521
V O L U M E 2 2 , NO. 4, A P R I L 1950 ture 3, where ethyl 6-nitrobenzothiazolyI-2-sulfide is the minor component. The only line recorded that might belong to the ethyl derivative is 3.41, but this also corresponds to the major line of the n-butyl derivative, the major component. It is safe to assume that if the line was due to the ethyl derivative there would, also, be present in the pattern a line equivalent t o the 5.25 spacing which is a very intense line, as can be seen from Table I. It may be concluded from the data that extreme purity of the crystalline samples was not necessary to obtain distinctive diffraction patterns. With improved techniques, it is possible that 25% of a second phase might be detected. The error in measurement of d increases from *0.05 A. a t 3.0 A. to 1 0 . 2 0 A. a t 16.0 A.
LITERATURE CITED
(1) Bmgg, W.L., J . Sci. Instruments, 24, 27 (1947). (2) Clarke, G. L., Kaye, W. I., and Parks, T. D., IND. ENG.CREM., ANAL.ED.,18,310-13 (1946). (3) Cutter, H. B., and Golden, H. R., J . Am. Chem. Soc., 69,831-2 (1947). (4) Edwards, Olive, and Lipson, H. J., J . Sci. Instruments, 18, 131 (1941). (5) Kersten and Maas, Rev. Sci. IpStruments, 4, 14 (1933). (6) McKinley, J. B., Nickels, J. E., and Sidhu, S. S.,IND.ENG. CHEY.,ANAL.ED., 16,304-8 (1944). (7) Matthews, F. W., and Michell, J. H., Ibid., 18,662-5 (1946). (8) Soldate, Albert, and Noyes, R. hl., ANAL.CHEM.,19,442-4 (1947). (9) Weissberger, Arnold, “Physical Methods of Organic Chemistry,” Vol. I, pp. 585-620, Kew York, Interscience Publishers, 1945. RECEIVED hfaY 6, 1949.
Polarography of Chloroform and Carbon Tetrachloride I. M. KOLTHOFF, T. S. LEE’, DANICA STOCESOVA, AND E. P. PARRY University of Minnesota, Minneapolis, Minn. Both carbon tetrachloride and chloroform can be polarographically reduced. Carbon tetrachloride gives two 2-electron reduction waves, the first corresponding to a reduction to chloroform and the second to a reduction to methylene chloride. Chloroform gives one wave which coincides with the second reduction wave of carbon tetrachloride. The height of the waves was found to be proportional to the concentration of the halogenated methane. Procedures are given for the determination of chloroform and carbon tetrachloride separately and in mixtures of the two. A solvent composed of 2 volume parts of methanol and 1 volume part of water with a supporting electrolyte of 0.1 M tetramethylammonium bromide was found to be most satisfactory.
N
0 systematic study of the reduction of halogenated methanes
a t the dropping mercury electrode has previously been made (9). Statements made by Matheson and Nichols (6) concerning carbon tetrachloride and by Stone (8) concerning carbon tetrabromide indicate that these compounds are polarographically reducible. I n the investigation described below it was found that carbon tetrachloride is reduced a t the dropping mercury electrode to chloroform, and chloroform is reduced to methylene chloride. Thus carbon tetrachloride yields two polarographic waves, the second of which is similar to the single wave given by chloroform. The dependence of the height of the polarographic waves on concentration and the dependence of the half-wave potentials on concentration and pH were studied. The results of this investigation lead to the conclusion that the polarographic method provides a simple and reliable means of identifying and determining carbon tetrachloride and chloroform. The polarographic method was used to determine the solubility of these two compounds in pure water and to determine small amounts of carbon tetrachloride in the presence of large amounts of chloroform. As will be shown in a subsequent publication, other halogenated methanes are also reducible a t the dropping mercury electrode. The ease of reduction is in the order: iodinated methanes > brominated methanes > chlorinated methanes. The ease of reduction also increases with the number of halogen atoms joined to the carbon atom-e.g., carbon tetrachloride is more easily reduced than chloroform. EXPERIMENTAL
Apparatus. A Sargent Model X I 1 HeyrovskG polarograph was used to record polarographic waves. The measurements of diffusion current constants and the determinations of carbon tetrachloride and chloroform were made with a manual polarograph ( 5 ) . Two capillaries were used in the investigation. The value of r n a / 3 t l /e of capillary A under the experimental conditions was 1
Present address, University of Chicago, Chicago 37, Ill.
2.060 mg.2/3sec.-1/2 a t -0.5 volt LS. S.C.E., 2.030 a t -1.2 volts, and 1.870 a t -2.0 volts. The value of m2 l 3 t 1’6 of capillary B was 1.791 at -0.5 volt, 1.770 a t -1.2 volts, and 1.625 a t -2.0 volts. A11 mercury electrode potentials were measured against an external saturated calomel reference electrode. I n the experiments carried out at 25’ C. the polarographic cell was immersed in a thermostated (*O.l”) water bath. For experiments a t 0 ” a bath of water and cracked ice was used. Reagents. Mallinckrodt’s reagent grade carbon tetrachloride and Merck’s reagent grade chloroform were used without further purification. In the early experiments the chloroform and carbon tetrachloride were purified by fractional distillation. However, the purified and the untreated reagent grade compounds gave identical results in polarographic experiments. (Reagent grade chloroform usually contains about 0.7y0 ethanol as an inhibitor for decomposition.) Reagent grade methanol was used. The tetramethylammonium bromide and calcium chloride used for supporting electrolytes were both polarographically pure. Procedure. Because carbon tetrachloride and chloroform are only slightly soluble in water, a solvent consisting of 2 volume parts of methanol and 1 volume part of water was used. Inasmuch as oxygen is easily reduced a t the dropping mercury electrode, it was necessary to remove dissolved oxygen from the solutions under investigation. Sodium sulfite, which is sometimes used for this purpose, xyas not effective because the alcohol greatly retarded the reaction of sulfite with oxygen. I t is not feasible to remove dissolved oxygen by bubbling nitrogen directly through the solutions of the halogenated methanes because of the high volatility of these compounds. The following technique was therefore adopted : Purified nitrogen previously saturated with the solvent was bubbled for 30 minutes through a polarographic cell, which contained the alcohol-water solvent and the supporting electrolyte. The flow of nitrogen was then interrupted and the nitrogen was allowed to pass sloi~lyover the surface of the solution. A small volume of a methanol solution of the halogenated methane was added from a pipet or microburet. The contents of the polarographic cell were stirred with a glass rod and the polarographic measurements were made immediately.
ANALYTICAL CHEMISTRY
522
Table I. Characteristics of Polarographic Waves of Carbon Tetrachloride and of Chloroform (at 25" C.)
CCII"
0.35 0.695 1.38 2.06 CHC1aC 0.33 0.665 1.33 a Capillary A used. b All values of diffusion Capillary B used.
...
...
-0.78 -0.75 -0.75 -1.69 -1.67 -1.88
I
4.076 4.23 4.24 4.23 4.35 4.37 4.29
-1.7fl -1.70 -1.73
..
..
..
7.47 8.10 8 28 8.17
.. .. ..
current corrected for reiidrial current.
-04
Concn.. Millimolar
Ill',
ra
Z,d/C,
ra./rnillimole
Ar. 5.11 CB('l3
-08
-12
-16
-20
-24
-02
-04
06
APPLIED P O T E N T I A L
Table 11. Dependence of Diffusion Current on Concentration (at 0" C.) Compound
/
0.632 1.26 1.88 2.50 3.11 3.73
4.59 4.82 4.85 4.87 4.75 4.74 Av. 4 . 7 7 a .411 values of diffusion current corrected for residual current. Capillary A used. b hIeasured a t - 1 2 volts u s . S.C.E. Measured a t -2.0 volts us. S.C.E. 2.OflC 6.06
9.14 13.18 14,80 17.70
I n the determination of chloroform and carbon tetrachloride the volume of methanol solution added should be less than 2 nil. (per 50 ml. of solvent) in order to have a negligible error due to oxygen. (Air-saturated methanol at 25" C. is about 0.002 M in oxygen.) Calibration curves are recommended in the determination of chloroform and carbon tetrachloride. This procedure effectively eliminates any error that might otherwise result from the presence of oxygen. An alternative procedure would be to apply a correction for the amount of oxygen introduced with the methanol solution. The correction can be found from a "blank" experiment, in which pure methanol is substituted for the methanol solution of carbon tetrachloride or chloroform. Great care must be taken in handling samples and solutions containing carbon tetrachloride and chloroform, to avoid serious loss by volatilization.
Figure 1. Polarogram of Carhon Tetrachloride and Chloroform I. 1.1 millimolar carbon tetrachloride 11. 1.0 millimolar chloroform 111. Residual current. Solvent 2 volume parts of methanol, 1 volume part of water, supporting electrolyte 0.1 M tetramethylammonium bromide IV. 1.53 millimolar lead nitrate in 0.1 .If potassium nitrate Temperature, 25O C. Caoillary A used
a small error in the value of the diffusion current, id, undoubtedly resulted from volatilization and loss of carbon tetrachloride, it is evident that the diffusion current is proportional to concentration. The value of the polarographic constant, id/Cm* 1st' 16, at 25'C. is about 4.2 pa. per millimole mg.Z/3sec.-l/*, compared with about 3.6 for benzoquinone in the same solvent. Inasmuch as the reduction of benzoquinone involves t R - 0 electrons and the diffusion coefficient of carbon tetrachloride is somewhat larger than that of benzoquinone, it can be concluded that two electrons are involved in the first step of the reduction of carbon tetrachloride. The value of the polarographic constant of the second LTave of carbon tetrachloridr is about 4.0, close to the value for the first wave; therefore, the second wave also corresponds to a reduction involving two electrons.
RESULTS AND DISCUSSION
Carbon Tetrachloride. I n Figure 1 (curve I) is shown a polarogram of carbon tetrachloride in the methanol-water solvent with tetramethylammonium bromide as supporting electrolyte. Carbon tetrachloride yields two waves of nearly equal height. The half-wave potentials of the two waves are -0.75 and -1.70 volts vs. the saturated calomel electrode. The waves are not nearly so steep as the n-ave for a reversible reduction, as can be seen by comparing the polarographic wave of lead in aqueous solution (curve IV) with the carbon tetrachloride waves. The equation of the wave is (4) 57
= TI/Z
-i -+ RT - In __ aF i id
where 01 has the value 0.23 instead of the theoretical value 2 for a reversible Pelectron reduction I n Figure 2 are shown polarograms of carbon tetrachloride solutions of varying concentrations. It can be seen that the half-wave potentials of the first and second waves are independent of the carbon tetrachloride concentration. The dependence of wave height on concentration is shown in Tables I and 11. The experiments of Table I were carried out a t 25" C., and although
o>
V.
APPLIED
POTENTIAL
(V.1
Figure 2. Dependence of Half-Wave Potentials of Carbon Tetrachloride on Concentration Solvent 2 t o 1 methanol-water. Electrolyte 0 . 1 M tetramethylammonium bromide. Temperature 25' C. Waves s t a r t a t - 0.20 volt I . 0.675 millimolar, sensitivity 1/100 11. 1.35 millimolar, sensitivity l / l O O 111. 2.01 millimolar, sensitivity 1/150 IV. Blank, sensitivity 1/70
The proportionality of diffusion current of the first carbon tetrachloride wave t o the concentration is also demonstrated in Table 11. These experiments were carried out at 0 " C. and the loss by volatilization was negligible. The effect of pH on the first carbon tetrachloride wave is shown in Figure 3. [The solutions for these experiments were prepared by adding 2 volumes of methanol to 1 volume of aqueous Britton-Robinson buffer solution ( 2 ) . The values of pH given in Figure 3 refer to the p H of the aqueous solution before addition of methanol.] The half-Tvave potential and the wave height do not change with pH in the range pH 4.6 to 11. I n solutions of low p H (4 or less) the reduction of hydrogen ion interferes with the carbon tetrachloride wave. Consequently,
V O L U M E 2 2 , NO. 4, A P R I L 1 9 5 0
523
the polarographic determination of carbon tetrachloride cannot be carried out in a solution more acid than pH 4. Chloroform. In Figure 1, curve 11, is shown the polarogram of chloroform in a methanol-water solution of tetramethylammonium bromide. The half-wave potential of chloroform is - 1.70 volts, the same as that of the second carbon tetrachloride wave. The dependence of the half-wave potential of the chloroform wave on concentration is shown in Figure 4 and Table I. It can be seen that the half-wave potential is independent of concentration of chloroform. The dependence of the height of the xave on concentration is shown in Tables I and .II. The data of Table I1 are more reliable because of the relatively low volatility of chloroform from the solution a t 0' C. The height of tlie wave is, within expel imental error, proportional to the concrnti ation.
If the potential of the mercury electrode is sufficiently negative, chloroform is also reduced. This reduction also involves two electrons. The over-all reaction is probably:
+ H' + 2e +CHzClz+ C1CHCI, + HzO + 2e --+ CH2C12 + C1- + OHCHC13
or
Inasmuch as the half-wave pote&ials of the carbon tetrachloride and chloroform waves are independent of pH, the electrode reactions cannot be reversible. Although the mechanisms of the electrode reactions are not known, it is probable that the reaction step involving the proton occurs subsequent to the ratedetermining reaction step. Determination of Carbon Tetrachloride and Chloroform. The recommended procedure is the following:
-
Mix 2 volumes of methanol with 1 volume of 0.3 M tetramethylammonium bromide solution in water and bring the temperature of the mixture to 2 5 " C. Place 50 ml. of the mixture in the polarographic cell, cool to 0" C., and pass nitrogen, previously saturat'ed with solvent a t 0" C., through the cell. After 30 minutes add exactly 1 nil. of a ure methanol solution of the sample to the cell as reconimencfed in the procedure above. Measure the diffusion current at -1.2 volts us. S.C.E. (carbon tetrachloride) or at - 2 . 0 volts (chloroform). Compare the value of diffusion current with a calibration curve that is established by treating known amounts of carbon tetrachloride Or chloroform in the same manner.
t,
,e,, 1 , ,
APPLIED
Figure 3.
, ,
POTENTIAL
a-
,
(
,
,
(YJ
Effect of pH on First Carbon Tetrachloride Wave
Solvent 2 to 1 methanol-water Electrolyte 0.1 M tetramethylammonium bromide. 2 millimolar carbon tetrachloride. Capillary B. Curves start at $0.1 volt I. p H 3.9 IV. p H 8.8 11. p H 4.6 V. p H 11 0 111. p H 5.9 e
Under certain conditions the chloroform wave exhibits a masimum in the region of greatest slope-Le., in the middle of the wave-which is most pronounced if the concentration of chloroform is high. The maximum also appears to be dependent on the capillary used, some capillaries yielding a maximum and others not yielding one. The maximum can be suppressed by addition to the solution of an electrolyte containing a divalent cation such as calcium. I n Figure 5 are shown the polarogranis of chloroform in solutions containing various amounts of calcium chloride. It can be seen that in a solution 0 05 IIf in calcium chloride the maximum is largely suppressed. However, the calcium ion itself yields a wave beginning a t about -2.1 volts, and thus makes it difficult to find accurately the height of the chloroform wave. Nevertheless, even in the presence of calcium, the concentration of chloroform can be found from the wave height with an accuracy of about 2%. The dependence of the half-wave potential of chloroform on pH was not investigated because of the relatively negative value of the chloroform half-wave potential. The diffusion currents of carbon tetrachloride and chloroform were found to be proportional to the 0.5 power of the height of the mercury column, as would be expected from the IlkoviE equation. It can be concluded that the reduction of these compounds a t the dropping mercury electrode is diffusion-controlled Electrode Reactions. The experimental results given above make it apparent that carbon tetrachloride is reduced a t the electrode to chloroform and that two electrons are involved in the reaction. The over-all electrode reaction is therefore probably:
+ H + + 2e +CHC13 + C1CCI, + HzO + 2e +CHCll + C1- + OHCCla
or
e-
o I
$4-
: 2-
I
-0.9
-1.2 -1.5 APPLIED
I
-1.8 -2.1 POTENTIAL
I
-2.4
Figure 4. Dependence of HalfWave Potential of Chloroform on Concentration Solvent 2 to 1 methanol-water. Electrolyte 0.05 -1.i calcium chloride. Temperature 2.3' C. Capillary B I. 1.33 millimolar 11. 0.665 millimolar 111. 0.33 millimolar
In the recommended procedure the loss by volatilization of carbon tetrachloride or chloroform is minimized. Moreover, because in each experiment 1 ml. of air-saturated methanol solution is used, the amount of dissolved oxygen introduced is constant. A convenient alternative procedure can sometimes be used, in which pure carbon tetrachloride or chloroform is dissolved in methanol, which has previously been rendered air-free by bubbling nitrogen through it. The data for the calibration curve are readily obtained by adding successive increments of the air-free standard solution from a microburet to 50 nil. of 0.1 M tetramethylammonium bromide in 2 to 1 methanol-ryater solution a t 0" C. If the calibration curve is prepared in this way, the sample must also be dissolved in air-free methanol. This procedure cannot be used if the sample before dilution contains less than about 10% carbon tetrachloride or chloroform. The concentration of carbon tetrachloride or chloroform in the methanol solution should be 0.02 to 0.2 31. The methanol of the original solution could undoubtedly be replaced by other common solvents such as ethanol, dioxane, or ethylene chloride. The calibration "curves" are plots of the observed diffusion cur-
ANALYTICAL CHEMISTRY
524 rent (not corrected for residual current) us. the amount or concentration of compound taken. The calibration curves are, within experimental error, straight lines. The accuracy of the recommended procedure can be seen from the results of Table 11, which correspond to an average deviation of less than 1% in the determination of carbon tetrachloride and less than 2% in the determination of chloroform. The presence of substances which are reducible a t the mercury electrode, such as aldehydes] organic nitro compounds, and many metal cations, interferes in the determination. The recommended procedure was applied to the determination of the solubilities of carbon tetrachloride and chloroform in water. The solubilities a t 25 C. were found to be 0.0053 M and 0.077 M , respectively. These values may be compared with the values 0.0050 M a t 25" C. (carbon tetrachloride) and 0.0646 M at 30" C. (chloroform) found by Gross and Saylor (3)and independently by van Arkel and Vles (1). Analysis of Mixtures of Carbon Tetrachloride and Chloroform. The polarographic method is also useful for the analysis of mixtures of the two halogenated methanes. Moderate amounts of chloroform do not interfere in the determination of carbon tetrachloride. On the other hand, carbon tetrachloride does make a contribution to the chloroform wave and it must be taken into account in the calculation of the amount of chloroform present. The total diffusion current a t -2.0 volts us. S.C.E. in the mixture is composed of the total diffusion current of carbon tetrachloride and the diffusion current of chloroform. The contribution made by carbon tetrachloride is found by multiplying the first diffusion current a t -1.2 volts us. S.C.E. by 9.925 X 1.93 = 1.79. The factor 0.925 accounts for the change of mz'3f1'6 between -1.2 and -2.0 volts, while 1.93 is the ratio of the total diffusion current constant to the first diffusion current constant of carbon tetrachloride (see Table I). The following procedure is recommended for a mixture of carbon tetrachloride and chloroform. Measure the diffusion current a t -1.2 volts (id,) and a t -2.0 volts ( i d l ) . hfultiply &, by 1.79 and subtract this from i d l . Compare the value thus obtained with the calibration "curve" to find the concentration of chloroform present. In a synthetic mixture composed of 25 parts of carbon tetrachloride and 75 parts of chloroform the results were accurate to within 2%.
I
I
w 0.3
Figure 5.
,
I
APPLIED
I
POTENTIAL
I
1
tW
Effect of Calcium Ion on Maximum in Chloroform Wave
Solvent 2 t o 1 methanol-water. Electrolyte 0.16 M tetramethylammonium bromide. Chloroform 2 millimolar. Temperature 26' C. Capillary B. Curves s t a r t at -0.9 volt I. 0.005 M IV. 0.03 M 11. 0.010 M V. 0.005 M . no chloroform 111. 0.015 I\,
The polarographic method has also been applied to the determination of carbon tetrachloride in the presence of a large amount of chloroform. I n Figure 6 is shown a polarogram of a mixture of carbon tetrachloride and chloroform (curve I), as well as a polarogram of a solution containing the same amount of chloro-
Table 111. Characteristics of Carbon Tetrachloride Waves in Solutions of High Chloroform Concentrations (Temp. 25' C., solvent 2 t o 1 methanol-water, 0.1 M in tetramethylammonium bromide) Concn. of cC1t4, Concn. of CHC1sa, r i i 2 of CCia id/C Millimolar .Millimolar (First Wave) (at 1.3 Volts) 2.0 0 0.78 7.5a 2.0 200 0.80 7.5 6.6 200 0.80 7.3 400 6.6 0.88 7.3 6.6 800 0.90 7.3 a Concentration of solution in polarographic cell. b Measurements made with capillary B. Diffusion current corrected for current due t o chloroform.
-
I
o
-0.2
-0.4
APPLIED
Figure 6.
-0.6 -08 POTENTIAL
-1.0
-12
-1.4
Polarogram of Mixture of Carbon Tetrachloride and Chloroform
Solvent 2 t o 1 methanol-water. Temperature 26' C. I. 0.002 .lf carbon tetrachloride and 0.2 .M chloroform 11. 0.2 M . chloroform
M calcium chloride. Capillary B 111. 0.002 ,Mcarbon tetrachloride IV. Blank 0.08
fork1 but no carbon tetrachloride (curve 11) The true diffusion current due to carbon tetrachloride is represented in Figure 6 by i d and is obtained by subtracting from the current of curve I (at 1.3 volts) the current of curve 11. For comparison purposes is shown a polarogram of a solution containing carbon tetrachloride only (curve 111). In Table 111 are given the results of the determination of i d / C of the first carbon tetrachloride wive in mixtures of varying composition. The diffusion current is proportional to the concentration of carbon tetrachloride and is practically independent of the concentration of chloroform. From Table 111 it also appears that the half-wave potential of the carbon tetrachloride wave is slightly more negative in solutions containing a large amount of chloroform than in solutions containing no chloroform. The change in half-wave potential is ascribed to the change in the nature of the solvent. Thus, in the last experiment of Table I11 the solvent contained about 6 volume % chloroform. The polarographic method therefore provides a means of determining a small amount (down to 1%) of chloroform in carbon tetrachloride. From the results of Table I11 it appears that an accuracy of 2 to 3y0 is attainable.
-
SUMMARY
Carbon tetrachloride is reduced at the dropping mercury electrode, yielding two waves for which the values of ?TI/; are -0.75 and - 1.70 volts FS. S.C.E., respectively. Chloroform yields one wave, for which the ~ I / value Z is -1.70 volts. The diffusion currents given by carbon tetrachloride and chloroform are proportional to concentration. Procedures for the polarographic determination of carbon tetrachloride and of chloroform yield results accurate to within 2%. Miutures of the two chlorinated methanes can also be analyzed.
V O L U M E 2 2 , NO. 4, A P R I L 1 9 5 0 ACKNOWLEDGMENT
Acknowledgment is made to the Graduate School of the University of Minnesota for a grant which enabled the authors to carry out this investigation. LITERATC R E CITED
Arkel, A. E. van, and Res, S. E., Rec. trau. chim.,55, 407 (1936). Britton, H.T. S., and Robinson, R. A., J . Chem. sot., 1931,1456. (:ross, P. M., and Saylor, J . H., J . Am. Chem. Sot., 53, 1744 (1931).
525 (4) Kolthoff, I. XI., and Lingane, J. J., “Polarography,” p. 195, New York, Interscience Publishers, 1941. ( 5 ) Ibid., p. 215. (6) Matheson, L. A., and Kichols, N., Trans. Electrochem. Soc.,73, 193 (1938). (7) Stackelberg, M.V.,and Straeke, W., 2 . Elektrochem.,53, 118 (1949). ( 8 ) Stone, K. G., J . Am. Chem. SOC., 69, 1832 (1947). Wawzonek, Stanley, ANAL.CHEM.,21, 61 (1949). RECEIVEDNovember 10, 1949. After this manuscript wa8 submitted, a publication by Stackelberg and Straeke appeared (7) in which the polarographic behavior of numerous halogenated hydrocarbons waa studied briefly.
Polarography of Reduced Glutathione and GlutathioneAscorbic Acid Mixtures Amperometric Method for Determination of Ascorbic Acid DALE AI. COULSON, WILLIAM R. CROWELL, AND SEYMOUR L. FRIESS University of California, Los Angeles, Calif. In oxygen-free solutions buffered at pH values between 3 and 4, glutathione alone or in glutathioneascorbic acid mixtures at concentrations from 10 to 300 micrograms per ml. can be determined with an accuracy of *4%, In the mixtures the character of the ascorbic acid wave is influenced markedly by the glutathione. In solutions containing 20 to 40 micrograms of glutathione and 9 to 80 micrograms of ascorbic acid per milliliter a method of measuring
I
S T H E determination of ascorbic acid in fruits and vegetables
the highest specificity is probably obtained by methods involving the use of 2,6-dichlorophenolindophenoldye and by those based on polarographic oxidation. When the solutions are too highly colored to permit direct titration with the dye solution, various potentiometric methods have been employed. Of these, that of Ramsey and Colichman (8) has the advantage of requiring only a stable potassium iodate solution as a standard oxidant, thus eliminating the necessity of frequent standardization of the dye solution. In the present work the authors have used an nmperometric method which has several advantages over the potentiometric procedure. The polarographic method has been employed by several workers (1-4, 6 , 7 , 9) with varying degrees of success. Results were satisfactory with certain fruits and vegetables, but there was indication of interference by other reducing agents in some cases. Recently Gillam ( 2 ) has made an extended study of the method in the determination of ascorbic acid in a number of fruits and vegetables and has developed a technique applying to quantities ranging from 4 to 85 micrograms per ml. with an accuracy of 3.3 to 4.3%. However, he noted that in certain materials interfering substances prevented the formation of a well defined limiting current, He also stated that the nature of the polarograms should show whether or not other reducing materials are present, and mentioned glutathione as a reductant that might have to be considered. S o data appear in the literature regsrding a study of the polarographic oxidation of glutathione or its effect on the polarographic determination of ascorbic acid. I t was the purpose of the present investigation to make such a study, to consider the practicability of making a polarographic determination of glutathione, and to compare results of ascorbic acid determinations obtained by polarographic., potentiometric, and amperometric methods.
ascorbic acid wave height is described which yields results within 0 to 2% of the weight value. Glutathione is found to be present in certain orange, guava, and potato samples. In most citrus fruit juices the content of glutathione and interfering materials is not high enough to affect the ascorbic acid wave seriously. An amperometric method for determining ascorbic acid is described and used in comparing polarographic with dye method results. MATERIALS, APPARATUS, AND PROCEDURES
Materials. The glutathione was Eastman’s No. 2585. An iodate titration of a vacuum desiccator-dried sample on the basis of oxidation to the disulfide showed a calculated value of 102% glutathione. A comparison of the glutathione polarograms with those of cysteine reported by Kolthoff and Barnum (6) indicated that no appreciable amount of cysteine was present. The ascorbic acid was Merck’s U.S.P. grade. An iodate titration of a dried sample showed 99.8% ascorbic acid. The indophenol dye was Eastman’s No. P3463. The buffer solutions were 0.2 M when all constituents had been added preparatory to analysis. At pH values from 2 to 3 and 6 to 7 they were prepared by partially neutralizing 0.8 M orthophosphoric acid with saturated sodium hydroxide solution. At pH values from 4 to 6 they were prepared by partially neutralizing 0.8 M acetic acid with the base. The natural products analyzed consisted of oranges, lemons, grapefruit, canned orange juice, guavas, and potatoes. Apparatus. In the potentiometric method, a k e d s & Northrup student’s potentiometer was used. In the polarographic work a Fisher Elecdropode was employed. A saturated calomel electrode was connected to the dropping mercury anode compartment by means of a 1 M sodium acetate agar bridge. For the amperometric method either a rotating platinum cathode or a fixed platinum cathode accompanied by constant stirring was found to be satisfactory. The anode consisted of a saturated calomel electrode which made connection with the titrated solution by means of a saturated potassium chloride agar bridge. Current measurements were made by means of the Elecdropode galvanometer. pH measurements vere made by means of a Model H Beckman pH meter. All polarographic measurements were made a t 25” * 0.1 C. in a water bath. Nitrogen was maintained oxygen-free by passage through a chromous chloride tower. Procedures. AMPEROVETRICTITRATIONS.Solution 1 consisted of 75 ml. of 0.3 N hydrochloric acid plus 10 ml. of 0.1 M potassium iodide solution and 10 ml. of ascorbic acid solution or unknown extract, Solution 2 was the same as 1 plus sufficient
