Polarographic Studies of Organic Peroxides in Nonaqueous Solutions W. H. LEWIS, F. W. QUACKENBUSH, AND THOMAS DE VRIES Purdue University, Lafayette, Ind.
A procedure is outlined for the polarographic study of water-insoluble materials in nonaqueous solutions. Current-voltage curves for the peroxides of fats, ethers, and hydrocarbons are given. In early stages of autoxidation of fats, a linear relationship exists between wave height and peroxide number. In very advanced stages of autoxidation, the polarograph does not reduce all the substances that are reduced by iodides in acidic solution. Three waves appear in the current-voltage curves of fat peroxides in neutral solution.
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N A study of the oxidation of fats, it was desirable to dis-
tinguish between reactive and stable peroxides. F a t peroxides are a poorly defined group of compounds which are thought to vary appreciably in reactivity. However, the methods commonly used which are based on the oxidation of iodide or ferrous ions, offer little opportunity to distinguish between reactive and relatively stable peroxides. A study was made with the polarograph, and when suitable conditions were established, several waves were obtained for the autoxidation products of fats. Procedures for the polarographic determination of a few organic peroxides in aqueous media have been described. Dobrinskaya and Neiman (2) determined methyl hydroperoxide in mixtures with aldehydes. Later Shtern and Pollyak (5) determined methyl hydroperoxide, ethyl hydroperoxide, ethyl peroxide, etc., in several aqueous electrolyte solutions. However, the peroxides of many larger relatively nonpolar molecules cannot be determined by similar procedures because of solubility limitations. In the present work nonaqueous media were used. EQUIPMENT
AND REAGENTS
The effect of traces of water in the solvents has not been carefully investigated. However, addition of 1% of water to each of the three supporting electrolyte solutions had little effect on the current-voltage curves of oxidized lard except to alter somewhat the ratio of the height of the three waves obtained. Consequently, the electrolyte should be anhydrous if quantitative results are to be obtained. That the solvents used were essentially anhydrous was shown by placing u few particles of anhydrous copper sulfate and 5 ml. each of methanol and benzene in a stoppered test tube and shaking occasionally. After standing for a day, the copper sulfate was still white. However, in the case of the methyl hydrogen sulfate supporting electrolyte solution 3.6 grams of water per liter were formed by the reaction of methanol and sulfuric acid. This water was not removed. Y
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The current-voltage curves were obtained with a Leeds & Northrop Electrochemograph. The m and t values for the capillary were determined a t 25' C. with an open electrical circuit and the tip dipping into a 0.3 M solution of lithium chloride in a mixture of equal volumes of absolute methanol and benzene. The values of m and t were 2.76 mg. per second and 2.10 seconds, respectively. Therefore, m2/3t ' / G was 2.13 mg.2/a t -lI2. At -0.70 volt with a closed circuit m and t were 2.10 mg. per second and 1.56 seconds. At this potential m2/a t 1 / 6 was 1.77 mg.2/3t-l/*. A short drop time was used because it completely eliminated oscillations in the polarograms recorded by the Electrochemograph. Two types of electrolysis cells were used. One utilized a mercury pool anode a proximately 6.2 sq. cm. in surface area. Results obtained wit{ this cell were satisfactory only in a neutral supporting electrolyte solution because the potential of the pool did not remain constant in acidic or alkaline solutions. In the alkaline solutions the peroxides reacted with the mercury of the pool. The other cell was an H-cell of the type described b Lingane and Laitinen (4),which utilized an external saturatedrcalome1 half-cell. The H-cell gave reproducible curves in all three electrolytes over the entire range of voltage which could be used with these electrolytes. The H-cell was used for all data reported herein except those dealing with gasoline peroxides. The polarograms of gasoline peroxides were obtained with a mercury pool anode before it was learned that the H-cell gave more satisfactory results. Solvents. The most suitable solvent investigated for use in studying autoxidation of fats was a solution of e ual volumes of methanol and benzene. Mallinckrodt's analytic3 reagent absolute methanol was satisfactory without further purification. Barrett & Company's thiophene-free benzene was also satisfactory as received. Two other brands of thiophene-free benzene 1
contained an impurity which interfered, but could be removed by vigorously shaking 1 liter of benzene with 100 ml. of concentrated sulfuric acid in a separatory funnel for 10 minutes a t room temperature, drawing off the sulfuric acid, and distilling the unreacted benzene.
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Residual Current Curves for Three Electrolytes
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Supporting Electrolytes. A supporting electrolyte solution must possess two properties if it is to be useful for the determination of fat peroxides. It must be capable of dissolving appreciable quantities of fat, and it must offer low electrical resistance, a property that was found very important for the production of polarographic waves with desirable characteristics. Solutions of high resistance gave waves which were drawn out over a considerable voltage range and usually had pronounced maxima. Reasonably satisfactory curves were not obtained with solutions which caused the minimum resistance of the unpolarized electrolysis cell appreciably to exceed 4000 ohms. A lower resistance is desirable. The resistance of the cells used was 3500 to 4000 ohms. Three supporting electrolytes, which differed markedly in
Present address, Research Laboratory, Kingan and Co., Indianapolis 6,
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Figure 2. Reduction Curves of Lard Peroxides in Three Electrolytes H-cell used for all three curves
acidity, gave information that was useful in studying the products of autoxidation of fats and related materials. Figure 1 shows the residual current curves for these supporting electrolyte solutions when a saturated calomel H-cell was used. Methyl Hydrogen Sulfate Supporting Electrolyte Solution. The strongly acidic supporting electrolyte used was a 0.2 M solution of methyl hydrogen sulfate in equal volumes of absolute methanol and benzene. It was prepared by adding 0.2 mole of analytical reagent grade sulfuric acid to 500 ml. of methanol and 500 ml. of benzene in a flask and refluxing the resulting solution for 30 minutes or longer. Titration indicated that the sulfuric acid was quantitatively converted to methyl hydrogen sulfate by this procedure. Lithium Chloride Supporting Electrolyte Solution. A satisfactory neutral supporting electrolyte was a solution of 0.3 M lithium chloride in equal volumes of absolute methanol and benzene. It was prepared by adding 12.72 grams of lithium chloride (c.P.) to a solution containing 500 ml. of methanol and 500 ml. of benzene. A study of various electrolyte concentrations showed that higher concentrations of lithium chloride decreased the resistance of the cell and somewhat improved the shape of the waves obtained. However, as the salt concentration was increased, the solubility of fat in the solution decreased. Because a 0.3 M solution of lithium chloride mould dissolve only about 2y0 of fat, more highly concentrated electrolyte solutions were not found practicable. Lithium Methoxide Supporting Electrolyte Solution. A 0.1 .If solution of lithium methoxide was prepared by immersing a 1- to 2-gram piece of pure lithium metal in methanol until the surfaces became shiny, and then quickly transferring the metal to a flask containing about 400 ml. of methanol into which hydrogen was continuously bubbled. After the reaction was complete, an aliqupt of the solution was diluted with water and titrated with standard hydrochloric acid. The lithium methoxide solution was then diluted to exactly 0.2 M with methanol. The 0.2 M solution was diluted with an equal volume of benzene. Sodium methoxide and lithium methoxide electrolyte solutions gave similar reduction curves with rancid lard. When a 0.1 M solution of either electrolyte was used, half-wave potentials were identical and wave heights were only slightly different. However, the maximum which appeared a t - 1.0 volt with some samples of rancid lard was larger and more frequently present when the sodium methoxide electrolyte was used than when the lithium methoxide electrolyte was used (Figure 2). Therefore, a 0.1 M lithium methoxide electrolyte solution was used in this study. A 0.1 M concentration of lithium methoxide was chosen because that concentration gave waves of desirable shape without excessive destruction of peroxides due to the high alkalinity of the solution. Lower concentrations resulted in cells of excessively high resistance. Higher concentrations decreased the height of the maximum which occurred a t -0.1 to -0.2 volt and also of the small wave on which the maximum appeared (Figure 2). PROCEDURE
Transfer 10 ml. of electrolyte solution to the cathode compartment of the H-cell. Bubble oxygen-free nitrogen through the
solution for 1 minute, then add 20 to 200 mg. of oxidized fat, depending upon the peroxide content of the fat. Fit a cork on thc capillary in the correct osition to stopper the H-cell loosely when the capillary is lowerecfinto the solution to be analyzed. Continue to bubble nitrogen through the solution for 2 minutes, then pass nitrogen over the surface of the solution during the remainder of the time required to obtain the polarogram. After the polarogram has been obtained, rinse the capillary and the H-cell with methanol. Connect the cathode compartment of the H-cell to a vacuum line and evacuate for at least 10 minutes. This step is essential if reproducible results are to be obtained. If the H-cell is not to be used again for several hours, fill the cathode compartment with aqueous potassium chloride solution. The peroxide numbers given in this paper were determined by the Wheeler method ( 6 ) , which is based on the oxidation of iodide ions in a chloroform-acetic acid solution. When the peroxide number of oxidized methyl linoleate was determined, the method was modified to exclude all oxygen from the reagents and the atmosphere over the reaction mixture, and the reaction time was extended to 30 minutes. The peroxide number as used in this paper is the number of millimoles of peroxide oxygen per kilogram of fat or per liter of gasoline or ether. EXPERIMENTAL
Fat Peroxides. The oxidized fats used in these studies were prepared by weighing 10 grams of fat into a 6-cm. Petri dish and placing it in a 100" C. oven for a suitable length of time. The concentration of fat used in the electrolyte solutions was varied with the degree of oxidation of the fat. In early stages of oxidation, 2% of fat was dissolved in the electrolyte solution. At this concentration it was possible t o obtain significant waves for autoxidation products before t:ie peroxide number reached 1,O.
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Figure 3. Relation between Wave Height and Concentration of Lard Oxidized to Peroxide Number 40
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Lithium methoxide supporting electrolyte. Wave height measured at -1.4 volts -I-x Lithium chloride supporting electrolyte. Wave height measured at -1.7 volts
The height of the waves given by 0 to 294 of rancid lard in either the lithium chloride or the lithium methoxide electrolytes was directly proportional to the concentration of rancid lard when the wave height was measured a t - 1.7 and - 1.4 volts, respectively (Figure 3). -4 similar relationship could not be accurately established in the methyl hydrogen sulfate electrolyte because when the wave a t about -0.8 volt was large, it did not reach a plateau before the hydrogen wave began. However, wave height was roughly proportional to the concentration of rancid fat. Solutions of oxidized fat in the electrolytes did not change significantly on standing 30 minutes a t room temperature. However, the characteristics of the curves were appreciably altered if the solutions were allowed to stand for several hours. Consequently, solutions were always prepared immediately before the current-voltage curves were obtained. Solutions of oxidized
ANALYTICAL CHEMISTRY
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fat in the strongly alkaline lithium methoxide electrolyte were observed to change less rapidly than the neutral or acidic solutions. However, because only two waves were obtained from lard peroxides in the lithium methoxide solution, one type of peroxide may have been almost instantly destroyed in the alkaline medium. This seems possible in light of the fact that benzoyl peroxide gave no wave in the alkaline electrolyte solution but did give two waves in either neutral or acidic solution (Figure 6). Typical currentrvoltage curves for oxidized lard in acidic, neutral, and alkaline supporting electrolyte solutions are shown in Figure 2. Each electrolyte solution contained 0.5% of lard oxidized to a peroxide number of 48. The curves have been corrected for the residual current obtained with the electrolyte alone. The curves of fresh fats did not differ significantly from the curves of the electrolyte alone. The potentials are referred to the saturated calomel electrode but are not corrected for the liquid junction potential. The potentials given elsewhere in this report, with the exception of the figure concerning gasoline peroxides, are also referred to the saturated calomel electrode and are uncorrected for the ZR drop of the cell or liquid junction potentials. Reference to the residual current curves of Figure 1 will show that the correct position for the beginving of the first wave on each of the three curves is somewhat in doubt because in each case the curves for the electrolyte solution alone began to change rapidly a t potentials which were more negative than the beginning of the wave for the peroxides. The small maximum a t -0.19 volt which follows the large maximum in the curve obtained with the lithium methoxide electrolyte is due to the characteristics of the recording instrument and does not accurately represent current-voltage relationships on this portion of the curve.
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PEROXIDE V A L U E
Figure 4.
Relation between Peroxide Number and Wave Height
Data obtained from methyl linoleate oxidized at 40" C. Concentration of methyl linoleate in 0.3 M lithium chloride, 1 %
Similar characteristic curves have been obtained with refined corn, cottonseed, soybean, peanut, and coconut oils. The method is currently being applied to the study of autoxidation of edible fats and related substances. Relation of Peroxide Number to Wave Height. The relation that exists between peroxide number and wave height is shown in Figure 4. On the vertical axis is plotted the height of the waves obtained when 1% solutions of oxidized methyl linoleate in the lithium chloride supporting electrolyte were analyzed. The polarograms of the peroxides of methyl linoleate were similar to those of lard peroxides shown in Figure 2. If the polarographic and the chemical methods deterniincd the same structures, a linear relation would exist. The lon-er line, which is nearly parallel to the horizontal axis of Figure 4, shows the relation between peroxide number and the height of the first small wave measured a t -0.18 volt. The broken line indicates the height
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Figure 5. Ether Peroxides Isopropyl ether, peroxide number 33, lithium chloride supporting electrolyte Isopropyl ether, peroxide number 33, lithium methoxide supporting electrolyte n-Butyl ether, peroxide number 13, lithium methoxide supporting electrolyte Saturated calomel H-cell used for all three curves
of the second small wave measured a t -0.30 volt. ,4 linear relation appeared to exist between the height of these two small waves and peroxide value. However, the reproducibility of the height of these waves was only about *20%. Consequently, deviations from linearity would not have been detected unless the deviations were rather large. A linear relation existed between the wave height measured a t - 1.50 volts and peroxide value until the peroxide value reached 275. Thereafter, the slope of the line decreased, indicating that the chemical method determined one or more structures which the polarographic method did not. The height of the large wave was reproducible to within * 5 % . Ether Peroxides. Peroxides were removed from samples of three ethers by the chromatographic technique described by Dasler and Bauer ( 1 ) . Autoxidation was accelerated by bubbling air through the samples during refluxing. Before oxidation, the ethers gave no waves with any of the electrolytes. The methyl hydrogen sulfate electrolyte gave no useful wave with ethyl, isopropyl, or n-butyl ether peroxides. The lithium chloride and lithium m e t h o d c electrolytes each gave waves with all these ~)croside-cont,ziniiiIg ethers. The curve given by isopropyl ether peroxides in the alkaline electrolyte differs uniquely from all other peroxides which the authors have studied, in that a negative current was obtained between about -0.1 and -0.26 volt (Figure 5). This was observed on two occasions with isopropyl ether from different sources. S o plausible explanation is offered for this anomalous result. The curve for n-butyl ether in lithium methoxide is included in Figure 5 to show the differences in these two ether peroxides. The general shape of the curve given by isopropyl ether peroxides in the lithium chloride electrolyte was also obtained with ethyl and n-butyl ethers. Benzoyl Peroxide. Benzoyl peroxide was included in this study because it has been used for comparative purposes in fat autoxidation studies. Figure 6 shows the curves obtained from 0.001 % benzoyl peroxide using three electrolytes. Apparently benzoyl peroxide was very rapidly destroyed in the alkaline electrolyte solution. Gasoline Peroxide. A sample of gasoline was distilled to remove inhibitors, Autoxidation was accelerated by bubbling air slowly through the solution while it was refluxed. Peroxide numbers and current-voltage curves were obtained at intervals. Some of the curves are shown in Figure 7. The mercury pool was used as the anode and the potentials given are referred to it. Much gum formation occurred before the gasoline reached a peroxide number of 43. I t is possible that these polymerization
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V O L U M E 2 1 , NO. 7, J U L Y 1 9 4 9 products may have been responsible for the pronounced changes in the character of the curves which were obtained from the highly oxidized samples. The three supporting electrolytes were not equally useful for polarography of gasoline peroxides. Unoxidized gasolines gave a wave when the methyl hydrogen sulfate electrolyte was used, and oxidized gasoline gave no additional useful waves. 'Cnoxidized gasoline gave no wave with either the lithium methoxide or the lithium chloride supporting electrolytes, but gasoline peroxides gave waves in each of these electrolytes. When 10 nil. of gasoline were added to 100 ml. of the electrolyte, 0.1 niillimole of peroxide oxygen per liter of gasoline could be detected. The gasoline for which the curves are given was probably n high olefin gasoline. Another brand of gasoline which was much more stable toward autoxidation gave very different curves. Yo further work was done with gasoline peroxides, because the authors were interested principally in showing that the method constitutes a useful tool for the petroleum laboratory as well as for the fats lahointorv. DI scus SIOA
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The procedure and reagents used in this study frequently do not give polarograms in which the waves are as well defined and as regular in shape as those usually obtained in aqueous solutions. Consequently, no attempt has been made to give precise values for half-wave potentials. The values given have been corrected for residual current but not for the I R drop across the cells. Because the resistance of the cells used was approximately 4000 ohms, the ZR drop would be approximately 4 millivolts per microampere.
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The procedure outlined has been used for a more extensive study of oxidized fats and fatty esters (3). It is suggested that this technique may profitably be used in the study of reaction mechanisms, thc mode of action of inhibitors, and the kinetics of autoxidations.
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Scvcral masiniuni suppressors were tried, but nolle \viis cff ective. Gelatin was not soluble to any appreciable extent i n the clectrolytes. Methyl red and methylene blue were completely ineffective. Salts such as aluminum acetate and trisodium phosphate were also ineffective. The waves for fat peroxides which appeared a t -0.12 and -0.23 volt in the lithium chloride electrolyte are too close together to provide good precision in measuring either wave. Howevcr, the sum of the heights of these two waves or the height of the third wave could be determined with a precision of * 5 % , unless very low concentrations were to be determined. The sensitivity of the method permits detection of less than 1 milliequivalent of peroxide per kilogram of fat. The solubility of gasoline or ethers in the electrolyte solutions is greater than the solubility of fats. Consequently, 0.1 milliequivalent of gasoline or ether peroxides per liter can be detected. The lithium chloride supporting electrolyte solution has proved to be the most useful of the three that were used. It gives waves that appear to correspond to the viavrs obtained i n the alkaline and acidic electrolytes without the interference of the hydrogen wave obtained in acidic solution or the large masimum and probably peroxide destruction obtained with the lithium methoxide solution.
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Figure 7. Gasoline Autoxidation Products Lithium chloride supporting electrolyte. Mercury pool anode. P.N. =Peroxide number
SU\I\lARY
h polarographic procedure is outlined which permits polarographic study of water-insoluble peroxides, such as those which form in fats, ethers, and hydrocarbons. With each of these substances, more than one wave is obtained for the autoxidation products. The precision of measurement is within *5% for fats with a peroxide number of 5 or higher. A linear relation exists between the peroxide number determined by a modification of the Wheeler method and wave heights until the peroxide number exceeds 250. Thereafter, wave heights do not increase as rapidly as peroxide numbers.
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Figure 6. Benzoyl Peroxide in Three Electrolytes
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ACKhOWLEDGMENTS
Thc authors wish to acknowledge the many helpful suggestions which were made by James B. Xartin of the Agricultural Cheniistry Department, Purduc Univcrsity, during the development of this method. They also thank H. A. Laitinen, of the Departiiient of Chemistry, University of Illinois, for suggesting the usc of a mixture of methanol and benzene as a solvent and for other helpful advice>. L I T E R i T U R E CITED
Daslq, Waldemar, and Bauer, C. D., IND.EXG.CHEM.,A N ~ L . ED.,18, 53 (1946).
Dobrinskaya, A. A , and Neirnan, M.B.,Zaaodskaua Lab., 8, 2SO (1939).
Lewis, W.R., and Quackenbush, F. TV., J . Am. Oil Chem. Soc., 26, 53 (1949).
Lingane, J. J., and Laitinen, H. .1.,IND.ENG.CHEY., ANAL. ED.,11,504 (1939). Shtern, V., and Pollyak, S., Acta Physicochim. U.R.S.S., 11, 797 (1939).
Wheeler, D. H., Oi2 & Soap, 9, 89 (1932). RECEIVED June IG, 1048. Journal Paper 354 of the Purdue University -4gricultural Experiment Station. The subject matter of this paper way undertaken in cooperation with the Committee on Food Research of the Quartermaster Food 6: Container Institute for the Armed Forces. Thc opinions or conclusions contained in this report are those of the authors. They are not to be construed as necessarily reflecting the views or endorsement of the War Department.
