Spectrophotometric Hydroperoxide Determination by the Use of Triphenylphosphine ROBERT A. STEIN and VlDA SLAWSON Department of Biological Chemistry, University o f California, 10s Angeles 24, Calif.
b A spectrophotometric analysis of hydroperoxides is described. The decrease in absorbance at 260 mp when triphenylphosphine is oxidized to triphenylphosphine oxide by a hydroperoxide is a quantitative measurement of the hydroperoxide present. Quantitative values for autoxidized fats, H Z 0 2 , t-butyl hydroperoxide, and benzoyl peroxide are compared with those obtained by iodometric and ferric thiocyanate methods. Di-s-butyl peroxide does not react with the phosphine under the conditions specified.
T
BE REACTION of triphenylphosphine
(TPP) with peroxides has been shown to yield triphenylphosphine oxide (TPPO) (6, 10). Hydroperoxides react rapidly with the phosphine (2),whereas diperoxides must first dissociate into alkoxy radicals before reaction occurs (10). I n this report we will show that under conditions that do not give an homolytic cleavage of diperoxides into radicals, i t is possible to measure hydroperoxides quantitatively. The ultraviolet absorption maxima of TPP and TPPO are sufficiently different so that the reaction may be followed spectrophotometrically. This method was used to determine a wide range of peroxide values in peroxidized fats, for hydrogen peroxide, benzoyl peroxide, and t-butyl hydroperoxide, and for demonstrating the lack of reactivity in di-s-butyl peroxide. EXPERIMENTAL
Reagents. TPP (Matheson Coleman & Bell, No. TX-1490) was recrystallized from absolute ethanol, ems=, 1.10 X l o 4 a t 260 mp [Literature value emax, 1.10 X lo4 a t 261 mp. ( B ) ] . TPPO was prepared by the oxidation of TPP by 30% H202 in CHClracetone solution 1:2. e, 1.52 X 10' a t 260 mp. Isopropyl alcohol, reagent grade (Baker and Adamson), was deoxygenated by bubbling nitrogen through it for 15 minutes on the steam bath. Cottonseed oil and corn oil were obtained from retail food stores. Soybean oil (Vegetable Oil Products, Wilmington, Calif.) was oxidized at 60' C. by aeration in the presence of 2 mg. per kg. each of CuCll and FeC18. &Butyl hydroperoxide which was synthesized (7) wm 1008
ANALYTICAL CHEMISTRY
assayed by iodometry and found to be 93.0%, Di-s-butyl peroxide was synthesized (11). Hydrogen peroxide was 30% (Merck Co.). Benzoyl peroxide (Matheson Coleman & Bell, No. BX470). Di-t-butyl-;a-cresol (Koppers Co., Inc.) was used as the antioxidant. Ultraviolet absorption spectra were measured in 1-cm. silica cells in a Beckman DU spectrophotometer. Reference Peroxide Methods. Iodometric 1: The method of Polister and Mead (9) was used with a modification to utilize isopropyl alcohol ae, solvent and a 12-minute reaction time a t 60" C. Iodometric 2: The official A.O.C.S. method (8). Ferric thiocyanate: The aerobic procedure of Glavind and Hartmann (4) was modified to use chloroform-methanol 8 :3 instead of chloroform-ethanol 8 :3. Triphenylphospbine Peroxide Method. The TPP reagent consists of 1 mg. of TPP and 0.1 mg. of antioxidant (di-t-butyl-p-cresol) in 1 ml. of isopropyl alcohol. This is conveniently prepared by weighing 25 mg. of T P P into a 25-ml. volumetric flask. The antioxidant, 2.5 ml. of a n isopropyl alcohol solution containing 1 mg. per ml. of di-&butyl-p-cresol, is added and the solution made up to volume with isopropyl alcohol. A peroxide sample containing about 2 x 10-2 meq. of peroxide is weighed into a 10ml. flask and dissolved in 1 ml. of isopropyl alcohol. After addition of 1 ml. of TPP reagent, the flask is stoppered, swirled to mix, and inverted once to rinse the neck. The mixture is allowed to stand for 20 minutes at room temperature and brought to volume by the addition of isopropyl alcohol. A secondary dilution of 1 ml. to 10 ml. is made for spectrophotometric reading. The
absorbance of the reaction mixture is measured a t 260 mp against isopropyl alcohol. A sample blank composed of a peroxide sample in isopropyl alcohol is measured at 260 mp to correct for the initial absorbance of the samples. A reagent blank consisting of 1 ml. of T P P reagent and 1 ml. of isopropyl alcohol and subsequently treated as a sample is prepared with each series of samples. Unless otherwise indicated, all figures represent the average of two determinations. Calculations. Decrease in the absorbance of the reaction mixture at 260 mp is used as B measure of hydroperoxide originally present. Since oxidation of TPP yields TPPO, which also has a significant absorbance at this wavelength, allowance must be made for contribution of TPPO to the absorbance of the total reaction mixture. Peroxide value (meq./kg.) = A , A* - A 21.1 sample weight
+
where A , and A are the absorbances of the reagent blank and the reaction mixture, respectively. A , is the absorbance of the sample blank calculated for the weight of sample in the reaction mixture. The 21.1, a constant, is a quotient obtained by dividing 2 X lo5 (to convert to meq./kg. for the dilution specified) by the difference between the molar absorptivity of TPP (1.10 X 104) and TPPO (1.52 X lo3) a t 260 mp. RESULTS A N D DISCUSSION
Comparison of Methods of Peroxide Determination. Soybean oil t h a t was autoxidized t o various peroxide values
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Table 1.
Sample 1
Peroxide Values of Oxidized Soybean Oils
TPP n 48
Meq. of peroxide per kg. of oil Iodometry 1 0 7
10.8 30.3 4a 5 6
7
8 9
64.7 90 .o 269 594 656 1561
98.5 278
577
678 1624
Mixtures of samples 1 and 5. b Single determination on 0.5-gram sample. a
2 0.6 12.2 30.2 263 380 (5789 398 (685b)
...
Ferric thiocyanate 1.03 16.8 44.4 96.9 170 488 1014 1190 2163
was analyzed by the TPP, two iodometric, and the ferric thiocyanate methods. T h e values for the two iodometric methods deviate from one another in a random manner. T o be particularly noted is the fact that with but one exception the phosphine procedure gave lower results than iodometry (Table I). It was previously noted (2) and verified in this study (see below) that a disubstituted peroxide does not react with TPP under the experimental conditions used here. Since the diperoxide does react with iodide t;o a slight extent, it may be considered that the differences in the values obtained by TPP and those obtained by iodometry are a measure of the disubstituted peroxides reduced by iodide ion. Thus, it may be concluded that the TPP method offers a more accurate measurement of hydroperoxide content than does iodometry. The values obtained by the ferric thiocyanate method itre much larger than those obtained by iodometry. The extreme susceptibility of this method to interference by atmospheric oxygen can result in values as high as three times those obtained by iodoinetry (1). Samples of commercial cotton seed and corn oils from different sources were analyzed for peroxide values to determine the importance of composition of the oil on the method (Table 11). The TPP values are not clmsistently lower than the iodometric values as is the case with the oxidiwd soybean oils. In fact, the TPP values are slightly larger than the iodome1,ricvalues for the cotton and corn oils. This may represent the extent to which iodine is absorbed by the double bonds of the oil during the analysis. 1 2 the cotton seed and corn oil, the peroxides developed spontaneously and a t these low levels are expected to consist of hydroperoxides which would be ,znalyzed by both TPP and iodometry. The comparable low levels of peroxide in the soybean oil were made by dilution of an oil with a peroxide value of 90 with unoxidized oil. At the level of oxidation represented by peroxide value of 90, polymeric peroxides are probably present (9). A reduction of these diperoxides by iodide but not by TPP will cause the observed difference in values. The Effect of Iteagent/Sample Ratio. Varied amounts of soybean oil were analyzed for peroxide by a constant amount of TPP in order to determine the influence of the reagent/ sample ratio on t h e reaction (Table 111). As long as 869b or less of the reagent was consumed during the reaction, the determination had a precision of 3~0.4. If more than 86% of the reagent is required to reduce all of the hydroperoxide, the reaction appears to be incomplete in the 20-minute
Table 11.
Oil Cottonseed 1 Cottonseed 2 Cottonseed 3 Corn 1 Corn 2 Single determinations.
Effect of
Oil Variety on Peroxide Value
Meq. of peroxide per kg. of oil Iodometry TPP 1 2 14.7 29.7 2.34 3.87 9.78
Table Ill.
Effect of Changing Reagent/ Sample Ratio Per cent of Sample wt. 4.06 pmole Peroxide
soybean
oil, gram 0.030
0.060
0.142 0.150 0.171 0,199 0.225 0.228 0.256 0.285 0.300 a
reagent consumed
value" nieq./kg.
10 20 50 52 60 69 76 78 86 91 95
27.4 27.4 28.5 28.0 28.5 28.1 27.6 27.8 27.3 25.9 25.6
Single determinations.
reaction time with the resultant low value for the peroxide. Reaction of Simple Peroxides. Using the concentrations of sample and TPP reagent specified above, hydrogen peroxide, t-butyl hydroperoside, and benzoyl peroxide gave low values. By increasing the concentrations of TPP and peroxide 10-fold, b u t leaving the antioxidant concentration unchanged, the reaction went t o completion and expected va,lues were obtained (Table IV). The reason for incomplete reaction of t b u t y l hydroperoxide at the lower concentration is attributed to the well known dependency of reaction rates on concentrations. Time studies with the concentration given in the experimental section showed that the apparent t-butyl hydroperoxide concentration increased
Table IV.
14.0 26.6 2.2 2.2 8.O
17.8 28.7 2.02" 1.41" 7.87
Ferric thiocyanate 26 .O 41 .O 2.87 1.92 13.4"
with reaction time longer than the 20 minutes specificd. The apparent concentration increased from 54% at 20 minutes to 89% a t 105 minutes. The fat peroxides did not show this slower reaction rate, nor did a 10-fold increase in reactant concentration result in higher peroxide values. Di-s-butyl peroxide, which is a simple model of the di-secondary peroxides found in autoxidized fats, was unreactive using conditions outlined here for the TPP reaction. This evidence suggests that the diperoxides in autoxidized fats are unaffected by this reagent. Effect of Added Antioxidant. Alcoholic solutions of TPP are oxidized slowly to the oxide presumably by atmospheric oxygen. This change is taken into account by B comparison of the reagent blank. However, the addition of a n antioxidant decreases this autoxidation t o a negligible amount, and a reagent blank is unchanged after as long as three hours. Noninterfering Compounds. Several compounds with functional groups representative of possible autoxidation products from fats were tested for reaction with T P P . 12-Ketooctadec10-enoic acid, 9,ll-octadecadienoic acid, 2-butanone, and isobutyraldehyde caused no measurable oxidation of TPP in concentrations of sample and reagent specified in the experimental part. Adherence to Beer's Law. Mixtures of TPP and T P P O obeyed Beer's law over a concentrabion range greater than t h a t covered by the peroxider determinations.
Analyses of Simple Peroxides
Per cent of theoretical Iodometry TPP 2
Ferric Peroxide thiocyanate TPPa 30% HzOz 81.7 96.0 48.3 98.3 &Butyl hydroperoxide 54.0 95.5 95.9 100.3b Di-8-butyl peroxide0 0 2.1'4 ... Benzoyl peroxide 75 .gd 98.6 ... 102.8d a Concentration of reactants were increased to 10 times the prescribed concentrations. b Reaction conducted in 95% ethanol. c Iodometry 1 gave 1.7%, constant boiling HI at 60" gave 6-lOY0 in 45 minutes, and K I in refluxing isopropyl alcohol containing 1.5% acetic acid gave 8% in 24 hr. d Single determination.
VOL. 35, NO. 8, JULY 1963
1009
LITERATURE CITED
( 1 ) Chapman, R. A,, McFarlane, W. D., Can. J . Research B 21, 133 (1943). (2) Denney, D. B., Goodyear, W. F., Goldstein, B., J . Am. Chem. SOC.82, 1393 (1960).
(3) Farmer, E. H., Trans. Faraday SOC. 42,228 (1946). (4) Glavind, J., Hartmann, S., Acta C h m . Scand. 9,497 (1955). (5) Horner, L., Jurgeleit, Ann. 591, 138 (1955).
m7.,
( 6 ) Jaffe, H. H., Freedman, L. D., J . A m , Chem. SOC.74. 1069 (1952). ( 7 ) Milas, N. i., Surgenor,’D. M., Ibid., 68,205 (1946). ( 8 ) “Official and Tentative Methods of
the American Oil Chemists’ Society,” 2nd edition. A.O.C.S. Official Method Cd 8-53, 1960, American Oil Chemists’ Society, Chicago. (9) Polister, B. H., Mead, J. F., J. Agr.
Food Chem. 2. 199 11954). (10) Walling, C., Based&, 0. H., Savas,
E. S.,J . Am. Chem. SOC. 82, 2181 (1960).
(li)-Welch, F., Williams, H. R., Mosher, H. S., Ibid., 77, 551 (1955).
RECEIVEDfor review December 21, 1962. Accepted April 29, 1963. Investigation supported by Public Health Service Research Grant H-4120 from the National Heart Institute, Xational Institute of Health, U. S. Public Health Service, Rethesda, Md.
Spectrophotometric Titration of Some Divalent Metals in Chloroform-Isopropyl Alcohol Solvent Mixture ROY K. BEHMl and REX J. ROBINSON Department o f Chemistry, University o f Washington, Seattle 5, Wash.
b Dimethylglyoxime was used as a titrant of micro amounts of nickel(l1) and cobalt(l1) in a solution of 1: 1 chloroform-isopropyl alcohol mixture. Spectrophotometric titrations of 0.4 to 4 pmoles of nickel(l1) in 40 ml. of solvent were done with an over-ail precision of 3 parts per thousand. Cobalt(l1) in amounts of 1 to 4.5 pmoles was titrated with a precision of 2.5 parts per thousand. Palladium(ll) and iron(l1) form chelates, but react too slowly for titrations. Copper(l1) could not b e titrated because a mixture of several species of chelates in equilibrium was formed. Copper(ll), iron (II), palladium(ll), and cobalt(l1) interfere in the titration of nickel(l1). These metals and nickel(l1) interfere in the cobalt titration.
T
HE METHOD of spectrophotometric titrations has been applied to the determination of metals with organic precipitating or chelating agents in nonaqueous solvents by Boyle and Robinson ( I ) , Marple, Matsuyama, and Burdett (S), and Takahashi and Robinson ( 4 ) . Nonaqueous solvents, especially organic solvents, are desirable because they are capable of dissolving the organic precipitating agent and the metal chelate formed. In addition, the metal ion can be titrated directly in the organic phase without further involvement with the aqueous phase. The solvent, when properly chosen, may also enhance the color development of the chelate. Organic precipitating agents are especially applicable for such titrations because of their specific and sensitive reactions with metal ions.
Present address, Eastman Kodalr Co., Rochester 3, N. Y.
1010
ANALYTICAL CHEMISTRY
Spectrophotometric end point detection can be used to good advantage either in the visible or ultraviolet region. The factors of solvent, reagent, and spectrophotometric titrations combine to provide a n effective means of titrating micro amounts of metal ions with high precision and accuracy. I n this work dimethylglyoxime was chosen as titrant because of its well known reactions with metal ions. A 1:1 mixture by volume of chloroform and isopropyl alcohol was chosen as solvent. EXPERIMENTAL
Reagents. Standard solutions of 0.1M nickel and copper were prepared by dissolving Baker’s analyzed reagent grade metal in a minimum amount of nitric acid and diluting t o volume with distilled water. A standard solution of 0.1M cobalt was made by dissolving Merck high purity nickel-free cobalt i n a minimum amount of hydrochloric acid and diluting t o volume with distilled water. A 0.01M palladium solution was made by dissolving Fisher Scientific Co. pure palladium chloride in distilled water containing a minimum amount of hydrochloric acid to facilitate solution by forming PdC14-2 and then diluting to volume with distilled water. A solution of iron was prepared 0.01M by dissolving Baker’s analyzed reagent grade iron wire in a minimum amount of perchloric acid and diluting to volume. Nickel and palladium solutions were standardized gravimetrically with dimethylglyoxime. The cobalt solution was standardized gravimetrically as cobaltous sulfate. Dilute standard solutions were prepared by appropriate dilution of the previous standard solutions. Solutions of gold chloride made with Fisher Scientific Co. c.P., AuC13.HCl 3H20 and chloroplatinic acid made with Baker’s 5% chloroplatinic acid solution, purity unspecified, were not standardized since
-
preliminary exploratory tests proved unsuccessful. Eastman Kodak White Label dimethylglyoxime, melting point 239’ C., was recrystallized four times from a 2 : l acetone-water mixture and was stored over anhydrous calcium sulfate. Standard solutions of dimethylglyoxime, ranging in concentration from 0.0008 to 0.005M, were prepared by weighing definite amounts, dissolving, and diluting to 500 ml. with chloroformisopropyl alcohol solvent. Standardization against standard metal solutions showed that dimethylglyoxime solutions were stable for a t least three weeks. Chloroform was Mallinckrodt reagent grade. Isopropyl alcohol was Eastman Kodak White Label, 98 to 99%. n-Butylamine (Matheson Coleman and Bell), boiling range, 76’ to 78” C., was distilled from an all glass apparatus. Cyclohexylamine (Matheson Coleman and Bell), was treated with sodium and distilled from an all glass apparatus, boiling temperature 135’ C. Acetic acid was Merck, reagent grade, 99.8%. Apparatus. A Beckman Model DU spectrophotometer was used for optical measurements, except for the spectrum of dimethylglyoxime which was taken on a Cary Model 11 recording spectrophotometer. Spectra were taken with pure solvent as reference in matched, I-em. silica cells. T h e metal chelate solutions were prepared by evaporating pipetted amounts of the aqueous metal ion solutions to crystals, dissolving, adding chelating agent, and diluting to volume with nonaqueous solvent. The spectrophotometric titrations were made with the D U spectrophotometer using the modified cell compartment as described by Boyle and Robinson ( I ) , eycept that magnetic stirring was used. The titrant was measured with a 10-ml. Fischer and Porter microburet having a Teflon stopcock plug. The buret was attached to a 1000-ml. flat-bottomed flask connected to two microl)nbble
