 470 430 450 470
Blank reference Water reference
a
b
-2.442 -3.789 -5.89 -2.437 -3.783 -5.85
4.867 7.55 11.72 4.802 7.49 11.62
I n addition to the various checks on the procedure provided by the use of two primary standard materials in the calibrations, an additional check was made by comparison of the method with A. 0. A. C. official gravimetric methods, for representative materials. The results in Table I11 are typical, and further confirm the soundness of the method. The precision and accuracy of the spectrophotometric analyses are satisfactory, considering that they require only a fraction of the time needed for the gravimetric determinations. TABLE111. SUMYARY OF TYPICAL RESULTS Materiala
Spectrophotometric Method 430 mp 450 mp 470 mp
Mg. P per
0 5 c i a l Method
10 grams of material
Apple vinegar 0.430 0.436 0.432 ... Dried grass 1 7.3 7 . 0 (volumetric) Dried grass 2 18.2 18.2 17.6 17.9 Kelp concentrate 25.7 26.1 26.3 27.8 Tomato concentrate 27.7 28.1 28.5 28.3 Milk powder 70.3 70.0 70.2 Spinach concentrate 72.5 70.8 70.6 Liver concentrate 189 195 193 193 Baking powder 1 303 309 310 303 Baking powder 2 324 320 318 314 Baking powder 3 841 839 834 828 Fertilizer 560 550 544 520 (not official) FeP0~2H20 (1 g. per liter) 1646 1650 1652 [I6581 (theoretical) a All samples were dried before analysis by 12 to 21 hours of vacuum desiocation, except vinegar and ferric phosphate, which also were not ashed.
...
...
.... ..
The authors are indebted to the Division of Chemistry of the Texas Agricultural Experiment Station for analyzed grass samples, to California Vegetable Concentrates, Inc., for the food concentrates, and to W. C. Craig for many gravimetric analyses.
Literature Cited (1) Gerritz, H.W.,IND. ENC).CHEM., ANAL.ED.,7, 167 (1935). (2) Johnson, C.R.,and Nunn, L.G., Jr., J . Chena. Education, 17, 628 (1940). (3) Misson, G.,Chem.-Ztg., 32,633 (1908). (4) Murray, W. M., Jr., and Ashley, S. E. Q . , IND.ENG.CHEY., ANAL.ED.,10,1 (1938). (5) Willard, H. H., and Center, E. J., Ibid., 13,81 (1941).
