Indirect Polarographic Determination of Phosphorus in Biological Material ADOLPH STERN Research Laboratory, Children's Fund of Michigan, Detroit, Mich.
P
ment was situated in a room, the temperature of which was very constant. When the temperature changed more than 0.5' C. a new calibration was performed immediately. Thus, in the course of the analyses calibration curves were obtained for various temperatures (range, 28.0" * 3" C. within one year). These curves could be used whenever the room temperature corresponded to the temperature at which a calibration had been made. Use of a thermostat thus can be avoided for practical analysis. Leybold's Handwriter ( I ) , which may be attached to the polarograph, was used for recording the current voltage curves. This device is useful in routine analysis and its recording of the current voltage curves is as accurate as the photographic method. With an adequate electrode and a galvanometer sensitivity as low as ~ / I W O ,oscillations are not noticeable. When higher sensitivities are employed! galvanometer oscillations can be practically eliminated by slight changes in the concentration of the colloid, the neutral salt concentration of the regulating solution, or the rate of droplet emission from the cathode. In routine analysis, it is advisable to check the calibration curve every day, before starting series of analyses, by determining the concentration of a known standard solution. The diffusion current was determined by measuring the wave heights of the current voltage curves, with the intersection points method frequently used in practical polarography (1). The molybdate waves are very well shaped and the correction for the residual current by this method is sufficiently accurate.
OLAROGRAPHIC methods of analysis possess attributes
which extend their value far beyond the direct determination of chemical elements in various materials. Especially in routine laboratory procedure, polarographic methods in combination with chemical methods result in shorter time requirements for analysis and the reduction of errors, owing t o the elimination of filtering, drying, and weighing procedures. In addition to developing polarographic methods for direct determination of mineral elements in biological material (4), a n indirect method for the determination of phosphate ions in biological material has been devised. Phosphate ions are not electroreducible a t the dropping mercury cathode. Uhl (6) has described a n indirect polarographic method for the determination of phosphate ions in pure solutions by precipitating the ions wit,h ammonium heptamolybdate. The resulting ammonium phosphomolybdate precipitate is of definite composition. Addition of identical quantities of a standard molybdate solution to solutions of different phosphate-ion concentrations reduces the concentration of molybdate in the supernatant liquid by the amount of molybdate combined with the phosphate ions precipitated. By polarographic determination of the molybdate ions in the standard solution and the molybdate ions remaining after the precipitation, it is possible to measure, indirectly, the phosphate ions precipitated. It was also proved by Uhl that small amounts of iron, aluminum, calcium, and magnesium added to the pure solutions do not interfere.
Determination of Molybdate Ions in Pure Solutions Nolybdate ions are electroreducible at the dropping mercury electrode and give a "polarographic wave". The wave appears in neutral and acid solutions, whereas in strongly alkaline solutions no wave can be observed, as Uhl (6) and Thanheiser and Rillems (6) stated. Thanheiser and Willems found that the molybdate waves in neutral and acid solutions are difficult to measure and they preferred, therefore, the determination of molybdate in steel by polarometric titration with lead. Uhl found that the height of the characteristic wave of molybdate ions in acid solution is proportional to the concentration only in the presence of free lactic acid and worked out a special procedure to adjust the acidity of the solutions so that the wave heights were proportional to the concentrations. In developing the indirect method for the determination of phosphate ions in biological materials, the most simple procedure for the determination of molybdate ions in pure solutions was the primary requirement. Investigation of the behavior, under different conditions, of the polarographic wave
Measureinents The current volta e curves were registered with a Leyboldtype Heyrovsky-Shifata polarograph (1, 2 ) , equipped with a ampere per mm. per highly sensitive galvanometer (2.4 X meter). The adjustment of the instrument was kept constant for all determinations. The wire resistance coil of the potentiometer --as connected with a storage battery of 4 volts. Solutions to be tested were placed in 25-ml. glass cups above a layer of mercury 4 mm. in depth, which served as the anode. The mercury reservoir of the cathode was maintained within 10.1 cm. of 80.0 cm. Emission of mercury droplets from the cathode, into the regulating solution used for the phosphate determination, was 5 drops per 10 seconds, with the dropping mercury cathode disconnect,ed from the polarizing e. ni. f. This drop rat'e was kept constant, Biter the determinations the capillary was allowed to release mercury droplets in air for about 15 minutes, then dipped into mercury and the connection to the mercury reservoir closed. With a satisfactory electrode it is possible to perform rout,ine analysis for months without difficulty. The det,erminat'ions were performed a t 26.0" C. The instru74
ANALYTICAL EDITION
January 15, 1942
due to molybdate ions showed that in a 3 N nitric acid solution containing 2 N ammonium nitrate the waves have a rounded maximum which makes quantitative determinations impossible. This maximum cannot be suppressed with gelatin because the rather high acidity of the solution causes precipitation of the colloid. In neutral solutions of 2 N ammonium nitrate containing some gelatin or methylcellulose, however, the molybdate wave shows the regular steplike form which may be used for quantitative determinations, the diffusion current being proportional to the concentration (Figure 1). The half-wave potential of the molybdate wave in 2 N ammonium nitrate solution containing 3 grams of methylcellulose per liter is -0.82 volt vs. the normal calomel electrode. The space requirement of the wave is rather large. Best results were obtained with a regulating solution which contained 3 grams of methylcellulose (or 1.0 gram of gelatin) in 1 liter of 2 N ammonium nitrate solution. By determining the wave heights for different molybdate concentrations and plotting them against the concentrations, a calibration curve is obtained which permits reading for unknown concentrations of molybdate solutions. Thus, molybdate ions can be determined within a concentration range of lo-' to 10-6 gram per milliliter with a n accuracy of *2 per cent, by using a suitable galvanometer sensitivity. Oxygen must be removed from the solution with nitrogen. The wave heights of molybdate ions in nitric acid solutions do not show a maximum if the solution contains a high amount of ammonium nitrate and, within certain limits, are independent of the acid concentration. Molybdate ions can be determined in nitric acid solutions within the same range of concentration with a n accuracy of *2 per cent if the solution contains 400 grams or more of ammonium nitrate per liter. The fact t h a t molybdate ions can be determined in acid solutions in the presence of a large excess of ammonium nitrate makes it possible to determine phosphate ions directly, without special adjustment of the p H or addition of free lactic acid. Uhl described a second wave caused by the reduction of molybdate ions, occurring shortly before the tangent potential of the high and well-formed wave is reached. I n nitric
75
Ede. . vs.normal C.E. FIGURE2. POLAROGRAPHIC WAVESOF MOLYBDATE SOLUTIONS I. 1 ml. of standnrd.molybdate solution in 50 ml. of regulating solution 11. Same solution after adding 1.0 X 10-4 gram of phosphorus (Pod) 111. Same solution after adding 2.05 X 10-4 gram of phosphorus (Pod Starting potential of each curve indicated on abscissa, and distance between each 10 mm. corresponds to 0.2 volt
acid, as well as in neutral solutions, under the conditions employed in this work a second wave could not be observed clearly. As may be seen in Figure 1, the sudden rise in current shortly before the tangent potential of the high wave is reached might indicate the existence of a second wave. This sudden rise in current is not observed a t all in solutions which contain a higher concentration of ammonium nitrate (Figure 2). Only one wave appears which can be used for quantitative determinations. The conditions under which a second wave would appear in measurable form, 3s well as the explanation of the nature of the reduction products of the molybdate ion a t the dropping electrode, require further study.
Determination of Phosphate Ions in Pure Solutions Different amounts of a standard phosphate solution (1 t o 2.5 X 10-4 gram of phosphorus) are placed in 50-ml. volumetric
E
E
E! 0
0.4
0.8
1.2
Total applied volts FIGURE 1. DETERMINATION OF MOLYBDATE I. 0.001 gram of molybdenum per milliliter of 3 nitric acid and 2 N ammonium nitrate solution
N
11. 0.001 gram of molybdenum per milliliter of 2 N ammonium nitrate solution containing 3 grama of methylcellulose per liter
flasks. Two milliliters of nitric acid (120 ml. of concentrated nitric acid diluted with 180 ml. of water) and 30 ml. of an ammonium nitrate solution (400 grams of ammonium nitrate in 1 liter) are added t o each and heated to 35" C. To each flask is added 1 ml. of a standard solution of ammonium heptamolybdate (3.1 grams in 100 ml. of water). A yellow precipitate appears immediately. Approximately 15 minutes later the flasks are filled t o the mark with ammonium nitrate solution and allowed to stand for several hours or overnight until the precipitation is complete. Ten milliliters of the supernatant solution are placed in the electrolytic cell and the polarogram is made with galvanometer sensitivities of l / i to ~ 1 / 2 ~ ~depending , upon the concentration. The decrease in the wave heights given by the supernatant molybdate solution, plotted against the increase in the concentrations of the original hosphate solutions, given directly as phosphorus, provides a caligration curve upon which the phosphate-ion concentration of unknown solutions can be read. The accuracy of the determination is 1 to 2 per cent. It is not necessary to weigh the ammonium heptamolybdate very accurately for preparation of the standard solution, if for a calibration and the corresponding series of determinations the same amount of the same standard solution is always employed. If another standard solution is prepared a new, complete calibration is essential.
76
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
Figure 2 shows three of the polarographic waves of solutions of different molybdate concentrations before and after the precipitation of phosphate and Figure 3 the calibration curve derived from the series of determinations. The heights of the waves corresponding to the various concentrations of phosphorus fall in a straight line which, when extrapolated to the ordinate, must intersect at a point identical with the wave height of 1 ml. of standard molybdate solution examined under the same conditions.
Vol. 14, No. 1
nitrate is not influenced by the presence of alkali metals, alkaline earths, or such elements as iron, copper, aluminum, and manganese, if the latter are not present in excessive quantities. Feces, foods, and urine usually contain heavy elements in small amounts in comparison with the amount of phosphorus present. Since a large excess of molybdate must be added to precipitate the phosphate ions completely, the amount of molybdate remaining in the supernatant liquid after the precipitation is also large and must be determined using a relatively low galvanometer sensitivity ( l / l o ~ to ‘/moo). With this sensitivity the galvanometer does not indiRESULTSOF PHOSPHORUS DETERNINA- cate a wave for any of the heavy elements which are present TABLE I. ILLUSTRATIVE TIONS UPON FOODS, URINE,AND FECES in low concentrations; hence, it is possible to determine moFoods Urine Feces lybdate ions directly in solutions containing all the elements PolaroPolaroPolaropresent in the ash of feces, urine, and common foods. The graphic Chemical graphic Chemical gra hic Chemical Sample method method method method metiod method conditions for the precipitation of phosphate ions with amMg./O.l Q. dru weight M g . / l . O ml. urine Mg./iO mg. dried feces monium heptamolybdate, however, must be well controlled. I 0.341 0.336 0.733 0.721 0.200 0.205 Arsenic acid, which would interfere with the precipitation, is I1 0.325 0.329 0.690 0.698 0.204 0.201 0.289 0.284 I11 0.623 0.620 0.245 0.232 not present and the silica content normally is too low to be of 0.769 0.326 0.318 0.761 0.216 IV 0.215 V 0.330 0.323 0.827 0.819 0.302 0.300 any significance. I n individual foods which may contain a 0.336 0.336 0.800 VI 0.804 0.285 0.283 high amount of silica, the silica must be removed. The very 0.340 0.344 0.834 VI1 0.846 0.282 0.272 small amount of chloride ions in the ash solutions does not interfere. The values obtained by indirect polarographic determinaDetermination of Phosphorus in Biological tion of phosphorus in food, urine, and feces (Table I) check Materials very closely with the results of chemical determination upon Phosphorus is one of the most important mineral elements the same samples by the method of Mackey and Butler (3). in the living organism. I n studies of mineral metabolism the Summary chemical determination of this element in food, feces, and urine is time-consuming and requires relatively large amounts The polarographic method for the direct determination of of material. For these reasons, a method whereby this elemolybdabe ions provides the basis for an indirect method for ment may be determined more rapidly and upon smaller the determination of phosphorus in biological materials. amounts of material considerably facilitates routine analyses. Phosphorus is determined with an accuracy of 1 to 2 per cent as the difference between the concentrations of molybdate To determine hosphate ions, carefully collected samples of before and after precipitation of the phosphate ions as amfeces, urine, and gods must be dried and ashed. For this work food and feces were oven-dried at 60’ to 75” C. and urine was dried from the frozen state under vacuum. Since minute amounts of hosphorus can be determined by the indirect polaroy p h i c metgod, only a small amount of dried material is required or analysis. It is difficult, however, if not impossible, to mix feces or food com osites so that a homogeneous sample yielding only 0.1 gram of &y material can be ashed. For the determination on feces, 5 grams of mechanically mixed, dried material were ashed; for the food composites, 20 grams of the ground and dried material were ashed. For analysis of urine the dried substance of 250 ml. of urine was used. Sam les were ashed in a platinum crucible a t 400’ to 500’ C. The asg was dissolved in concentrated hydrochloric acid (5 ml.) and filtered into a 100-ml. volumetric flask, the residue wm reashed and added to the solution, and the flask was filled to the mark with distilled water. The amount of acid added should always be the same, so that the acidity of the regulating solution used later on for the determination of the remaining molybdate ions will be changed only slightly and always by approximately the same amount. Since these ash solutions contain a high concentration of phosphorus, only a small portion is needed for the larographic determination. The 100 ml. of ash solution of gces (5 grams of dried material) contain 0.10 to 0.15 gram of phosphorus; 5 ml. of this solution are diluted to 25 ml. with distilled water and 1 ml. of this solution (0.1 to 0.3 mg. of phosphorus) is taken for analysis. Portions of the ash solutions of foods and urine are diluted to the same concentration. The remainder of the ash solutions may be used for the determination of other mineral constituents. Phosphorus in the ash solutions is determined as in pure solutions, using 1 ml. of ash solution instead of the standard phosphate solution. The 1 ml. of ash solution should not contain more than 1 to 3 X gram of phosphorus. The calibration curve obtained with pure solutions may be used for the determination of phosphorus in ash solutions, proving that the ash solutions do 0 I 2 3 not contain interfering substances. Calibration using “internal X gram P per 50 m l . standards” gives the same results. Recoveries are made within the same range of accuracy as the determinations themselves, 1 CURVEFOR DETERFIGURE 3. CALIBRATIOX to 2 per cent. MINATION OF PHOSPHORUS The polarographic determination of molybdate ions in niDerived by plotting wave heights of curves shown in Figure 2 (and others in series) against amounts of tric acid solutions containing a large amount of ammonium phosphorus precipitated from solutions
ANALYTICAL EDITION
January 15, 1942
77
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
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.
(1939).
Literature Cited
(6) Uhl, F. A., 2.anal. Chem., 110,102 (1937).
H., “Chemische Analysen mit dem Polarographen”,p. 18, Berlin, Julius Springer, 1937.
(1) Hohn,
PREBENTED before the Division of Micro Chemistry a t the 100th Meeting of the AKERICANCHEMICAL SOCIETY,Detroit, Mich.
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 10s DM, 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
