20
ANALYTICAL EDITION
trations of water in the alcohol dilute the caustic soda solution and introduce serious error. Dehydrating such alcoholic mixtures does not offer serious difficulty because the two alcohols are very similar in physical and chemical properties. Dehydration with quicklime was found t o be easy and effective. For example, a mixture of 93 per cent of eth 1 alcohol and 7 per cent isopropyl alcohol (technical purity in goth cases) was diluted 1 t o 1 with water and distilled through a bead-packed glass
Vol. 4, No. 1
column, collecting the first 50 per cent cut. This cut was shaken with an excess portion of quicklime and placed in an Engler flask. A small portion was distilled off and collected for analysis. It was found to contain a quantity of alcohol which, calculated on a basis of the original undil4ted sample, showed an allowable error of 0.25 per cent isopropyl alcohol. RECEIVRD November 9, 1931.
Determination of Small Amounts of Ethyl and Butyl Alcohols M. J. JOHNSON,University of Wisconsin, Madison, Wis.
M
ANY m e t h o d s have been devised for the d e t e r m i n a t i o n of ethyl and butyl alcohols in mixtures. A~~~~ the recent those of Bogin ( 2 ) a n d of D o n k e r (9) make use of the Of
A R A P I D method for the determination of small amounts (3 to 15 mg.) of ethyl or butyl alcohol or mixtures of the two is described. I t is particularly useful in the analysis of bacterial cultures where only small samples are available. The alcohols are oxidized by means of an acid dichromate solution, and the acids produced are distilled directly from the Oxidizing solution according to a procedure which makes use of the Duclaux principle. The whole determination may be completed in 40 minutes.
in water* The methods of W e r k m a n and O s b u r n (6) and of van der Lek (4) involve t h e oxidation of the alcohols to acids, which are then determined by a suitable method. In the course of an investigation reported elsewhere, it became necessary to determine ethyl and butyl alcohols in small samples of bacterial culture. Since a large number of determinations were to be made, a rapid method was desired. No method making use of solubility differences could be considered because of the large amount of sample required, and methods involving oxidation of the alcohols, steam distillation of the resulting acids, and determination of acetic and butyric acids in the distillate are likely to be slow and cumbersome. It was thought that a method might be devised whereby the acids could be distilled directly from the oxidizing solution in a modified Duclaux procedure. Such a procedure, if feasible, would eliminate the steam distillation and greatly shorten the time necessary for the determination. . After more than a hundred trials in which the effect of a number of variables was studied, a satisfactory procedure was developed. The method t o be described has been in use for more than a year, during which time approximately 150 routine determinations have been made. It has been used in a study of the acetone-butyl alcohol organism ( 3 ) and has given satisfactory fermentation balances.
EXPERIMENTAL PROCEDURE REAGENTS.The following reagents were used in this method: 1. Oxidizing solution. Mix equal volumes of exact1.y 3 N potassium dichromate solution and exactly 10 N sulfuric acid. Carbon dioxide-free water should be used in making these solutions. 2. Barium hydroxide. Solution 0.1 N o r 0.02 N strength. 3. Indicator solution. To 0.1 gram of phenol red (phenol sulfonphthalein) add 2.85 cc. of 0.1 N sodium hydroxide and make up to 500 cc. APPARATUS. The pieces of apparatus employed were as follows: 1. Alcohol-distillation apparatus. The distilling vessel is a Pyrex test tube, 38 by 200 mm. It is connected to a small
condenser. The lower end of the condenser tube is connected by a short piece of rubber tubing to a glass tube which extends to the bottom of the receiver flask, of 10 cc. capacity, and recalibrated to deliver 10 cc. 2. Acid-distillation apparatus.
A drawing of this a paratus is given in Figure 1. &e distilling vessel, A , is a Pyrex test tube, 20 by 200 mm. The condenser tube is made from 8-mm. tubing. The bend at B is made as close above the rubber stopper as practicable (1 cm.). The end of the condenser tube, C, is drawn out to a tip to facilitate collection of the distillate continuously rather than drop by drop. The receiver is a 10-cc. volumetric flask. Two of these are needed. The flame of the microburner heating the test tube is kept constant at any desired size by means of the bubbling bottle D and the manometer F . The tuhes entering the bottle should be of ample size (7 mm.). The excess gas escaping from the bottle is burned. The jet should not be constricted. The size of the flame of the burner may be varied by adjusting cock E or by changing the head of water in the bubbling bottle. The burner should be so adjusted that the rate of distillation is fairly rapid, but not so rapid as to permit any mechahical carrying over of liquid. This rate is such that 10 cc. of distillate are collected in 7 or 8 minutes. Once the apparatus is adjusted, the reading on the manometer F , and consequently the rate of distillation, should be kept constant. The manometer is filled with paraffin oil or some other nonvolatile liquid. A cylindrical shield, G, protects the flame from air currents. 3. Buret. This should be graduated at 0.01-cc. or at 0.05-cc. intervals, depending upon the strength of alkali used. A sample of culture containing not more than 15 mg. of total alcohols is placed in the distilling tube of the alcoholdistillation apparatus, made slightly alkaline, and diluted to approximately 20 cc. A few glass beads are added, and the sample is distilled into the 10-cc. receiver Aask. About 1 cc. of carbon dioxide-free water is first placed in the flask so that the end of the condenser tube is below the surface throughout the distillation. The distillation should be carried out a t a constant rate to prevent sucking back of distillate. The last part of the distillate is collected with the end of the condenser tube above the surface of the distillate. The distillation is continued until the receiver Aask is full to the mark. While this distillation is in progress, exactly 10 cc. of oxidizing solution are pipetted into the distilling tube of the aciddistillation apparatus. Four small glass beads are added, and the 10 cc. of alcohol distillate are rinsed into the tube with 5 cc. of carbon dioxide-free water. The tube, which should now contain exactly 25 cc. of reaction mixture, is closed with a rubber stopper and the stopper wired down. The tube is heated in a boiling water bath for exactly 5 minutes, and is
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January 15, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
then cooled under the tap, opened, and connected to the aciddistillation apparatus. Two 10-cc. fractions of distillate are collected. This distillation should always be made at the same constant rate. Each fraction is rinsed into a 25-cc. Erlenmeyer flask with carbon dioxide-free water, and titrated with barium hydroxide. A 0.1 N barium hydroxide solution is usedif the buret is graduated a t 0.01-cc. intervals, and a 0.02 N solution if the graduations are spaced 0.05 cc. apart. Phenol red is used as indicator, and the end point is taken at a permanent pink color (pH 8.0 to 8.2). A blank determination is run on the reagents alone, and in all cases the titrations obtained are corrected for this blank. The correction should be about 0.01 cc. 0.1 N for each fraction. A complete determination may be made in 35 or 40 minutes. STANDARDIZATION AND CALCULATION. The procedure is standardized on pure ethyl and butyl alcohols. Since the largest source of error in the method is the variability of recovery in the alcohol distillation process, it is best to obtain the constants of the apparatus for ethyl and butyl alcohols without going through the alcohol-distillation step. The recovery of alcohols by distillation may be determined subsequently.
21
The acetone is determined independently. I n determining the acetone correction, it is essential to use pure acetone. The acetone on the market usually contains about 0.5 per cent of ethyl alcohol. The calculation of the quantity of each alcohol in a sample is illustrated by the following example: A sample containing 5 mg. of butyl alcohol, 2 mg. of ethyl alcohol, and 5 mg. of acetone was subjected to oxidation and distillation.
-
TITRATION Corrected for Correoted for blank 6 mg. acetone Cc. 0.1 N Cc. 0.1 N 0.650 0.639 0.380 0.367
I
FRACTION
Actual Cc. O S N 0.660 0.390
a b
- 4.99 B = (1.334 X 0.639) - 0.367 0.0975 0.639 - (0.098 X 4.99) E= 0.0758
Obtained :
=
4.99 mg. of butyl alcohol 1.98 mg. of ethyl alcohol
CONSTANTS OF ETHYL AND BUTYL ALCOHOL TABLEI. TITRATION SAMPLE Ma
OBSERVED TITRATION (CORRECTED ROR BLANK) 1st fraction 2nd fraotion Cc. 0,1 N Cc. 0.1 N
TITRATION PER SAMPLE 1st fraction 2nd fraction Cc. 0.1 N CC.0.1 N MQ. OF
ETHYL ALCOHOL
15 10 5 2
1.138 0.769 0.378 0.163
1.622 1.011 0.608 0.200
16 10 5 2
1.486 0.981 0,490 0.196
0.497 0.333 0.167 0.069
75 25
0.172 0.067
0.192 0.066
0.0769 0.0769 0.0766 0.0765
0.1014 0.1011 0.1016 0.1000
0.0990 0.0981 0.0980 0.0980
0.0331 0.0333 0.0334 0.0345
0.0023 0.0023
0.0026 0.0026
BUTYL ALCOHOL
When the sample is subjected to the preliminary alcohol distillation, however, the recovery of such small quantities of alcohol is not complete. It has been found by experiment that 2 per cent of the ethyl alcohol and 6 per cent of the butyl alcohol present are lost in this preliminary distillation. The results calculated from the fractional distillation must therefore be divided by 0.98 and 0.94 to give the correct figures for ethyl alcohol and butyl alcohol, respectively.
ACE T0N E
If 15 cc. of carbon dioxide-free water, containing a known quantity of a single alcohol, are added to 10 cc. of oxidizing solution and the mixture heated and distilled, it will be found that the ratio of the titration values of the two distillate fractions is constant for each alcohol, and that the titration values are directly proportional to the quantity of alcohol present in the sample. If a sample containing both alcohols is oxidized, it will be found that each alcohol behaves, on oxidation and distillation, as if the other were not present. I n Table I are given titrations obtained by oxidizing and distilling various quantities of ethyl and butyl alcohols. From the data of Table I the following relationships may be obtained: a = 0.07583 b = 0.1011E
++0.098B 0.0333B
where a and b are the titration values of the first and second fractions, respectively, expressed as cubic centimeters of 0.1 N base and E and B are the quantities of ethyl and butyl alcohols, expressed in milligrams, present in the sample. If we solve the above equations for B and E, we obtain 1.334 a - b 0.0975 a - 0.098 B E = 0.0758 B=
From these formulas, the amount of each alcohol present in an unknown sample may be calculated. Since cultures producing ethyl and butyl alcohols usually produce acetone also, and since acetone is oxidized to a small degree by the dichromate mixture, it is necessary to apply to the titration values a correction proportional to the' amount of acetone present.
FIGURE1. ACID-DISTILLATION APPARATUS ~ V A L Y S I S OF KNOWNMIXTURES. I n Table I1 are given the results obtained in a series of determinations where known amounts of acetone, ethyl alcohol, and butyl alcohol were added t o 1per cent peptone solution, and then determined according to the procedure outlined. In the calculations it was assumed that the losses of ethyl and butyl alcohols in the preliminary distillation were those given above. The recoveries are typical of those obtained in a large number of determinations and show that the method is accurate to within 0.1 mg.
TABLE11. RECOVERY OF ALCOHOLS FROM PEPTONE SOLUTION IN PRE~ENCE OF ACETONE 7
Ethyl alcohol Mg. 10 2
SAMPLE Butyl alcohol Mg.
5
1 10 6
1
1
?
Acetone Mg. 6 5 5 5
-FOVNDEthyl alcohol
Butyl alcohol
Mo.
Ms.
9.94 2.03 6.02 1.02
1.04 10.07 4.97 1 .00
22
ANALYTICAL EDITION
Vol. 4, No. 1
FACTORS INFLUENCING DETERMINATION
USE OF METHODAS QUALITATIVE TEST The method just described was adopted after a large This method should not, of course, be used for quantitative number of experiments in which the size of sample, volume measurement of the alcohols present in a culture unless it is of distillate fractions, oxidation time, dichromate concentra- known that no alcohols other than ethyl and butyl are tion, and distillation time were varied. The effect of some of present. these factors will be discussed in detail. In a culture whose alcoholic constituents are unknown, the RATEOF DISTILLATION.In Table I11 the effect of rate of method may or may not be a valuable qualitative tool. If a distillation upon the ethyl alcohol constants is shown. When determination is run on an unknown culture, and the calculathe rate of distillation is slow, there is more condensation in tion from the observed titrations indicates the presence of a the upper part of the distilling tube, and the resulting quantity of ethyl alcohol and of no butyl alcohol, the data fractionation causes a change in the distilling constants. It is may be taken as qualitative and quantitative evidence of the evident, then, that the rate of distillation should be held at presence of ethyl alcohol in the culture, since it is extremely unlikely that two other alcohols, such as methyl and butyl, some constant value. are present in the exact proportion to produce the same titraTABLE111. EFFECTOF DISTILLATION TIMEON TITRATION tion ratio as ethyl alcohol. If, on the other hand, a determinaCONSTANTS tion on an unknown sample yields a result indicating the presence of both ethyl and butyl alcohols, it does not follow (Eaoh sample oontained 10 mg. of ethyl aloohol) TIM=OF -TITRATIONthat these two alcohols are the ones actually present. In LLATION 1st fraction 2nd fraction general, the method has the same defects, when used qualiSeC. cc. 0.1 N cc. 0.1 N 4 0.762 1.011 tatively, as the Duclaux method for acids. While the method -+‘ 7 56 0.769 1.011 as described is intended for use in determining ethyl and butyl 0.746 1.006 9 0 9 4 8 0.743 1.006 alcohols, it may be modified for use in the determination of other pairs of alcohols. TIMEOF OXIDATION.Five minutes of heating are necessary SOURCESCOP ERRORAND POINTS OF TECHNIC to oxidize the ethyl alcohol. The butyl alcohol is completely oxidized to butyric acid in the first minute or two, and in the Any substance which is readily volatile under alkaline course of five minutes a portion of this is further oxidized to conditions and which will yield carbon dioxide or a volatile acetic acid. That a constant proportion is thus oxidized is acid on oxidation with acid dichromate will interfere with the shown by the data of Table I, where the ratio of the first determination; for instance, several amines and to some extitration to the second is constant. The time of oxidation tent acetyl methyl carbinol. ahould be held constant so that an unvarying proportion of The constants given in this paper hold only for the apparathe butyric acid is oxidized to acetic. tus for which they were determined. If a new apparatus is CONCENTRATION OF OXIDIZING SOLUTION AND SIZE OF constructed, the constants must be redetermined. Time will SAMPLE.It will be noticed that the amount of dichromate be saved if the volumetric flasks used to collect the acid added is greatly in excess of the amount required to oxidize the distillate are calibrated with the inside surface wet. They alcohol present. This large amount of dichromate is necessary need not hold precisely 10 cc. provided the flasks used in a in order that the initial and final dichromate concentrations determination are the same as were used in standardizing may be nearly the same. If the dichromate concentration is the apparatus. All glassware, including the inner surface of low, the change in concentrationis sufficient to affect the oxida- the condenser tube of the acid-distillation apparatus, should tion of butyric acid to acetic acid materially, and thus to give a be kept scrupulously clean, since, when small volumes of “titration constant” for butyl alcohol which is not independ- liquid are handled, droplets adhering to glassware introduce a ent of the size of sample. Even with the dichromate con- very appreciable error. centration given, if the quantity of alcohol in the sample is too LITERATURE CITED great, the amount of dichromate destroyed is large enough to change its concentration sufficiently to cause a change in the (1) Bogin, C . D., IND. ENO.C H ~ M16, . . 380 (1924). titration constant. A tendency in this direction may be seen (2) Donker, H. J. L., “Bijdrage tot de k e d s der boterzuur -, butylalcohol - en acetongistingen,” p. 57, Delft, 1926. in Table I. Fifteen milligrams of butyl alcohol are enough to M. J., Peterson, W. H., and Fred, E. B., J . Bioi. Chem., change the first distillation constant from 0,0980 to 0.0990. (3) Johnson, 91, 569 (1931). .If 15mg. of butyl alcohol are present in a sample, the change in (4) Van der Lek, J, B., “Onderzoekingen over de butylalkolgiating,” the titration constant is sufficient to reduce the apparent p. 22, Delft, 1930. ENQ.CHEM.,Anal. ethyl alcohol content of the sample by 0.14 mg. In some (5) Werkman, C. H., and Osburn, 0.L., IND. Ed., 3,387 (1931). cases, this error would be important. For this reason the sample should never contain more than approximately 15 RIBCEIVIBD July 27, 1931. This work was supported in part by & grant from the speoial researoh fund of the University of Wisoonsin. mg. of alcohols. CHOICEOF INDICATOR. The titration of the distillate fractions is made somewhat difficult because of the carbon dioxide produced in the oxidation of butyric acid. In dilute Correction solution, carbon dioxide is completely neutralized at a pH of IN THI PAPER entitled “Determination of Organic Halogen approximately 8.2. When a fading end point, such as is given b Liquid Ammonia-Sodium Process’’ [IND. ENQ.CHEM.,Anal. by carbon dioxide, is encountered in a titration, a two-color 3,274 (1931)]+,the first word of ;he fourth line under “Modiindicator is for most observers easier to use than a one-color fied Procedure” is “monobutylamine.” This should be triindicator. Phenol red acquires a red color a t pH 8.0, so that butylamine, Inasmuch as monobutylamine forms a salt under conditions of the procedure, this error is rather serious should it is a satisfactory indicator from the standpoint of pK range. the anyone attempt t o use the method as described. Phenolphthalein, o-cresolphthalein, thymol blue, and phenol THOMASH. VAUGHN red have all been used as indicators, but phenol red seems to J. A. NIEUWLAND give the most reliable and most easily reproducible results. UNIVERSXTY OF NOTRE DAME The quantity of indicator solution added should, of course, be NOTREDAME, IND. October 15, 1931 kept constant.
&.,
,
