Absorptiometric Determination of Hydrogen Peroxide in Submicrogram Amounts with Leuco Crystal Violet and Peroxidase as Catalyst Horacio A. Mottola, Benny E. Simpson, and George Gorin Chemistry Department, Oklahoma State Unicersity, Stillwater, Okla. 74074
AN ACCURATE, convenient, selective, and very sensitive method for the determination of hydrogen peroxide is described. The method is based on the formation of crystal violet from its leuco base, catalyzed by the enzyme peroxidase from horseradish. Numerous methods are available for the spectrophotometric determination of hydrogen peroxide in microgram amounts. These methods may be grouped into the following categories: (a) oxidation of metal ions or metal chelates ( I , 2 ) ; (b) oxidation of iodide ion ( 3 ) ; (c) oxidation of organic compounds, some giving dyes (4-7); and (d) production of fluorescence or luminescence (8). In most of these methods, the sensitivity is limited to solutions containing more than 10-jM hydrogen peroxide. To attain greater sensitivity requires rather specialized equipment, as in the case of luminescence-fluorescence, or complicated, time-consuming procedures. The exceptionally high molar absorptivity of crystal violet makes the present method applicable directly to submicrogram amounts of hydrogen peroxide. The procedure is simple and the apparatus needed is generally available. Enzymes are efficient, specific catalysts that are finding increasingly wide application in analytical chemistry (9). In the present case, the peroxidase makes possible a rapid reaction at low concentrations and also affords good selectivity. Other methods using this enzyme have been described; the chromogenic reagents were o-tolidine (4), benzidine (9, and o-dianisidine (7). In our hands, the colored products first formed from o-tolidine and o-dianisidine faded rather rapidly. o-Dianisidine gave subsequently a more stable colored product, but its molar absorptivity is not sufficient to provide a sensitive method. EXPERIMENTAL
Reagents. All chemicals used, except enzyme and leuco crystal violet (LCV), were of AR grade. The water used to make solutions of the reagents was purified by redistilling distilled and deionized water through a borosilicate-glass still equipped with a quartz immersion heater. Peroxidase isolated from horse radish (HRP) was obtained from Sigma Chemical Company (lot # 127B-1620, approxi(1) A. Egertan, A. Everett, G. Minhoff, S. Rudiakanchawa, and K. Salvoja, Anal. Chim. Acta, 10, 422 (1954). (2) C. E. Meloan, M. Mauck, andC. Huffman, ANAL.CHEM., 33, 104 (1961). (3) A. 0. Allen, C. J. Hochanadel, J. A. Ghormley, and T. W. Davis, J. Phys. Chem., 56, 575 (1952). (4) L. L. Solomon and J. E. Johnson, ANAL.CHEM., 31,453 (1959). (5) Y . Avi-dor, E. Cutolo, and K. Paul, Acta Physiol. Scand., 32, 314 (1954). ( 6 ) T. Hauser and M. Kolar, ANAL.CHEM., 40, 231 (1968). (7) M. Kminkova, M. Gottwaldova, and J. Hanus, Chem. Ind. (London), 1969, 519. (8) W. A. Armstrong and W. J. Humphreys, Can. J. Chem., 43, 2576 (1965). (9) G. G . Guibault, ANAL.CHEM., 40,459R (1968). 410
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mate activity: 200 purpurogallin units/mg). LCV (K & K Laboratories) was purified by dissolving it in aqueous hydrochloric acid and extracting it with benzene, evaporating the extract, and recrystallizing the residue three times from benzene. The final product had a melting point of 173-175 "C; the reported value is 177 "C (10). Apparatus. Quantitative absorptiometric measurements were performed with a Beckman D U spectrophotometer equipped with a Gilford Model 222 photometer and light source stabilizer. Matched 1-cm silica or glass cells were used. Absorption spectra were recorded with a Beckman DB spectrophotometer and Sargent SRL strip chart recorder. The pH measurements were made with a Corning Model 7 pH meter. Solutions. LCV, 50 mg, was dissolved in about 80 ml of 0 . 5 x (v/v) HC1 and diluted to 100 ml with acid of the same strength. The buffer was made by mixing equal volumes of 2 M sodium acetate and 2M acetic acid then adjusting the pH to 4.5 with glacial acetic acid. A stable solution of hydrogen peroxide, O.lM, was prepared by diluting 30% hydrogen peroxide (Fisher, A. R.) and standardized iodometrically. Aliquots of the stock solution were diluted volumetrically to obtain the concentrations necessary for calibration. A solution of HRP was prepared as needed by dissolving 10 mg of the enzyme preparation in 10 ml of water. All solutions, except the buffer, should be refrigerated during storage. Procedure. Add 1 ml of LCV solution to a sample containing from 0.6 to 5.0 pg of hydrogen peroxide in a 10-ml volumetric flask; then add 0.5 ml of HRP solution and 4 ml of buffer and dilute the mixture to 10 ml with water. A blue color develops almost immediately upon addition of the HRP, and the mixture turns violet when the buffer is introduced. Measure the absorbance of the sample at 596 nm against a reference prepared in the same manner but with no hydrogen peroxide. The concentration of hydrogen peroxide is determined by comparing to a calibration curve. RESULTS AND DISCUSSION
The procedure was developed with reference to the following considerations. It has been reported (11) that the intensively colored violet form of crystal violet (CV+) predominates above pH 3.4; in acetate buffer of pH 5, it has a maximum at 590 nm and a molar absorptivity coefficient at 1.00 X 1 0 6 W 1cm-I. As the pH is lowered, HCV2+is formed, blue in color, withpK 2.39, and then H2CV3+,yellow, p K 0.91. I n the conditions of the determination, control and sample solutions became turbid at pH above 5 ; a pH of 4.5 was accordingly chosen. The sensitivity was found to increase slightly as the dye concentration was varied from 2 X 10e5M to 2.7 X 10-4M,but no appreciable variation was found around 10-4M; a concentration of 1.3 X 10-4M was used in all other experiments. The amount (10) P. M. Heertjes, J. C. van Kerkhof, and K. A. de Vries, Rec. Trav. Chim., 62, 745 (1943). (11) R. Cigkn, Acta Chem. Scand., 12, 1456 (1958).
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Table I. Effect of Added Substances on the LCV and Iodide Methods Foreign species Hydrogen peroxide, pg (8.0 X 10-4M) Method Added Found Valine iodide 4.31 4.25 Valine LCV 1.54 1.50 iodide 4.31 4.21 Adenine Adenine LCV 1.54 1.48 Tyrosine iodide 4.31 4.17 Tyrosine LCV 1.54 1.52 Tryptophan iodide 4.96 0.25 LCV 1.54 1.42 Tryptophan
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Figure 1. Absorption spectrum of the product of the reaction between hydrogen peroxide and LCV in presence of HRP. Experimental conditions according to procedure. Amount of H202added: 1.00 X 10-5M. Spectrum of blank obtained against water as reference
of color developed was not affected by changing the concentration of enzyme from 2 to 16 units/ml of reaction mixture, although the color developed more slowly at low enzyme concentration, as would be expected. Figure 1 shows the spectrum of the product obtained in the experimental conditions specified in the procedure. The maximum is at 596 nm. The absorption spectrum for a blank sample (against purified water as reference) is also shown in the figure. The absorbance readings for the blank showed no change for at least 4 hours with the sample exposed to laboratory fluorescent light and daylight. A calibration curve obtained in the range of 0.6 to 5 pg of hydrogen peroxide showed that a straight line can be drawn through the experimental data although this line does not pass through the origin but cuts the abscissa near the origin. The molar absorptivity based on HP02and calculated from the slope of the line, is 75,000. The negative intercept may be due to the presence of a species that reduces hydrogen peroxide, or to the occurrence of some competitive reaction, which converts CV+ into a product of lesser absorbance, and which is faster at low dye concentrations. Behavior of this type has been observed in the oxidation of malachite green cation by periodate with manganese(I1) as catalyst (12). Braude (13) has estimated, on the basis of certain theoretical considerations, that molar absorptivities cannot exceed about (12) H. A. Mottola and H. Freiser, ANAL.CHEM., 39, 1294 (1967). (13) E. A. Braude, J. Chern. SOC.,1950,379.
lo5 for molecules of moderate size. The sensitivity that can be realized with crystal violet is therefore near the maximum. If one expresses sensitivity in the manner suggested by Sandell (14),-i.e., as pg of substance/cm2 giving an absorbance of 0.001-the result for the present method is 4.5 X For comparison, the sensitivity of the iodide method (3) is 1.4 x lo+, that of the o-dianisidine method (7) only 1.2 X 10-2. The precision of the method is illustrated by the following results. In eight determinations at 10-5 M concentration and five determinations at 5 X 10-6M concentration, the deviations from the mean were all less than 2 %. Table I summarizes some results obtained in the presence of various substances, added at a concentration one hundred times greater than that of the hydrogen peroxide. For comparison, determinations were also done by the iodide method. Adenine, valine, and tyrosine did not interfere in either case, tryptophan interfered with the iodide but not with the LCV method. Peroxidisulfate and chromate did not interfere with the LCV method. Cerium(1V) interferes but its effect may be eliminated by previous precipitation of cerium(1V) hydroxide. Periodate oxidized LCV in the conditions of our experiment and, surprisingly, the reaction is catalyzed by HRP-in the absence of enzyme, the reaction was very slow. Because manganese(I1) catalyzes the periodate oxidation of other leuco bases, it was thought that the catalysis might be due to traces of this metal ion in the enzyme; however a test for the presence of manganese(I1) in HRP gave negative results.
RECEIVED for review October 27, 1969. Accepted December 23, 1969. This work was supported in part by the Research Foundation, Oklahoma State University. One of us (B. E. S.) was a participant of an Undergraduate Research Program, held in Summer 1969, sponsored by the National Science Foundation. G. G. held Career Development Award GM 13,489 from the National Institutes of Health. (14) E. B. Sandell, “Colorimetric Determination of Traces of Metals,” 3rd ed., Interscience, New York, N. Y.,1959, p 83.
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