V O L U M E 23, NO. 1 2 , D E C E M B E R 1 9 5 1 marked by rubbing a series of dots on the glass with a Carborundum hone and then impregnating the dots with achinamarking pencil. Because of the heat and handling, marking with a wax pencil is not very satisfactory. The method, because of its reasonable reproducibility of results and the very small amount of time required, has been helpful in recent research on the treatment of these wastes. LITERATURE CITED
1767 American Public Health Association, “Standard Methods for Examination of Water and Sewage,” 9th ed., 1946. (3) Happold and Key, Biochem. J . , 31,1323 (1937). (4) Ingols and Murray, Water and Sewage Works, 95, No. 3, 113-17 (2)
(1948). ( 5 ) Madison, Mellon Institute, 37th Annual Report, p. 20. (6) Moore, Kroner, and Ruchhoft, ANAL.CHEM., 21,953-7 (1949). (7) Rhame, Water and Sewuge Works, 94, No. 5, 192-4 (1947). (8) Shaw, IND.ENQ.CHEM., ANAL.ED.,1,118 (1929).
(1) Adeney, “Principles and Practice of the Dilution Method of Sew-
age Disposal,” Cambridge, England, Cambridge University Prees.
RECEIVEDOctober 4, 1950. Contribution from the Fellowship on Gas Purification sustained a t Mellon Institute by Koppers Co., Ino.
Polarographic Determination of Tartrates in Wines A. P. RIATHERS, J. E. BECK, AND R. L. SCHOENEMAN Alcohol Tux Unit Laboratory, Wushington, D . C . For the past 100 years there has been a divergence of opinion as to the occurrence of tartaric acid in fruits and berries other than grapes. Various governmental agencies have held that the presence of tartaric acid in certain food and beverage products is an indication of adulteration or mislabeling. There is no suitable method in the literature for determining small amounts of tartaric acid in the presence of fairly large amounts of other frui t acids and solids. Tartaric acid is quantitatively precipitated and interfering substances are removed from wine by the
N
0 SUITABLE method is described in the literature for the
quantitative determination of small amounts of tartaric acid in the presence of relatively large quantities of other fruit acids and solids in wines-e.g., 20 mg. of tartaric acid, 200 mg. of other acids, and 10 grams of solids per 100 ml. of wine. The Association of Official Agricultural Chemists method ( 1 ) is satisfactory for determination of tartrates in grape wine or wine containing more than 20% grape wine. Slathers (6) reported a colorimetric test for tartrates which has sufficient sensitivity to detect traces of tartrates in mine, but the method has not been standardized for quantitative work. Tartaric, malic, citric, and oxalic acids have been used extensively as supporting electrolytes and complexing agents in polarographic studies of metallic ions. Rleites (7-9) has dealt with the polarographic characteristics of copper(I1) in tartrate, citrate, and oxalate media. Lingane ( 4 ) has discussed the polarographic behavior of arsenic, antimony, bismuth, tin, lead, cadmium, zinc, and copper in acidic, neutral, and alkaline tartrate media. Lingane ( 5 ) reports a polarographic investigation of oxalate, citrate, and tartrate complexes of ferric and ferrous iron and shows the influence of pH on the polarographic waves. Furness, Crawshaw, and Davies ( 2 ) describe an indirect polarographic determination of ethylenediaminetetraacetic acid by forming the copper complex, precipitating the excess cupric ion with magnesium oxide, and measuring the height of the polarographic wave of the complex. As the tartrate ion does not give a polarographic wave, a number of tartrate complexes were investigated to find one suitable for an indirect polarographic determination. Kolthoff and Lingane (3)note that antimonyl tartrate produces a fairly well-defined wave with Eli2 = - 1.07 volts vs. the saturated calomel electrode with the main wave preceded by a smaller wave a t -0.5 volt. I n the preeence of sodium methyl red, three waves are obtained which upon being made more alkaline give a single well-defined wave a t a potential of -0.94 volt us. S.C.E. Various metallic tartrate complexes give well-defined polaro-
procedure presented. A tartaric acid-antimony complex may be polarographed and a characteristic reduction wave obtained for use in quantitative work. Tartaric acid was found only in wines derived from grapes. The scope of the polarographic method is broadened by showing measurement of ions which in themselves are not reducible under conditions ordinarily employed in polarographic work. Most a-hydroxy acids can be determined from suitable complexes. There may be application to certain acids produced in metabolic processes.
graphic waves, but in most cases metallic complexes of isocitrate, citrate, malate, or all three give waves that cannot be readily separated from that of the tartrate complex. The antimony tartrate complex, however, is adaptable to the problem a t hand. The present work describes a method for quantitatively precipitating tartrates from wine and preparing the antimony tartrate complex for polarographic analysis. APPARATUS AND REdGENTS
Polarograph. A Heyrovskg polarograph, Model XII, manufactured by E. H. Sargent and Co., was used for polarographic measurements. The drop time for the capillary was t = 2.5 seconds a t E d e = 0 volt cs. S.C.E. in base solution. The temperature of the cell was maintained a t 25” C. by means of a water bath. A saturated calomel electrode was constructed from a 50ml. Erlenmeyer flask by attaching a right-angle side arm of 10mm. glass tubing near the top of the flask, to serve as an agar bridge. The electrolytic cell consisted of a 50-ml. beaker with a Transite cover containing the necessary openings for dropping electrode, saturated calomel electrode, and nitrogen tube. Reagents. Bismuth nitrate solution, 20 grams of bismuth nitrate pentohydrate dissolved in 10 ml. of concentrated nitric acid and diluted to 250 ml. with distilled water. Sodium hydroxide solution, 10%. Antimony chloride solution, 3 grams dissolved in anhydrous ethyl alcohol and made to 100 ml. with anhydrous ethyl alcohol. hlethyl red indicator solution. Gelatin solution, 0.3y0in water. Buffer solution, 17 grams of sodium formate, 5 grams of glycine, formic acid, and Rater sufficient to bring pH to 3.4 and volume to 500 ml. ANALYTICAL PROCEDURE
Treatment of Wine Sample. Dilute 10 ml. of wine in a Babcock bottle with 25 ml. of water, add 5 ml. of bismuth nitrate solution, centrifuge, and discard the supernatant liquid. To the precipitate add 20 ml. of water and 2 ml. of bismuth nitrate solution, shake, and place in boiling water for 15 minutes. Cool, centrifuge, and discard the supernatant liquid. To the bottle add 20 ml. of cool water and 1ml. of bismuth nitrate solution, shake, centrifuge, and discard the supernatant liquid. Dissolve the precipitate in
ANALYTICAL CHEMISTRY
1768 0.5 ml. of concentrated hydrochloric acid, add 2 ml. of antimony chloride solution and 10 ml. of water, and adjust the pH to about 4.5 with 10% sodium hydroxide. Add 5 ml. of buffer, place the Babcock bottle in boiling water for 15 minutes, cool, centrifuge, and decant the supernatant liquid into a 100-ml. graduate. Repeat, beginning with the addition of hydrochloric acid. Add 2 ml. of gelatin solution to the graduate and bring the volume to 60 ml. with water. (In adjusting to about pH 4.5, methyl red indicator may be added directly to solution if the wine colors do not obscure the end point; otherwise 0.5 ml. of hydrochloric acid plus 2 ml. of antimony chloride in aqueous solution is titrated to the methyl red end point and this definite amount of sodium hydroxide is used.)
100
-
this buffered solution. No wave was obtained in c before the appearance of the hydrogen wave a t - 1.5 volts. The influence of pH on the polarographic ave of the antimonyl tartrate complex is shown in Figure 2. The test solutions are the same as that used in Figure 1, a, except that the acidity was adjusted with hydrochloric acid or sodium hydroxide to the values shown. The wave at pH 2 is ill defined. It is seen that a t the lower pH values the waves are displaced to more positive potentials. The waves a t pH 3 and 4.5 are ne11 defined, but that at pH 3 can be measured more easily as the top of the wave is lees steep. ;it pH 5.9 the two waves of the antimonyl tartrate are again ill defined. 4 t pH 7 , not shom-n in this figure, only a single wave is obtained. The principal organic acids found in fruit, berry, and grape wines are tartaric, citric, isocitric, and malic. Figure 3 shows the
E E 50
100
-
-
E
E
i so
C
I
I
0.5 VOLTS
vs.
LO S.C.E.
-
I
1.5
Figure 1. Effect of Complexing Tartaric Acid and Antimony Xlirroamperes mm. X 0.0037 X shunt (applies to all figures) Shunt value 50 a. Antimonyl tartrate, pH 3.6 b . Antimony, pH 3.6 C. Tartaric acid, pH 3.6
Polarographic Analysis. A portion of above solution is polarographed in the applied voltage range of 0.0 to - 1.5volts us. S.C.E. A desirable shunt value is determined by manually operating the instrument. The polarographic wave a t -0.95 volt us. S.C.E. in approximately pH 3.5 solution is measured. The tartaric acid value is obtained by comparison of the wave height with a standard curve prepared from two known tartaric acid concentrations in a wine previously found to be free of tartaric acid. Suitable tartaric acid concentrations for the preparation of the standard curve are 25 and 250 mg. per 100 ml. of wine.
I
I
Figure 2. Influence of pH on Polarographic Wave of Antimonyl Tartrate Shunt 50 a. pH 2 b . pH 3
c. d.
pH 4.5 pH 5.9
EXPERIMENTAL
To demonstrate that characteristic polarographic waves may be obtained from the antimonyl tartrate complex, the following work was performed. Buffers of varying pH value were used, but the pH a t which polarographic measurements were obtained is shown for each figure. To 10 mg. of tartaric acid in a Babcock bottle were added 1 ml. of concentrated hydrochloric acid, 4 ml. of antimony chloride solution, 20 ml. of water, and methyl red indicator. The acidity was adjusted to the end point of methyl red with sodium hydroxide and 10 ml. of buffer solution were added. The sample was placed in boiling water for 15 minutes, cooled, and centrifuged, and the supernatant liquid was decanted into a 100-ml. graduate. To the graduate was added 2 nil. of gelatin solution and the volume was brought to 60 ml. with \vater. Figure 1, a, shows the polarogram obtained on the above solution, Figure 1, b, is the polarogram obtained when the tartaric acid is omitted and c that obtained when the antimony is omitted, The antimonyl tartrate complex shows two well-defined waves a t -0.95 and -0.46 volt DS. S.C.E. A small wave is obtained in b because the antimony is not completely precipitated in
IOC
6 E
sa
C
Figure 3.
Polarograms of Fruit Acids
Shunt 50, pH 4.2 Antimonyl tartrate b . Antimonyl citrate C. Antimonyl isocitrate d . Antimonyl malate a.
V O L U M E 23, NO. 1 2 , D E C E M B E R 1 9 5 1 polarograms obtained when 10 mg. of each of these acids were treated in the same manner as in Figure 1, a. The citric, isocitric, and malic acids show only a single wave, all appearing at approximately the same voltage as the first wave of the tartaric acid. Figure 4 shows the polarograms that mere used for comparison standards. The wines were treated according to the analytical procedure for wines described above. Figure 4, a, shows the waves obtained from grape wine containing 205 mg. of tartaric acid per 100 ml. (determined by the A.0..4.C. procedure); b, a n apple wine to which 250 mg. of tartaric acid per 100 ml. were added; and c, the same apple wine to which were added 25 mg. of tartaric acid per 100 ml. A shunt value of 100 was used for sam-
1769 ples a and b and 50 for sample c. The heights of the waves at E' '2 = -0.95 volt us. S.C.E. in pH 3.5 solution were as folloivs: a 31 mm., b 38 mm., c 7 mm. Figure 5 shows the polarograms obtained with several different types of wine. The apple wine was the same as used for Figure 4, b and c, but contained no added tartrates. These wines were all fermented by Peter Valaer of this laboratory in glass fermenters, so that no chance for external tartrate contamination could occur. The characteristic antimony tartrate wave was absent in all cases,
100.
E E IOC
so. E
E
sc O
/
I
0.0
03 VOLTS
1.
1.1
Figure 6 . Polarograms of Commercial Wines a. I
I
a5
0.0
VS.
1.0 %&E.
1.1
1.0
VOLTS VS. S.C.E. Figure 4. Polarograms for Comparison Standards
Shunt 50 Loganberry wine, pH 3.85
b. Cherry wine,,pH 3.80 C.
d. e.
f.
Elderberry wine, pH 3.80 Apple wine, pH 3.7 Blackberry wine, pH 3.19 Peach wine, pH 3.80
Figure 6 shows the polarograms obtained on a number of commercial wines. No indication of tartaric acid was seen i n these samples,
a. Grape wine, pH 3.5, shunt 100 b . Apple wine, pH 3.5, shunt 100 C. Apple wine, pH 3.5, shunt 50
DISCUSSIOX
I
! SO
a: d) Figiire 5. a. b. C.
I
I
VOLTS vs. S.C.E. I'olarograms of Laboratory Wines
S h u n t 50 Raspberry wine, pII 3.85 d. Bolsenberry wine, pH 3.95 e. Blackberry wine, pH 3.9 f.
Elderberry wine, pH 3.9 Cherry wine. pH 3.8 Apple wine, pH 3.7
The polarographic wave height of tartaric acid at constant pH appears to vary linearly with concentration, but two treatments of the bismuth tartrate precipitate as described do not recover 100% of the tartaric acid present. The percentage loss of tartaric acid seems to be slightly greater in the more dilute solutions, probably because of occlusion or coprecipitation, but the tartrate recovery is fairly reproducible. With careful work it is believed that the tartrate determination will give a t least 90% accuracy. At l o a tartrate concentration this compares very favorably v ith the present methods, which often fail to detect tartaric acid in small amounts or give small positive values when tartrates are ahsent. Spoiled mine often gives a colloidal suspension in the bismuth precipitation step, rendering an accurate tartrate determination difficult. d few laboratory-fermented wines in which the juice was fermented without addition of sugar and water contained so much acid that the sample size had to be cut to 5 ml. in order to secure complete precipitation of the organic acids with the bismuth nitrate reagent. The wines showing abnormally high acid content were loganberry, gooseberry, and currant. As no commercial m-ines of such abnormally high acid content have been found, the amounts of reagents specified above will probably be found sufficient. Fairly well-defined polarographic aves may sometimes be ob-
1770
ANALYTICAL CHEMISTRY
tained on wines without preliminary treatment by adding the antimony chloride and buffer directly to the wine. A number of other buffers were tested, but the polarographic waves obtained were not as well defined. Most of the buffers which did not precipitate the antimony gave excellently defined waves a t higher p H values, but when the solution was much above p H 5 the polarographic waves of other acid complexes were displaced to more negative potentials and interfered with the antimonyl tartrate wave. A pH range between 3 and 4 was found best for determining tartrates in the presence of other fruit acids. Wine samples treated according to the analytical procedure outlined using pH 3.4 buffer gave solutions of approximately pH 3.5 for polarographic analysis. The necessity of glycine in the solution is questionable, but where this compound or some other compound containing an amino acid group is absent the waves are often poorly defined. The glycine may be replaced by aniline, glutamic acid, or mphenylenediamine. Perhaps a complex of the aniline-antimonyl tartrate type is formed in solution. Lead, bismuth, cerium, and zirconium were tested as precipitating agents for tartaric acid in aqueous and alcoholic solutions. The bismuth reagent prepared as above was found to give the most quantitative precipitations of tartaric acid, only about 0.0001 gram per 100 ml. remaining in solution. It had the added advantage that the bismuth tartrate could be readily hydrolyzed to free the tartaric acid. One of the advantages of antimony as a complexing agent for tartaric acid is the ease with which the excess can be hydrolyzed and precipitated. LITERATURE CITED
(1) Assoc. Offic. Agr. Chemists, ed., p. 174. 11.23, 1950.
“Official Methods of Analysis,” 7th
(2) Furness, W , Crawshaw, P., and Davies, W. C., Analyst, 74, 629 (1949).
(3)
Kolthoff, I. &I., and Lingane, J. J., “Polarography,” revised ed., p. 262, New York, IntersciencePublishers, 1946.
100 ’
E P
50-
nn Figure 7.
0.5
1.0
IS
Polarograms of Tartaric Acid at Varying pH Values
Concentration of tartaric acid in stock solution 1 mg. per ml. Shunt value 100 Tartaric acid was not carried through precipitation procedure specified for wines, but antimony and buffer were added directly a. p H 2 c. pH 5.0 b . pH 3.5 d. pH 7.0
(4) Lingane, J. J., IND.ENO.CHEM.,ANAL.ED.,15, 583 (1943). (.5) Lingane, J. J.. J . Am. Chem. Soc., 68, 2448 (1946). (6) Mathers, A. P., J . Assoc. Ofic.Agr. Chemists, 32,417 (1949). (7) Meites. L., J . Am. Chem. Soc., 71,3269 (1949). (8) Ibid., 72, 180 (1950). (9) Ibid., p. 184. RECEIVED March 22, 1951.
Study of Saponification Reaction Rate of Ethyl Acetate by High Frequency Titrimetry F. W. JENSEN, G. M. WATSON, AND J. B. BECKHAM Agricultural and Mechanical College of Texas, College Station, Tex.
The possibilities of following the rate of saponification reactions with a high frequency titrator were investigated. The saponification reaction rate of ethyl acetate was followed successfully with a high frequency titrator after calibration curves were determined to correct for the nonlinearity of the instrument’s performance. A method of study of saponification reaction rates is indicated which uses simple equipment, and no internal indicators or electrodes. With further investigation it should prove useful for studies of reaction rate.
H
IGH frequency and other types of titrators are becoming increasingly useful and common as new applications are discovered and reported with augmenting regularity (1-3,6, 8, 16). The purpose of this project was to determine the applicability of a tuned-grid, tuned-plate oscillator as described by Jensen and Parrack ( 5 ) to the study of reaction rates of saponification reactions, and to develop a general method of procedure for the kinetic investigation of such reactions which could be applied with most of the different types of titrators available today. Because of the exploratory nature of the work, the saponifica-
tion reaction of ethyl acetate was chosen as a standard. This reaction has been studied by several investigators (9, 12-15) a t different temperatures with good agreement of results. Essentially three methods, with refinements, have been used by these investigators in their experiments with ethyl acetate. The first is the ordinary titration method ( 4 ) ,where the composition of the reacting mixture is followed by analysis of measured samples withdrawn from the system a t definite time intervals. The precision of this method is limited by the mechanical manipulation of transferring a definite volume of solution quantitatively
