Vanadium Determination in Biological Materials at Nanogram Levels by a Catalytic Method Ross M. Welch and William H. Allaway U . S. Plant, Soil, and Nutrition Laboratory, S W C - A R S - U S D A , Tower Road, Ithaca, N . Y . 14850 and Department of Agronomy, Cornell University, Ithaca, N . Y. 14850 A sensitive and accurate spectrophotometric method for measuring amounts of V as low as 5 ng in biological materials is described. Samples are digested with HNO3-HCIO4and V i s separated from interfering elements by complexing with 8-quinolinol in chloroform at pH 4.0. It is then released from the complex by readjusting the pH to 9.4 with an aqueous “,NO3 buffer. Vanadium is then determined in the aqueous extract by its catalytic effect on the rate of oxidation of gallic acid by acid-persulfate. The extraction procedure eliminates all interfering elements except Mo. Mo interfered with V determination when it exceeded 10 pg per sample. Comparisons with a neutron activation method were made. Recoveries of V added to plant samples were between 94 and 98%. Recoveries of radioactive 48V added to standard V solutions and carried through the extraction procedure were between 90 and 100%.
THEPOSSIBILITY that low levels of V may be beneficial to biological organisms or essential for life has created the need for a method sufficiently sensitive to measure nanogram amounts of V in biological materials (1-10). This is particularly true where a number of different analyses must be made on a limited amount of material. Neutron activation analysis as well as spectrographic and spectrophotometric methods have been developed for measuring V levels in biological tissues (11-14). Of these methods, neutron activation analysis is probably the most suitable for measuring nanogram quantitiesofV(11,13). Fishman and Skougstad (15) have developed a spectrophotometric (catalytic) method for determining submicrogram quantities of V in water. Their method involves the oxidation of gallic acid by acid-persulfate, a reaction catalyzed by nanogram amounts of V. A method utilizing this reaction was (1) F. Hudson, “Vanadium Toxicology and Biological Significance,” Elsevier Publishing Company, Amsterdam, London, New York, 1964. (2) H. A. Schroeder, Air Quality Monograph No. 70-13, American Petroleum Institute, Washington, D.C., 1970. (3) H. A. Schroeder, J. J. Balassa, and I. H. Tipton, J. Chronic Dis., 16, 1047 (1963). (4) E. J. Underwood, “Trace Elements in Human and Animal Nutrition,” Academic Press, New York, N.Y., 1971. (5) H. L. Cannon, Soil Sci., 96, 196 (1963). (6) P. F. Pratt, “Diagnostic Criteria for Plants and Soils,” University of California, Riverside, Calif., 1966, p 480. (7) K. Schwarz and D. B. Milne, Science, 174, 426 (1971). (8) L. L. Hopkins, Jr., and H. E. Mohr, Fed. Proc., 30, 462 Abst. (1971). (9) W. H. Allaway, Adcan. Agron., 20, 235 (1968). (10) H. J. M. Bowen, “Trace Elements in Biochemistry,” Academic Press, London, 1966. (11) R. J. Soremark, J . Nutr., 92, 183 (1967). (12) R. Z. Bachman and C . V. Banks, ANAL.CHEM.,41, 112R (1969). (13) J. P. F. Lambert, R. E. Simpson, H. E. Mohr, and L. L Hopkins, Jr., J. Ass. Oflc. Anal. Chern., 53, 1145 (1970). (14) N. A. Talvitie, ANAL.CHEM.,25, 604 (1953). (15) M. J. Fishman and M . W. Skougstad, ibid., 36, 1643 (1964).
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first reported by Jarabin and Szarvas (16). However, this catalytic method has not been applied successfully to analysis of biological materials for V because other transition elements and halides may interfere. Therefore, to utilize the high sensitivity and low detection limits of the catalytic method described by Fishman and Skougstad (15), a technique for separating V from halides, Fe, and other transition elements is needed. The procedure presented herein satisfies such a need. The method makes use of the complex formation of V with 8-quinolinol as developed by Talvitie (14) to separate V from other interfering elements. Acid digests (HN03-HC104) of the tissue are lirst treated with 8-quinolinol at pH 4.0 to form the 8-quinolinol complex of the dioxovanadium cation (V02+). Extraction into chloroform separates V from interfering anions. The ionic form of V is then shifted from the VOz+ cation to the V03- anion and separated from interfering cations by extracting the chloroform phase with aqueous NHlN03 buffered at pH 9.4. Thus separated, V is used as a catalyst for the oxidation of gallic acid by acid-persulfate. The oxidation product develops a yellow to red color. The rate of color development depends upon the concentration of V present, and the intensity can be measured spectrophotometrically. EXPERIMENTAL
Reagents. Prepare stock solutions containing 0.025, 0.100, or 0.25 pg V per ml of solution. Prepare the following reagents : Hg(NO& solution (0.035 %), gallic acid solution (2 %), 8-quinolinol solution (0.5 in chloroform, wt/vol), glacial acetic acid, redistilled “OB, and HC10, (70%). Use reagent grade chemicals, double distilled water, and HC1rinsed glassware. Prepare the NH4N03buffer solution (pH 9.4-9.5) by dilutand 100 ml of 4N H N 0 3 to 2 1. ing 200 ml of 4N “,OH with water. Prepare the acid-persulfate solution by dissolving 2.5 grams of (NHJ2S208in 25 ml water, heating just to a boil and adding 25 ml of concentrated H3P04. Let stand in a closed container for approximately 24 hours but not more than 48 hours before using. Prepare the gallic acid solution fresh before each analysis by dissolving 1 gram of gallic acid in 50 ml of near boiling water and filtering to remove any undissolved gallic acid crystals. Procedure. Weigh out a sample of biological material containing from 0.025 to 0.250 pg V. Place in a 100-ml micro-Kjeldahl flask containing a glass boiling bead. Add 10 ml of “Os. Slowly bring to boil and continue heating for approximately 15 min and add 2 ml of 70% HC104. Bring to a boil and distill off HN03. Continue boiling for 20 min after white HC10, fumes initially appear. Remove from heat. Some samples high in fats will char or turn black when HC104 fumes appear. If this happens, cool and add 2 ml of H N 0 3 . Return to heat and repeat procedure above. Several additions of H N 0 3 may be needed for samples high (16) 2. Jarabin and P. Szarvas, Acta Unir. Debreceit. Ludocico Kossuth Nominatae, 7, 131 (1961); Chern. Abstr., 57, 9192e (1962).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
Table I. Comparison of the Catalytic Method with Neutron Activation Analysis for Animal Diets Catalytic Activation Sample methode method No. Material V, nglg V, nglg Animal diet 8 7 A-VR-1 Animal diet 8 7 B-VR-1 Vitamin mix 26 65 C-GBI a Values are averages of duplicates.
7000r
o 5 ml
H a o 4000
Table 11. Recovery of V from Timothy Grass Dry ,weight, V added, V found,. V recovered, lrg fig lrg gram 0.25 0.0 0.083 ... 0.25 0.10 0.181 0.098 0.25 0.25 0.318 0.235 a Values are averages of duplicates.
0 in fats until digestate remains clear during oxidation by HC104. Transfer the contents of the digestion flask to a 125-1111 separatory funnel with water. If salts precipitate during the digestion and are not redissolved when water is added, filter the digest solution during transferring. Add 5 ml of glacial acetic acid and adjust pH to 4.0 with N H 4 0 H . Add 10 ml of 8-quinolinol (0.5 % in chloroform, wt/vol). Extract by shaking for 5 min and then draw off the chloroform phase into a second separatory funnel. Add another 10 ml of the 8-quinolinol reagent to the aqueous phase and repeat the extraction. Combine the chloroform phase into a second separatory funnel. Add another 10 ml of the 8-quinolinol reagent to the aqueous phase and repeat the extraction. Combine the chloroform phases and discard the aqueous phase. Add 25 ml of the NH4N03 buffer solution (pH 9.4-9.5) to the combined 8-quinolinol-chloroform phases and extract for 30 min. Readjust pH of aqueous phase to and record the volume of ",OH 9.4-9.5 with 4N ",OH added in order to calculate the total volume of the aqueous phase. Extract for an additional 1.5 hr. Pipet two 10-ml aliquots of the aqueous phase into matched 50-ml absorption cells (approx. 20 mm in diameter) or other suitable absorption cells or containers. Add 1 ml of Hg(NO,)? solution to each cell and place in a water bath at 30 + 0.5 "C. Equilibrate at this temperature for approximately 30 min. Add 1 ml of acid-persulfate solution (equilibrated at 30 "C) and mix thoroughly. Return to water bath and proceed to the next sample. Next add 1 ml of a 2 solution of gallic acid (temperature equilibrated) and mix thoroughly. Return to water bath and proceed to the next sample, allowing approximately 30 seconds or longer between gallic acid additions to permit accurate control to the reaction time for each sample. Sixty minutes after adding the gallic acid, measure the absorbance at 415 nm using a reagent blank as a reference. If the sample solutions are colored or turbid after the addition of the acid-persulfate, they must be decolorized or filtered before the addition of the gallic acid. Standards and reagent blanks should be carried through all stages of the extraction procedure. Standards should bracket the absorbance of the unknowns because the slope of the standard curve changes from linear to curvilinear when V levels exceed 0.08 pg. The amount of V in an unknown sample is then determined by reference to the corresponding absorbance on the standard curve. RESULTS
Comparison with Neutron Activation Analysis. The concentrations of V in two diets and a vitamin mix were de-
10 20 EXTRACTION TIME ( M I N 1
30
Figure 1. Radioactive 48V retained in aqueous phase (pH 4.0) plotted as a function of extraction time with 5 , lo-, and 20-ml volumes of 8-quinolinol (0.5% in chloroform, wt/vol)
termined by both the catalytic method described above and by neutron activation analysis. The latter analyses were performed by Union Carbide Corp., using the method of Lambert et al. (13). As shown in Table I, the concentrations of V in the diets were similar regardless of the method used. In the vitamin mix, however, the level of V determined by neutron activation was about two times greater than the amount obtained by the catalytic method. The reason for this disagreement in results is not known. The vitamin mix was repeatedly analyzed by the catalytic method, and all values were within ~k0.005pg of the value shown in Table I. Recovery of Added V. The recoveries of 0.10 and 0.25 pg of V added in 1 ml of NH4V03 solution to 0.25-g samples of dried timothy grass tissue prior to wet digestion are shown in Table 11. The recoveries are within 0.002 pg and 0.015 pg of theoretical for the 0.01-pg and 0.25-pg additions, respectively. Radioactive V Studies. Experiments using &V were conducted to determine the efficiency of extraction of V from digest solutions (adjusted to pH 4.0) with 8-quinolinol in chloroform, and its subsequent release from the 8-quinolinol complex into the ",NO3 buffer (pH 9.4). One-milliliter aliquots of a 0.2-pgiml solution of NH4V03labeled with 48V were added to solutions containing 25 ml of water and 5 ml of glacial acetic acid adjusted to pH 4.0 with ",OH. The solutions were then extracted with either 5-, lo-, or 20-ml volumes of 8-quinolinol in chloroform. After extracting for either 5 , 10, 20, or 30 rnin, ,*V in aliquots of the aqueous phase was measured using a gamma spectrometer. As shown in Figure 1 , maximum extraction efficiency was obtained after 5 min of extraction with 20 ml of 8-quinolinol in chloroform. It was concluded that by extracting twice for 5 rnin with 10 ml of 8-quinolinol in chloroform, maximum recoveries of V would be obtained. The proper pH for the most efficient extraction of V with 8-quinolinol in chloroform from aqueous solutions was determined by Talvitie (14) to be between pH 3.58 and 4.51. In preliminary work, the use of formic acid to buffer the digest at pH 4.0 resulted in low recovery of V in the chloroform
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
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Table 111. Elements Not Interfering with the Determination of 0.10 pg V at the Amounts Listed Amount added, Element added pg per sample Cr(V1) 10 Fe(II1) 100 Co(I1) 100 Ni(I1)
Mn(I1)
100 100
Table IV. Effects of Various Amounts of Mo on the Determination of 0.10 pg of V Mo added, V determined, pg per sample pg per sample 0 1 10 50 100 500
0.10 0.103 0.128 0.148 0.240 > O . 50
phase. This is apparently due to the formation of V 0 2 + cations because of the reducing properties of formic acid. The release of V from the 8-quinolinol complex into the pH 9.4 N H 4 N 0 3buffer was also studied by using 48Vlabeled solutions. Extraction time and pH level proved to be critical in the release of V from the 8-quinolinol complex. Apparently the release of V from this complex into the pH 9.4 NH4N08buffer as the VOs- anion is relatively slow and very sensitive to changes in pH. Maximum recoveries were obtained only after 2-hr extraction periods. The pH of the N H 4 N 0 3buffer had to be readjusted to pH 9.4 with 4N N H 4 0 H after 30 min of extraction time in order to assure maximum recoveries of V. Borate buffers proved to be no better in maintaining the pH at 9.4 than did the NH4NOB buffer. The per cent recoveries of 48V carried through the extractions varied between 90 and 100%. Talvitie (14) also has reported that the release of V from the 8quinolinol complex was complete when the extraction solutions were adjusted to pH 9.4 with an aqueous NH4N03 buffer. Interferences. Table I11 summarizes the results of an experiment in which various elements, known to interfere with the catalytic determination of V, were added to a standard solution containing 0.10 pg V. After extraction, these elements at the levels listed did not interfere significantly with the determination of 0.1 pg of V. Fishman and Skougstad (15) and Jarabin and Szarvas (16) have investigated the possible interferences of numerous elements on the determination of V by its catalytic reaction with gallic acid. Halides such as Br and I and various transition elements interfere seriously with the determination of V by their methods. By adding Hgz+ ions to the gallic acid reaction mixture, Fishman and Skougstad (15) were able to complex interfering halides and minimize their interference. The extraction procedures outlined above for separating V from wet digests of biological materials have drastically reduced the number of interferences from various transition elements and halides in the determination of V. With the exception of Mo, no element tested at relatively high levels interferes significantly with the measurement of V by the method described here. Table IV shows the results of an experiment in which various amounts of Mo (VI) were added to standard solutions con1646
Table V. Analysis of Biological Material
Sample
Weight, g
V found,.
@gig
Dry weight Alfalfa (stems and leaves) Blackeyed peas (stems and leaves) Corn (leaves) Tomato (stems and leaves) Rat Rat Rat Rat 0
1 .o 1 .o 1 .o 1 .o
Wet weight liver 2.825 kidney 1.090 muscle 1.455 bone 2.520 Values are averages of duplicate subsamples.
0.115 0.298 0.244 0.463 0.020 0.002 0.019 0.009
taining 0.10 pg of V. The molybdic acid reagent used was analyzed for V impurities by X-ray fluorescence spectrography and no detectable amounts of V were measured. These results indicate that more than 10 pg of Mo per sample will significantly interfere with the determination of 0.10 pg of v. In most biological materials, Mo is usually less than 1 ppm. However, grasses and legumes grown on soils containing very high levels of available Mo may contain up to 300 ppm on a dry-weight basis. It may be necessary for persons working with biological materials that are suspected of containing high levels of Mo to screen their tissue for Mo content by emission spectrography or other suitable means before determining V catalytically. A rapid and nondestructive means of determining Mo concentrations in plant materials by X-ray fluorescence spectrography has been reported by Kubota and Lazar (17). Determination of V in Some Biological Materials. The results shown in Table V summarize the data obtained for the determination of V concentrations in various types of biological material. The plant samples were obtained from field-grown plants that were dried at 50 O C and ground in a Wiley mill to pass a 20-mesh chrome-plated screen. Grinding of plant material in this fashion did not contaminate samples with measurable amounts of V. When brass screen sieves were used, however, substantial quantities of V were added to the plant material during the grinding process. The samples of rat tissue used were obtained from 7-week-old laboratory rats which had been fed an experimental diet primarily composed of corn and Torula yeast with no added V. The levels of V in the plant samples ranged from 0.115 to 0.463 pg/gram on a dry-weight basis. These values are within the range of values reported by Fleming (18) for V concentrations in various pasture plants measured spectrographically. The present values are also in general agreement with the reported ranges of V concentrations found in various fruits and vegetables by Soremark (11) who used a neutron activation technique. Schroeder and his colleagues (3, 19), using a spectrophotometric method, have reported V concentrations in various rat tissues and organs. The range of V concentrations reported by Schroeder et al. (19) in rat tissue was much higher ( L e . , 0.39 to 3.38 pg/g wet wt) than shown in Table V. In their experiments, however, the rats were supplied V at a dietary level of from 1.4 to 3.2 pg/g on a wet-wt basis. (17) J . Kubota and V. A. Lazar, in “Instrumental Methods for Analysis of Soils and Plant Tissue,” Soil Science Society of America, Madison, Wis., 1971, pp 67-82. (18) G. A. Fleming, J. Sci. Food Agr., 14, 203 (1963). (19) H. A. Schroeder, and J. J. Balassa, J . Nutr., 92, 245 (1967).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
Precision. Seven 0.25-gram subsamples of timothy grass were analyzed for V content. The standard deviation was k0.049 pg of V. This standard deviation is relatively small compared to the mean value of 0.584 pg of V per g of dried tissue. Fishman and Skougstad (13, using the catalytic method without prior separation of V from other elements, have reported standard deviations of 0.2 pg and 0.6 pg of V for natural water samples containing 0.8 and 18 148 of V per liter, respectively. DISCUSSION
The catalytic procedure for V determination is applicable to a wide range of biological materials. The range of V levels that can be measured by this method can be extended tenfold by reducing the reaction time from 60 min to 10 min. Although the standard curve is not linear over this higher range of V levels, it does provide satisfactory results if a standard curve is constructed each time a set of samples is analyzed. In routine work, the time required from the start of the acid digestion of the biological material to the completion of the analysis is about 12 hr. The number of samples that can be analyzed at one time is dependent upon the capacity of equipment for digesting the samples and for shaking separatory funnels .
The shifts in ionic form of V with change in pH may be quite slow. In studies of recovery of **Vin various steps of the procedure, it was necessary to heat the tagged solutions to ensure that the 4*V added from strong acid solutions of the radioactive material had reached the same ionic form as the inactive V present. The ternary acid digestion with "02, HC104, and HzS04 is not practical for the oxidation of organic material because of V impurities in reagent grade In preliminary work with this method, dry ashing of the biological material was used satisfactorily. Wet ashing as described here is used at this Laboratory because it permits determination of certain other trace elements on aliquots of the digest. ACKNOWLEDGMENT
The authors are indebted to L. L. Hopkins, Jr., and H. E. Mohr for supplying the animal diets and the neutron activation analyses. RECEIVED for review February 17, 1972. Accepted April 18, 1972. Trade names and company names are included for the benefit of the reader and do not imply any endorsement or preferential treatment of the product listed by the U. S. Department of Agriculture.
Influence of Amalgam Formation on Cyclic Voltammetry Floyd H. Beyerlein' and Richard S. N i c h o l s ~ n ~ ~ ~ Chemistry Department, Michigan State Unicersity, East Lansing, Mich.48823
The theory of cyclic voltammetry has been extended to include amalgam formation on the assumption that the effects of finite electrode volume are negligible. This assumption is justified on the basis of perfect agreement between the theory and experimental data. For the single scan experiment, theoretical results are presented in terms of a semiempirical spherical correction term. For the cyclic experiment, results are summarized in tabular form. Results of the theory show that the ratio of anodic to cathodic peak currents is always greater than unity, and that enhanced peak potential separations occur under some conditions.
DURINGTHE COURSE of research involving stripping analysis and the use of hanging mercury drop electrodes (HMDE), we have noted some apparent anomalies in careful comparisons between experiment and theory for cyclic voltammetry. The most likely explanation seemed to be that the theory did not include the effects of amalgam formation, and therefore is not rigorously applicable to the HMDE when amalgam formation occurs. The importance of considering sphericity and amalgam formation has been demonstrated by other workers for both chronoamperometry ( I ) and ac polarography (2). Present address, S . C. Johnson and Sons, Inc., Racine, Wis. 53403.
Present address, National Science Foundation, Washington, D.C.20550. Author to whom correspondence should be addressed. 2
(1) W . Stevens and I. Shain, ANAL.CHEM., 38,865 (1966). ( 2 ) J. R. Delmastro and D. E. Smith, ibid., p 169.
These facts prompted us to attempt calculations for cyclic voltammetry that would be applicable in the case of amalgam formation at the HMDE. The mathematics of this problem can be greatly simplified by ignoring the finite volume of the electrode, and considering only the effects of the convergent nature of the diffusion process. Reinmuth (3) has discussed this problem quantitatively, and shown that for typical electrodes the effects of finite volume are important only for electrolysis times of the order of 40 seconds. For cyclic voltammetry this corresponds roughly to scan rates of 8 mV/sec, or slower. Hence, the use of Reinmuth's mathematical approach (3) seemed justified, and is the basis of the calculations reported below. CALCULATIONS The proper boundary value problem for cyclic voltammetry with reversible amalgam formation at a spherical electrode was formulated and then transformed to an integral equation by standard methods ( 4 ) . The approximation developed by Reinmuth (3) was incorporated in this derivation. The solution of the integral equation is a function we shall label x(y,q5), This function is related to current by the following equation i = nFA z//aDo Co* x(y,q5) where the variables y and q5 are defined as y =
z/o,lz/o,
(1) (2)
(3) W. H. Reinmuth, ANAL.CHEM., 33, 185 (1961). (4) R. S . Nicholson and I. Shain, ibid., 36,706 (1964). ANALYTICAL CHEMISTRY, VOL. 44, NO. 9. AUGUST 1972
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