S. T. SCIIICKTANZ AND A. D. ETIENNE, U. S. Treasury D epartm ent Laboratory, W ashington, D. C.
I
N THE production of ethyl alcohol by fermentation there is produced a group of higher alcohols generically known as “fusel oils”, which constitute an essential part of the congeners of alcoholic beverages. Although the fusel oil fraction is necessary for odor and taste characteristics, the amounts present are extremely small and range between 70 and 250 grams per 100 liters of distilled spirit.
Since the fusel oil fraction is used as a step in the assay analy
sis of distilled beverages, many tests have been developed for their quantitative determination. The preferred method of Allen-Marquardt, which is the official method and adopted as the standard by the Association of Official Agricultural Chemists, estimates the fusel oil content by extraction with carbon tetrachloride and subsequent oxidation to the respec
tive acids, which are then quantitatively estimated by titra
tion with standard 0.1 N sodium hydroxide. Modifications of this method have been described by Tohnan and Hillyer (6) and Mitchell and Smith (6). Herzfeld and Rose (7, 9) describe a method by which the fusel oil is determined by a measure of the increase in the volume of chloroform used as the extractant.
Komarowsky (4) determined the fusel oils by means of a color reaction. Many authors (1, 3, 5,10) have investigated the reactions responsible for the color produced by the com
bination of the alcohols of higher molecular weight with cyclic aldehydes such as salicylaldehyde, vanillin, veratic alde
hyde, and p-dimethylaminobenzaldehyde. Penniman, Smith, and Lawshe (8) describe a method of analysis which appears to yield a true value for the fusel oil content of a distilled
spirit. However, their high values may be in error because during the treatment of the beverage with sulfuric acid and alkaline silver nitrate there is produced some acetic acid which reacts with the cyclic aldehydes and produces addi
tional color (2).
In the present method, the fusel oil, separated from the dis
tilled spirit by distillation and subsequent extraction with carbon tetrachloride, is determined by esterification with acetyl chloride (12). After the reaction is completed, the excess acetyl chloride is decomposed and titrated. The difference in titer between the sample and a blank gives the moles of acetic acid removed by esterification with the higher alcohols.
Reagents
Eastman’s reagent grade acetyl chloride is used in preparing a 0.23 M (molal) solution in dry toluene. The pyridine solution is made approximately 0.50 M in toluene. Both solutions may be kept safely in regular well-ground glass-stoppered bottles.
Apparatus
The distillation unit recommended bjr the official Allen- M arquardt method is used to separate the fusel oil fraction and other volatile constituents from the solids of the whisky.
The reaction flask, shown in Figure 1, is made from a 125- ml. Erlenmeyer flask to which is attached a standard-taper ground joint No. 15. The neck of the flask is elongated as shown in order to assure no loss of reagents during pipetting.
In Figure 2 is shown the dehydration and dealcoholizing still used to remove the ethyl alcohol and water from the
ANALYTICAL EDITION 391 composite carbon tetrachloride ex
tracts. The still is packed with small glass helixes 0.64 cm. (0.125 inch) in diameter.
P rocedure
The sample of beverage to be ana
lyzed (50 ml.) is placed in a 500-ml.
Erlenmeyer flask equipped with a ground j oint. Thirty milliliters of 0.1 N sodium hydroxide and a few pieces of silicon carbide (10-mesh) are added, and the flask is attached to the Allen- Marquardt distillation unit. When 45 ml. of distillate have been col
lected in a 125-ml. separatory funnel, the distillation is stopped and 25 ml. of distilled water are added to the residue. Distillation is then continued until the total volume of dis
tillate is approximately 65 ml. To the distillate are now added 10 ml.
of distilled water and 10 grams of sodium chloride and the contents are well shaken to dissolve most of the salt before beginning the extraction with carbon tetrachloride.The extraction consists of four con
secutive treatments with 40-, 30-, 20-, and 10-ml. portions of carbon tetra
chloride, shaking at least 15 seconds upon each addition of carbon tetra
chloride. The carbon tetrachloride extract is collected in the reaction flask (Figure 1) to which have been added a few pieces of silicon carbide to ensure even boiling. The reaction
flask is attached to the still (Figure 2) and 50 ml, of distillate are collected by allowing the still to reflux for 5 minutes before removing the first fraction and then removing nine more con
secutive fractions at 5-minute intervals. The reaction flask is allowed to cool 1 minute, removed while still warm, loosely stoppered, and cooled for from 3 to 5 minutes in an ice bath.
To the flask are now added 5 ml. of pyridine solution and 10 ml. of acetyl chloride solution from precision pipets, being sure during the pipetting procedure that the respective reagents are introduced well down in the flask.Immediately following the addition of the acetyl chloride solution the flask should be tightly stoppered. It is advisable to put a very small amount of lubricant on the stopper (just enough to make a seal but not enough to allow the stopper to blow out of the flask during the following heating period). The stoppered flask is then shaken and placed in a wrater bath (12) kept at 60° C. The flask is allowed to remain in the bath for 30 minutes with shaking every 5 minutes. It is then placed in an ice bath for 5 minutes, after which 25 ml. of water are added, washing down the neck of the flask during the addition. After the flask has been shaken to decompose all pyridine salts and to extract the acids into the aqueous layer, an excess (1 to 3 ml.) of 0.100 N sodium hydroxide is added from a buret, the flask is shaken vigorously, 4 or 5 drops of phenolphthalein are added, and the solution is back-titrated to a light pink with 0.100 N sulfuric acid.A blank should be run with each set of experiments and the alcoholic hydroxyl groups in the sample estimated as the difference in alkali used between the sample and that of the blank. One milliliter of 0.1 N sodium hydroxide is equivalent to 0.0001 mole of fusel oil or 0.0088 gram of fusel oil or 17.6 grams of fusel oil per 100,000, calculated as amyl alcohol.The accuracy of the method was tested by using a carbon tetra
chloride solution containing a known amount of sec-butylcar- binol. The latter had a refractive index at 25° C. of 1.4084 and boiled between 129.35° and 129.55° C. In all experiments the results indicated a recovery of approximately 96 per cent, which may be assumed to be quantitative, since the alcohol used had a boiling range of 0.2° C. and undoubtedly contained a small percentage of inert substance.
Because of the small concentrations of amyl alcohol used, it was found necessary to heat the reaction mixture at least 30 minutes during esterification. In Table I are given the results obtained by heating for 20 and 30 minutes, respectively.
Heating for longer than 30 minutes is apparently not nec
essary. In one experiment sec-butylcarbinol was ex
tracted from an ethyl alcohol solution with carbon tetra
chloride and subsequently freed from ethyl alcohol and water by distillation prior to the esterification process. One set of samples was heated for 30 minutes and another set for 40 minutes, yet the same number of moles of alcohol were re aldehydes or esters does not interfere with the determination.
In Table II are given the results obtained when equal molal quantities of acetaldehyde and ethyl acetate dissolved in carbon tetrachloride were added to the carbon tetrachloride solution of sec-butylcarbinol prior to the determination.
T a b l e I I . E f f e c t o f A l d e h y d e s a n d E s t e r s Sec-Butylcarbinol E thyl A cetate A cetaldehyde Alcohol Found
Mole M ole M ole Mole
Further confirmation, presumably, of the negative effect of esters was obtained by determining the fusel oil content of a whisky by distilling immediately upon the addition of al
kali, and by refluxing the whisky for 0.5 hour with alkali prior to distilla
tion. The results are given in Table III and indicate either that com
plete saponification had occurred during the dis
tillation process, or that esters do not interfere evident that the results obtained on sec-butylcar loss was proportional to the volume of solution
satu-392 INDUSTRIAL AND ENGINEERING CHEMISTRY YOL. 11, NO. 7 rated salt and then extracted, while in another set 50 ml. of
100-proof ethyl alcohol containing sec-butylcarbinol were di
luted with 110 ml. of saturated salt solution. The moles of alcohol recovered were 0.000459 and 0.000411, respectively, which indicates a loss of approximately 10 per cent of sec- butyl alcohol in the solution having the greater volume.
T a b l e III. E f f e c t o f R e f l d x i n g Treatm ent Prior to D istillation Alcohols Esterified D istilled im m ediately
Reflux 0.5 hour prior to distillation
0.00102Mole that all of the fusel oil fraction was recovered by the modified method of distillation and that the loss in fusel oil presumably occurs in the extraction process.
Comparison of A. O. A. C. and Acetyl Chloride M ethods
The acetyl chloride-pyridine method of analysis offers con
siderable advantage over the official A. O. A. C. method. The time required for a complete analysis by the acetyl chloride method is approximately 3 hours by virtue of the elimination of the lengthy oxidation procedure. The method is not af
fected by aldehydes, which if present in the whisky must be removed prior to the analysis according to the official method.
Since esters do not affect the results, saponification prior to the initial distillation is not necessary. By obtaining only 65 ml. of distillate instead of 110 ml., the time required for the initial distillation is almost halved. Further, it is not neces
sary to saturate the distillate to a definite salt concentration, since an excess of salt does not affect the results of the new method. Errors in the regular extraction procedure are re
duced, since after the initial extraction of the alcohols with carbon tetrachloride, the present procedure removes traces of water and ethyl alcohol by simple distillation rather than by subsequent extraction with saturated sodium chloride solution followed by extraction with saturated sodium sulfate solution.
T a b l e IV. E f f e c t o f M o d i f i e d M e t h o d (M ixture of sec-butylcarbinol in 100-proof ethyl alcohol)
A lcohol Alcohol T reatm ent of Sample
M odified distillation and extraction N o distillation, only extraction
Present
During the distillation procedure used for the dehydration and dealcoholization of the carbon tetrachloride extract, the ternary azeotrope ethyl alcohol, water, and carbon tetra
chloride are removed first. Dry ethyl alcohol remaining after the water has been removed then forms a binary azeotrope with carbon tetrachloride which has a boiling point approxi
mately 11.5° C. below that of carbon tetrachloride itself, and is readily removed. It is fortunate that carbon tetrachloride does not form any azeotropes with alcohols boiling above n-butyl alcohol by virtue of the wide difference in boiling points between carbon tetrachloride and the higher alcohols.
The azeotropic power as exhibited by carbon tetrachloride with alcohols is conducive to a simple distillation procedure for the removal of extraneous amounts of ethyl alcohol and water. The method allows for a complete removal of the lower alcohols, since distillation can proceed long after the ethyl alcohol has been removed, by distilling only carbon tetrachloride, without fear of the loss of any of the higher al
cohols.
The presence of isopropyl, «-propyl, ieri-butyl, and iso
butyl alcohols and their removal during the distillation pro
cedure would indicate a decrease in value for fusel oils. It may be assumed that a similar decrease in fusel oil value is obtained by the official method, since the percentage amounts of alcohols lower than the amyls present in fusel oil are small, and, too, that since the solubility of the low boiling alcohols is more closely related to the solubility of ethyl alcohol than the amyls, the extraction of the carbon tetrachloride extract with saturated sodium chloride and sodium sulfate solution would remove a considerable portion.
After removal of the water and alcohol from the carbon tetrachloride extract, the fusel oils present require only 30 minutes for complete esterification; whereas an oxidation procedure requires at least 8 hours, plus a subsequent distilla
tion. The subsequent titration of the excess acetyl chloride is analogous to that of the official method, using 0.1 iV sodium hydroxide as titer and phenolphthalein as indicator. How
ever, since the results depend on the determination by differ
ence between the amount of acetyl chloride added and the amount remaining after esterification, it is necessary to know accurately the amount of acetyl chloride added.
Ta b l e V . Co m p a r is o n o f Me t h o d s
Treatm ent Allen-M arquardt A cetyl Chloride Residue
In Table V is given a comparison of the results obtained by the two methods using a standard sample of sec-butylcarbinol in solution in 50 per cent ethyl alcohol. The samples of sec-butylcarbinol in 50 per cent ethyl alcohol were subjected to the following procedure prior to the oxidation and esterifi
cation reactions.
Fifty milliliters of sec-butylcarbinol solution were diluted with saturated salt solution and sodium chloride to a density of 1.1. This was then extracted with 40-, 30-, 20-, and 10-ml. portions of carbon tetrachloride and the extract washed three and two times with saturated sodium chloride and sodium sulfate solutions, respectively. The solutions were then subjected to distillation in the dehydration stills and 50 ml. distilled off as distillate. Because of the large excess of water present, the acetyl chloride method could not be applied successfully to determine the amounts of alcohols contained in the distillates.
However, the usual method of procedure as outlined in the Allen-Marquardt method allows a considerable amount of ethyl alcohol to remain in the carbon tetrachloride extract, which in the usual procedure attributes considerably to the final evaluation of fusel oil content.
T a b l e VI. C o m p a r is o n o f M e t h o d s Treatm ent Allen-M arquardt A cetyl Chloride Residue
In another experiment using a standard sample of whisky, it was again evident that results obtained by the Allen-Mar
quardt method are too high, owing to the existence of some ethyl alcohol in the carbon tetrachloride extract. The results of the experiments are given in Table VI. The samples were subjected to the same treatment prior to distillation as the sec-butylcarbinol solution, except that the esters in the whisky were destroyed by the usual method subsequent to the distillation to remove extraneous solid or nondistillable ma
terial from the fusel oil fraction.
It is again evident that considerable amounts of ethyl al
cohol are present in the carbon tetrachloride extract and con
393 tribute to the value of fusel oil as normally determined.
However, the acetyl chloride method indicates the presence of considerably more alcoholic hydroxyl groups than the Allen-Marquardt method. The major portion of the differ
ence lies in the distillate, and indicates that the oxidation procedure is not capable of oxidizing quantitatively ethyl alcohol to the corresponding acid. This fact was corroborated by another set of experiments in which carbon tetrachloride containing a small amount of ethyl alcohol was subjected to both procedures. By the Allen-Marquardt method 0.00074 mole of alcohol was recovered; by the acetyl chloride method, 0.000975 mole. This is in agreement with the conclusions of Schidrowitz and Kaye (11), who indicated that ethyl al
cohol was not oxidized quantitatively during the oxidation procedure.
In the Allen-Marquardt method a blank must be run to determine the acidity produced by the carbon tetrachloride in the oxidation procedure.
T a b l e VII. G e n e r a t i o n o p A c id s CCU Used
M l.
2550
100
Acid Found Mole
0.0 0 00 2 0
0.000025
0.000020
Several experiments were made to determine the true cause of the generation of acids during the oxidation procedure.
The results, given in Table VII, indicate that the acids are not generated by virtue of impurities in the carbon tetrachloride but by the partial decomposition of the carbon tetrachloride itself.
It is evident that the evaluation of fusel oil in distilled spirits
depends upon the method used for the determination. Each method produces accurate values which are relative, but not in agreement when compared with values obtained by different procedures. Each procedure, used previously, is not specific for alcoholic hydroxyl groups alone, but is affected by the presence of other reactive groupings such as aldehydes, un- saturates, and esters. The acetyl chloride esterification procedure is normally not affected by the presence of these groupings and tends to give values which are specific for hy
droxyl groups.
However, according to the method as outlined for the acetyl procedure, any alcohol below n-butyl will be excluded from the fusel oil value. In view of these considerations, the results obtained by the acetyl chloride method are designated as an “amyl alcohol” value and not as a “fusel oil” value.
Literature Cited
(1) Bleyer, B., Dicmair, W ., and Frank, E., Z. Untersuch. Lebensm., 66, 389 (1933).
(2) Budagyan, F., and Ivanova, N., Ibid., 63, 200 (1932).
(3) Fellenberg, Th. von, Chem.-Ztg., 34, 791 (1910).
(4) Komarowaky, Ibid., 27, 807, 1086 (1903).
(5) Iiorenmann, J., Z. anal. Chem., 88, 249 (1932).
(6) Leach, “Food Inspection and Analysis”, p. 780, New York, John W iley & Sons, 1930.
(7) Lunge, G., ‘‘Technical M ethods of Chemical Analysis", p. 726, New York, D . Van Nostrand Co., 1914.
(8) Penniman, \V. B. D„ Smith, D. C., and Lawahe, E. I., Ind. Eng. Cheji., Anal. Ed., 9, 91 (1937).
(9) Rose, Stutzer, and Windisch, Arb. kaiserl. Gesundh., 5, 391 (1889).
(10) Ruppin, E., Z. Untersuch. Lebensm., 66, 389 (1933).
(11) Schidrowitz and Kayo, Analyst, 30, 190 (1905).
(12) Smith, D. M ., and Bryant, W. M. D., J. Am. Chem. Soc., 57, 61 (1935).