ent. The steric effects of the large trimethylsiloxy group are probably an important factor in the poor reaction with silanol on treated silicas. Apparently the amide reagent can only react with the exposed, unhindered silanol groups on the surface of a treated silica. This type of numerical data could be valuable if compared to the total hydroxyl present by the condensation method ( 2 ) , and might be correlated to residual surface activity and efficiency of trimethylsiloxy treatment. The residual titration reaction previously mentioned was less pronounced when titrating treated silicas, as shown in Figure 1, Curve 4.
The visual indicator was partially adsorbed upon the surface of the silicas as the titrations proceeded. It was necessary to add excess dye at the beginning of the titration so that the end point was visible 8 to 10 minutes later. The excess dye had no apparent effect upon titration results. Treated silicas adsorbed the indicator to a lesser extent than did untreated samples. Estimates were made of the reproducibility of titration results. Relative standard deviation of 3 to 6 x was typical. RECEIVED for review May 6, 1968. Accepted July 30, 1968.
Quantitative Spectrophotometric Determination of Hydrogen Peroxide with para-Dimethylaminobenzaldehyde Bibhuti R . DasGupta and Daniel A. Boroff Research Laboratories, Laboratory of Immunolog)~,Albert Einstein Medical Center, Plziladelphia, Pa. 19141 p-DIMETHYLAMINOBENZALDEHYDE has been widely used as a reagent for qualitative and quantitative analysis of various compounds. We have observed that a color, X/max 525 mp, results when H202 is treated with this reagent in p H 5.8 buffer. This color producing reaction has not been previously reported in the literature. The purpose of this report is, primarily, to utilize the observed novel reaction, which requires a simple procedure, as a quantitative measure of HP02. The color formation, within a certain range, is a linear function of HPOPconcentration. The reaction can measure as little as 3 pmole of H202. The assay requires preparation of a calibration curve with standardized H202 and can be performed in the presence of a number of substances without interference. EXPERIMENTAL
Materials. p-Dimethylaminobenzaldehyde (PDMB), mp 73-75 "C, and H 2 0 2 ,3 0 x Reagent, A.C.S., were obtained from Matheson Scientific Co. p-Dimethylaminobenzoic acid, m p 240 "C (dec) was purchased from Aldrich Chemical Co. Ethyl alcohol ( 9 5 7 3 and other compounds were of analytical grade. Buffers were prepared by titrating the acidic and basic conjugates of the same molarity to the desired pH-e.g., 29.5 ml of 0.1M citric acid was added to 200 ml of 0.1M sodium citrate to pH 5.8 =t 0.02 (Radiometer pH meter 26). Citrate-phosphate buffer was prepared with citric acid and Na2HP04. Spectrophotometric measurements were carried out with a Zeiss Spectrophotometer model PMQ 11, employing 1-cm absorption cells. A fresh solution of PDMB prepared in 9 5 z ETOH was kept away from light and was used within two hours. H ? 0 2 , after standardization against K M n 0 4 , was diluted with distilled water to various concentrations. Procedure. To 4.8 ml of 0.1M citrate buffer pH 5.8, in a 15 x 125-cm screw-capped tube, were added 0.4 ml of PDMB (20 mg/ml) and 1.0 ml of H2O2 solution. The tube, securely capped, was vigorously shaken for five seconds and placed in a water bath at 45 "C. After five hours the absorbance of the solution was measured at 525 mp against either a blank containing all reagents except HqO?, or just water. RESULTS
Effect of Time and Temperature on Color Formation. The results of the color-forming reaction carried out at 25", 35", 45", and 55 "C are shown in Figures 1 and 2. Each reaction tube, after removal from the water bath, was cooled 2060
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ANALYTICAL CHEMISTRY
Table I. Yield of Color at Various pHs PH 5.5 5.8 6.2 6.5 Absorbance 0.330 0.385 0.375 0.334 at 525 mw 0.329 0.386 0.380 0.332 0.328 0.385 0.374 0.333 Mean absorbance 0.329 0.385 0.376 0.333 Reaction conditions: 0.1M citrate buffer was used in all cases except at pH 6.5 where 0.1M citrate-phosphate buffer was used. Standard reaction conditions with approximately 20 pmole HZOn.
to 25" i 2 "C within one minute (see legend of Figure 2). Figure 1 shows the absorption spectra of the color formed at 45 "C in five hours with 10 pmole and 30 pmole H z 0 2 in the presence of a constant amount of PDMB (curves A and D, respectively). Maximum absorption was obtained in both cases between 515 and 525 mp. The reaction carried out with 20 pmole H 2 0 2at 35 "C for 24 hours also produced a color with a similar absorption profile between 480 and 580 mp (curve C). A similar reaction at 45 "C for 24 hours changed the pattern of the peak (curve B ) showing a shift toward the shorter wavelength. When measured against water, the absorbance of the reagent blanks containing all reagents except HZ02never exceeded 0.003 between 480 and 580 mp. Dependence of the reaction rate on temperature was found to be as follows: At 25 "C the initial reaction proceeded slowly, accelerating with the time (Figure 2). At 35 "C the rate of the reaction was essentially uniform over a seven-hour period. The reaction rates at 45" and 55 "C were initially fast but then decreased with time. U p t o three hours, higher temperature afforded a more intense color; but at the end of seven hours, reactions at 45" and 35 "C produced more color than at 55"; and at the end of 24 hours, the reaction at 35 "C resulted in more color than what was obtained at 45". Because enough color developed in five hours (convenient for an eight-hour working day) at 45 "C,this combination of temperature and time was chosen as the standard reaction condition. For the determination of H z 0 2 at a concentration less than 10 pmole H20z, the 24 hour reaction at 35 "C was found more suitable.
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Figure 1. Absorption spectra of color due to reaction of p-dimethylaminobenzaldehyde and H?Os Absorption spectra were recorded against reference solutions containing reaction mixture less HzO?
Reaction conditions as in text except temperature and time. All tubes con. point on the curves represents average of three readtained 20 pmole H z ~ zEach ings against water. Tubes withdrawn from water baths at 55 ', 45 ', and 35' C were shaken in ice water for 20, 15, and 5 seconds, respectively, followed by holding them in 25' water for additional 40,45, and 55 seconds
Effect of p H on Color Formation. The reaction carried out at various p H indicated that the formation of color was sensitive to the pH of the reaction medium. Data in Table I indicate that pH 5.8 was optimum. The reaction carried out in water instead of buffer yielded less color and increased the acidity of the reaction mixture. An increase in acidity was observed either by a decrease in p H (about 0.4 unit) o r by titration with NasCO:,. Effect of Various Concentrations of PDMB on Fixed Concentrations of HsOz. Figure 3 shows the amount of color formed by various concentrations of PDMB with fixed concentration of H,O,. Absorbance vs. concentration of PDMB did not yield a linear relation. Even 16 mg PDMB did not result in saturation in color formation. Higher concentration of PDMB could not be used because of solubility difficulties. Based on these observations, 8 mg of PDMB in 0.4 ml EtOH was decided as the optimum concentration of these reagents. Effect of Various Concentrations of H202. F o r a constant amount of PDMB, increasing the concentration of H 2 0 2 (up t o 45 pmole was tested) produced increasing amount of color. Color generation under standard conditions followed Beer's law between 2.5 and 30 pmole Hz02 (0.021 and 0.575 absorbance, respectively). Above a concentration of 30 pmole, deviation from linearity was noted. Doubling the concentration of PDMB did not yield twice the amount of color. Formation of color at 35 "C for 24 hours also followed Beer's law between a concentration range of 10 and 2 prnoles of H202. Under this condition 3 pmole H 2 0 2 (0.070 absorbance) could, therefore, be conveniently assayed. Reproducibility. Variations in the color yield within a n experiment and between different batches of H 2 0 2solutions were tested. H202 solutions from three sealed bottles were
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( rng ) Figure 3. Color formation as a function of varying concentration of p-dimethylaminobenzaldehyde on fixed concentration of Hz02 All reactions at standard conditions except for indicated amount of PDMB/0.4 ml ETOH. Amounts of H202in the upper and lower curves were approximately 30 and 20 pmoles, respectively. Each point on the curves represents an average of three readings VOL. 40, NO. 13, NOVEMBER 1968
2061
Table 11. Effect of Various Substances upon Color Formation in the Reaction between p-Dimethylaminobenzaldehyde and Hydrogen Peroxide Material Absorbance Material Absorbance Material Absorbance 0.385 Methyl alcohol 0.276 Lysine. HCI Control 0.378 0.389 Histidine. HCI Glucose 0.389 0.308 KCI Arginine.HCI 0.293 Fructose 0.385 KBr 0.382 0.038 Glycine 0.360 Tyrosine KI 0.377 0.382 Phenylalanine Leucine 0.387 0.372 KN03 Tryptophan 0.364 Isoleucine 0.385 0.355 (NH&S04 Proline Cysteine 0.366 0.392 NaH2P04 0.302 Methionine 0.009 Alanine Sodium thiosulfate 0.310 0.386 0.389 Valine Na-acetate Bovine serum albumin 0.375 0.385 Lysozyme 0.177 Glutamic acid 0.380 Pyridine 0.387 Ribonuclease 0.163 Aspartic acid 0.377 Acetone 0.384 0.241 Serine 0.371 Dioxane 0.383 Starch ( I ) Threonine 0.290 0.367 Ethyl alcohol Assay: Buffer 4.8 ml, material to be tested 0.5 ml (salts 0.1 mmole, organic solvents 0.5 ml, sugars and amino acids 0.5 mg, starch and proteins 100 pg each, and water in case of control) PDMB 8 mg in 0.4 ml EtOH; H?O?20 pmole in 0.5 ml. The reactants were mixed in the above mentioned order, incubated at 45 “C for five hours. All absorbance readings, at 525 mp against water, were the average of at least two readings.
diluted to approximately 0.02M and the color was developed in standard reaction conditions. The mean absorbance of 10 replicates from one batch of peroxide was 0.377 (standard deviation = 0.0011)and the color yield from the other two batches of peroxide was 0.396 and 0.385 absorbance units (mean of five replications in both cases). Influence of Various Substances on Color Formation. Table I1 shows the effect of the presence of various salts, water miscible organic solvents, sugars, amino acids, protein, and a polysaccharide on color formation. Although an excess of ethyl alcohol inhibited color formation, 0.4 ml of it in the standard reaction procedure was necessary to keep PDMB in solution. DISCUSSION
Based on H202, molar absorptivity of this assay under standard conditions is 99 which becomes 191 when the reaction is carried out at 35 “C for 24 hours. The present method requires preparation of a calibration curve with suitable dilutions of a standardized HzOzsolution, as most other methods do, for the determination of micromole quantities of H2O2- e.g., in spectrophotometric methods utilizing KI and ammonium molybdate ( 2 3 , chromate-0dianisidine (4, 1,2-di-(4-pyridyl) ethylene (9,and in fluorometric methods with 6-methyl-7-hydroxy-l,2-benzopyrene (Scopoletin) (6,7), or diacetyldichlorofluoracein (8). Although the color reaction is highly reproducible, small variations in color value may result among different batches (1) 0. Smithies, Biocl7em. J . , 71, 585 (1959). (2) W. A. Patrick and H. B. Wagner, ANAL.CHEM., 21,1279 (1949). (3) T. C. J. Ovenston and W. T. Rees, Analyst (London), 75, 204 (1 950). (4) F. Buscarons, J. Artigas, and C. Roda-Rodriguez, Anal. Clzirn. Acta, 23, 214 (1960). ( 5 ) T. R. Hauser and M. A. Kolar, ANAL.CHEM., 40, 231 (1968). (6) W. A. Andreae, Nature, 175,859 (1955). (7) H. Perschke and E. Broda, ibid., 190,257 (1961). (8) A. S. Keston and R. Brandt, Anal. Biochem., 11, 1 (1965).
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ANALYTICAL CHEMISTRY
of H 2 0 2unless the exact concentration of H 2 0 2in the stock solution, used for the preparation of the calibration curves, is established. We have observed that the reaction generates titratable acid, but the colored product is not p-dimethylaminobenzoic acid. This acid is a white solid and its alcoholic solution in p H 5.8 buffer is water clear. Under standard reaction conditions, 0.04 mmole H202 in the presence of various concentrations of p-dimethylaminobenzoic acid failed to produce any color. It appears, therefore, that the aldehyde moiety is essential for color formation. Two reactions which may have some similarity with the proposed system are as follows: Most aromatic aldehydes are known to form a yellow precipitate or color in the presence of H 2 0 2 and p-phenylenediamine under acidic conditions (9). A mixture of 2% PDMB in 15% HCI and 3% H 2 0 ? in the presence of Kynurenine or its analogues - e.g., anthranilic acid, p-aminoacetophenone - produces a yellow color which on heating at 70 “C for 20 minutes becomes violet (10). However, no color formation was noted by Kikkawa (10) when only PDMB and H20z were mixed. ACKNOWLEDGMENT
The authors thank Allan Day, Department of Chemistry, University of Pennsylvania, Philadelphia, Pa., for helpful discussion, and Edmund Sambuco, who assisted in this work as a participant in the Secondary Science Training Program (1967)sponsored by the National Science Foundation. RECEIVED for review May 16, 1968. Accepted July 5, 1968. This work was supported in part by the National Institutes of Health Grant AI 04180,and the National Science Foundation Grant GB-6719. (9) F. Feigl in “Spot Tests in Organic Analysis,” 7th ed., F. Feigl, Ed., Elsevier, New York, N. Y . , 1966, p 198. (10) H. Kikkawa, Generics, 26, 587 (1941).
