Indirect Determination of Inorganic Phosphate by Atomic Absorption Spectrophotometric Determination of Molybdenum SIR: Of the elements commonly classified as nonmetals, only B, Si, 49, Se, and Te can be determined directly by atomic absomtion spectrophotometry. Indirect methods appear necessary for the application of this analytical tool to other nonmetallic elements. These methods might make use of stoichiometrically formed complexes with metals capable of being isolated and determined by atomic absorption and related quantitatively to the complexed nonmetal. Such an example is the determination of inorganic phosphate by direct determination of molybdenum ( 2 ) in the phosphomolybdic acid complex extracted into an organic solvent from the aqueous environment. The application of similar techniques in which the nonmetal is complexed, then precipitated, extracted, or isolated by some other means, is easily envisioned. In spectrophotometric determinations of phosphate, Berenblum and Chain (1) demonstrated that extraction of phosphomolybdic acid into an organic solvent has the advantages of increased sensitivity and independence from errors due to slight changes in acidity or the presence of certain interfering substances. Numerous modifications of this method have been devised. All are basically similar and use ultraviolet or visible light absorption by the complex as a basis for quantitative measurement. We have investigated the adaptation of Such a method to analysis by atomic absorption spectrophotometry and have found that inorganic phosphate can be rapidly and accuratel? determined in this manner.
EXPERIMENTAL
Stock Solutions. Acid molybdate reagent was prepared by dissolving 58.4 grams of (T\rTH4)6M01024.4 H 2 0 in 200 nil. of concn. HC1, diluted to 1 liter with glass distilled H 2 0 (0.048M ammonium molybdate). Citrate reagent was prepared by dissolving 143 grams of C a 8 O 7 H . 20 in 1 liter of glass-distilled HzO (pH adjusted to 2.9 with NaOH; 0.6831 citrate buffer). Phosphate standards included 0.01OM K2HP04 and dilutions. Reagents and standards were stored in polyethylene bottles to prevent silicate contamination. Practical grade 2-octanol (b.p. 177-80' C.; Distillation Products, Inc.) was satisfactory. Other chemicals were analytical grade. A Beckman Model 76 pH meter was used for all pH measurements. Procedure. Phosphomolybdic acid was extracted into 2-octanol which, following phase separation, was a5pirated directly into the atomic absorption spectrophotometer. Acid molybdate (0.5 ml.), 2-octanol (5.0 ml.), H20, and sample (total volume of H 2 0 and sample, 5.0 ml.) were added to a test tube which was capped with a polyethylene cover and shaken manually for 15-20 seconds. The cap was then removed, citrate buffer (1.0 ml.) added, and the tube recapped and shaken. Extraction mixtures were usually centrifuged briefly a t low speeds t o facilitate phase separation. The absorption due to molybdenum in the organic phase was determined with a Perkin-Elmer Model 303 atomic absorption spectrophotometer and expressed as pg. phosphorus based on stock phosphate solutions as standards. Instrument operating conditions included analysis a t wavelengths of 313.3, 379.8, 386.4 and 390.3 mp, slit 3 (0.3 mm.), and a lamp current of 30 ma. (molybdenum lamp). An air-acetylene burner (0.5- X 110mm. slot) was used with an air supply of 28 p.s.i. (flowmeter, 6.5) and an acetylene supply of 8 p s i . (flowmeter, 10.0), giving an aspiration rate of 0.8 ml./minute a t 23' C. The sensitivity (absorbance/pg. P) was 0.026 a t 313.3 mp in the linear region of the standard curve. RESULTS AND DISCUSSION
pg. PHOSPHORUS
Figure 1. Standard curve for analysis of inorganic phosphate The absorbance a t 313.3 mp was determined on samples containing known amounts of potassium phosphate b y the method described in the Experimental section
Figure 1 illustrates a standard curve obtained a t 313.3 mp, showing linear response to about 10 pg. of phosphorus. The linear range of a stanard curve may be extended to quantities exceeding 10 fig. of phosphorus by selecting a less sensitive wavelength. We have used the following wavelengths (mp) given in order of decreasing relative sensitivities (in parentheses) :
313.3 (100) > 379.8 (58) > 386.4 (42) > 390.3 (30) The described procedure permits an accurate determination of as little as 0.2 pg. of phosphorus (0.04 pg./ml. of 2octanol). With the volume of 2-octanol reduced from 5 ml. to 2 ml. and using 313.3 mp as the wavelength for absorption measurements, 0.03 pg. of phosphorus has been detected (0.015 pg./ml. of 2-octanol). Modifications which increase instrument sensitivity to molybdenum, such as using a three-slot Holing burner (5) or a nitrous oxide-acetylene fuel supply ( 8 ) ,may also lower the limits for phosphate detection and determination. Figure 2 shows the effect of pH on the extraction of phosphomolybdic acid into 2-octanol. Curve 1 was obtained by subtraction of Curve 2 (no phosphate) from the total observed absorption in the sample. The aqueous phase of the assay miyture described by this procedure has a buffered pH of 1.01.1, which falls within the pH limits for maximal extraction (0.9-1.2) indicated in Figure 2. The assay mixture mill tolerate addition of 0.5 ml. of l N NaOH or 2.0 ml. of 1N HC1 without significant change in extraction characteristics. Care must be taken that the acid-base
APPARENT pH Figure 2. Effect of pH on the extraction of phosphomolybdic acid into 2 -octono1 The pH of assay mixtures containing 12.4 pg. P (as potassium phosphate) was varied b y addition of either 1 N N a O H or 1 N HCI prior to citrate addition and analyzed as described in the Experimental section. HCl (0.1 ON) was used as a reference to standardize the p H meter a t p H 1.1
VOL. 38, NO. 12, NOVEMBER 1966
1759
tolerances are not exceeded with addition of the sample to be assayed. Solvents other than Zoctanol may be used, but optimal pH values will vary with the chain length, stereoisomer, and degree of oxygenation of the organic molecule. The described procedure was designed to be used for determining changes in phosphate concentrations of reaction mixtures containing enzyme systems-e.g. adenosine triphosphatase and oxidative phosphorylation activities. Such systems contain acid-labile organic phosphates which undergo molybdenum (7) and acid-catalyzed hydrolysis. As in the procedure used by Marsh (4) citrate was added to bind excess molybdate (3) and prevent molybdenumcatalyzed hydrolysis of these labile organic phosphates. Once the free molybdate was bound by citrate, subsequent formation of phosphomolybdic acid was completely inhibited and introduction of errors due to acid hydrolysis or accidental contamination of the sample was prevented. Citrate also lowered blank values by decreasing the amount of free molybdic acid in the organic layer. Phosphomolybdic acid became adsorbed to denatured protein and was not
extracted from the aqueous phase by 2-octsnol. Therefore, i t was necessary to eliminate all protein from samples before addition to the assay mixture. Perchloric acid was used to precipitate the protein which was then removed by centrifugation. When added to the reaction mixtures perchloric acid does not interfere with the assay if the final concentration is kept within the acid tolerance of the procedure. A constant temperature must be maintained in the samples and standards during aspiration into the spectrophotometer. A change of 1' C. (in the range of 17-40') altered the rate of Z-octanol uptake sufficiently to cause a 2% change in the absorption reading. Shielding the samples from flame heat may be required if an extended aspiration time is necessary. An extensive study of interfering ions has not been attempted. Presumably, ions reported to interfere with similar extraction techniques (3, 6) will exhibit comparable effects in this system. In the presence of 0.4 pmole phosphate, equimolar concentrations of sodium silicate or disodium arsenate resulted in absorbance readings which were 6 and 4% higher, respectively, than with phosphate alone.
The absorption readings from phosphomolybdic acid in 2-octanol were quite stable with time, showing only slight decreases after standing up to 3 days. The small changes that occurred on standing had no effect on the determination of phosphate concentrations, however, because of comparable changes in the standards. The described method works well with the biological systems mentioned and its application to the analysis of inorganic phosphate in other systems is anticipated. LITERATURE CITED
(1) Berenblum, I., Chain, E., Biochem. J . 32, 295 (1938). (2) David, D. J., Analyst 86, 730 (1961). (3) Davies. D. R., Davies, W. C., Biochem. J. 26, 2946 (1932). (4) Marsh. B B., Bwchem. Biophys. Acta 32, 357 (1959). (5) Sprague, S., Slavin, W., At. Absorption Newsletter 4, 293 (1965). (6) Wadelin, C., Mellon, M. G., ANAL. CHEM.25, 1668 (1953). (7) Weil-Malherbe, H., Green, R. H., Biochem. J . 49, 286 (1951). (8) Willis, J. B., Nature 207,715 (1965).
W. s. Z.4CGG R.J. KNOX Bureau of Sport Fisheries and Wildlife Western Fish Nutrition Laboratory Cook, Wash.
Application of Controlled Potential Techniques to Study of Rapid Succeeding Chemical Reaction Coupled to Electro-Oxidation of Ascorbic Acid SIR: The theory of stationary electrode polarography has been extended recently to the case of a reversible electron transfer followed by an irreversible coupled chemical reaction (8). .R - ne-
e0
k -+
Z
(1)
By applying cyclic voltammetric experiments, the value of k can be evaluated from the ratio of cathodic to anodic peak currents observed a t a particular frequency. In addition, Schwarz and Shain (14) have developed the theory and application of a cyclic s t e p functional controlled-potential technique in which the ratio of the cathodic to anodic currents for a particular frequency reflects the kinetics of the succeeding chemical reaction. Each of these techniques was employed here to obtain kinetic data for the rapid decomposition in aqueous solution of the oxidized form of ascorbic acid. There is nearly unanimous agreement in the recent literature that the electron transfer step in the electro-oxidation of 1760
ANALYTICAL CHEMISTRY
ascorbic acid is fast and that the initial electrode product undergoes an irreversible hydration reaction (3,4,9,12, 16),as in the equations HO
0
OH
\ / c=c
i i
C CHCHOHCHzOH
0
0
involved correlations of relatively subtle changes in the polarographic half-wave potential with drop time and temperature with the kinetics of the succeeding
- 2e-
0
\ / / c-c I \ - 2H+ e C CHCHOHCHzOH // \ / 0
0
HzO
L k
HO
OH
\/
c-c
CI
0
//
CHCHOHCH20H I
/\/
0
At least t w o previous attempts have been made to evaluate the kinetics of disappearance of the initial electrode product (3, 4). One of these studies
0
reaction (4). A rate constant of 3.33 x lo3 second-' was obtained. However, Ihe possibility for large experimental error was great. Furthermore, the
