THE DETERMINATION OF ETHYL ALCOHOL*

BY THEODORE E. FRIEDEMANN AND ROSALIND KLAAS

(From ihe Department of Medicine of the University of Chicago, Chicago)

(Received for publication, April 13, 1936)

Of the various chemical methods described in the literature for the determination of ethyl alcohol (1) the majority are based upon the oxidation of the alcohol to acetic acid by means of potassium dichromate in the presence of a high concentration of sulfuric acid. Typical of these methods are the ones devised by Widmark (2) and by Nicloux (3). Widmark reports that it is not possible to calculate the amount of alcohol from the amount of dichromate used, for the oxidation does not lead exclusively to the formation of acetic acid; varying amounts of carbon dioxide and acetaldehyde are simultaneously produced. With solutions of pure alcohol ranging in concentration from 1.43 to 5.16 mg. per cc., he finds that 1 cc. of 0.01 N sodium thiosulfate solution corresponds to 0.1000 to 0.1241 mg. of alcohol. He adopts 0.113, the average of 54 determinations, as the factor to be used in the calculation of the number of mg. of alcohol present in a sample, but it is evident that considerable error is involved in the use of this factor. Further- more, Widmark’s method is limited in its application to samples which contain only relatively small amounts of other volatile substances.

Nicloux attempts to choose conditions such that a maximum yield of acetic acid and a minimum of carbon dioxide and acetalde- hyde are produced. He reports a high degree of accuracy under the conditions which he finds optimum, but his method involves the use of three carefully prepared and standardized solutions and

* This study was supported by grants from the Bartlett Memorial Fund and the Douglas Smith Foundation for Medical Research, the University of Chicago.

The authors gratefully acknowledge the assistance of Mr. Theodore Brook and Mr. William G. Motel.

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Determination of Ethyl Alcohol

heating the reaction mixture for 1 hour in a bath thermostatically regulated to maintain a temperature of 85”.

After extensive experience with chromic acid we have turned to the use of potassium permanganate as the oxidizing agent. Chap- man and Smith (4) were probably the first to report that, in the presence of alkaline potassium permanganate, ethyl alcohol is oxidized to oxalic acid.’ Barendrecht (7) determined relatively small quantities of alcohol, 8 to 10 mg., by introducing the sample into a boiling solution of alkaline permanganate. Friedemann and Ritchie (8), in a preliminary study published in 1933, applied a similar principle to the determination of very small quantities of alcohol. Since then, we have studied the method intensively, modified it, and applied it to a variety of biological materials.

In this method the sample is introduced directly into a Kjeldahl flask and distilled from tungstic acid and mercuric sulfate, which precipitate the proteins and prevent foaming. If certain volatile substances other than ethyl alcohol are present, the first distillate is further distilled from alkaline mercuric oxide. The final dis- tillate, or an aliquot thereof, is then oxidized at 100” with potas- sium permanganate in the presence of sodium hydroxide. After cooling and acidification of the mixture, the excess permanganate is determined iodometrically.

While the oxidation of ethyl alcohol does not proceed quantita- tively to carbon dioxide and water under the conditions chosen, yet the yield of other products is low and, what is more im- portant, remains remarkably constant within the limits specified (see Table I). Hence, the use of an empirically determined factor introduces no appreciable error. The final low acidity permits an accurate iodometric determination of the residual oxidizing agent, which is difficult in the case of the iodometric determination of excess dichromate. The epd-points are always sharp, even with the 0.005 N solutions. A further advantage of the use of potassium permanganate lies in the fact that 1 cc. of 0.01 N solution is equiva- lent to 0.042 mg. of alcohol, whereas with dichromate 1 cc of 0.01 N is equivalent to 0.113 mg. (2). The larger volume of oxidizing agent per given amount of alcohol makes possible greater

For a discussion of the reaction of alcohol with permanganate, see Morris (5). A more complete review of the literature is given by Evans and

Day 03).

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T. E. Friedemann and R. Klaas

accuracy in burette readings, and hence, greater sensitivity to small variations in amounts of alcohol present. Moreover, the time required for a complete determination is relatively short, and no special apparatus is required. Finally, the fact that the method can readily be adapted to many types of biological materials per- mits it a wide range of usefulness.

Description of Method

ApparatusFor the distillation process, a still of small capacity with a minimum of rubber connections is desirable. All of the re- sults reported in this paper were obtained withunits constructed as in Fig. 1. The still consists of a Kjeldahl flask,2 a glass condenser jacket, and a metal tube. The latter consists of an upper portion of $ inch tin-coated copper tubing and a lower portion of & inch tin- coated brass tubing. A glass collar, for the protection of the receiver from dust and from water which may have collected on the con- denser, is made from the upper part of a 6 ounce wide mouth bottle; this is held in place near the end of the delivery tube by means of a loosely fitting rubber stopper. A number of units may be conveniently mounted on a rack of the type described by Friedemann and Graeser (9). Microburners are used as a source of heat.

For work of greater accuracy an all-glass still, with ground glass connections, should be used.

All glassware is carefully cleaned with chromic acid cleaning solution, rinsed well (the final rinsings are with distilled water), and dried on a wire rack in such a way that contamination is avoided. The use of greasy or dusty glassware results in the introduction of large and variable errors.

Reagents

Distilled water. The water used should be free from organic impurities. It should be stored in a large glass bottle and deliv- ered therefrom through a glass siphon with a minimum of rubber connections. Water from wash bottles should never be used.

Sodium tungstate. A 10 per cent solution. Mercuric sulfate. 100 gm. of mercuric sulfate are dissolved in

2 The rubber stopper should be boiled in dilute alkali and then in dis- tilled water before using.

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50 Determination of Ethyl Alcohol

1 liter of 2 N sulfuric acid. To prevent precipitation of basic mercuric salt, the mercuric sulfate should be added to 500 cc. of water to which have been added 56 cc. of concentrated H&304 After heating until solution of the salt is complete, the volume is brought to about 1000 cc.

Calcium hydroxide suspension. 200 gm. of ordinary unslaked lime are slaked with a minimum of water. Then 1 liter of water is added. After a vigorous shaking, the.smooth suspension is poured off from the lumps. This is shaken well before using. Lime often

FIG. 1. Single unit of apparatus for determination of alcohol

contains considerable quantities of organic impurities. For more accurate work we recommend the use of Ca(OH)n prepared from chemically pure CaO. 100 gm. of CaO (reagent quality) are slaked with a minimum of water. To this is then added 1 liter of water. The resulting creamy suspension is kept in a glass stoppered bottle protected from dust.

5 N sodium hydroxide. Reagent quality should be used. The solution should be stored in a glass-stoppered bottle carefully protected from dust. To prevent resuspension of settled particles

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T. E. Friedemann and R. Klaas 51

a layer of coarse broken glass, about 1 inch deep, may be intro- duced.

Potassium permanganate. A solution of 33 gm. per liter is digested 5 or 6 hours on a steam bath and then filtered by suction through asbestos. The first portion of filtrate is discarded. A stock solution, slightly stronger than 0.1 N, is prepared by diluting 100 cc. of the cooled solution to 1 liter. Further dilutions are made when needed.

10 N sulfuric acid. Potassium iodide. Small crystals; analytical reagent. Starch solution. Sodium thiosulfate. The standard 0.1 N solution is diluted to

give solutions that are exactly 0.02,0.01, or 0.005 N.

Procedure

Distillation-The sample is measured (or weighed) into a 300 cc. Kjeldahl flask. To this are added distilled water (sufficient to make the total volume 50 or 60 cc.), 5 cc. of sodium tungstate, 5 cc of mercuric sulfate, and a small amount of powdered talc. The flask is rotated to insure a thorough mixing of the contents, con- nected to the still, and the mixture brought slowly to the boiling point (there is usually little or no foaming). During the course of 15 to 20 minutes, 30 to 35 cc. are distilled. A 150 cc. fat extraction flask or a 100 cc. volumetric flask may be used as the receiver. In the case of the latter, the volume is adjusted to the mark at the end of the distillation.

With certain samples a second distillation may be necessary in order to remove volatile substances which would interfere in the subsequent oxidation procedure. The initial volume is made about 100 CC. ; 60 to 70 cc. of distillate are collected in a Kjeldahl flask containing 5 cc. of mercuric sulfate. A volume of calcium hydroxide, sufficient to impart a deep orange color to the mixture, and powdered talc are added. The flask is rotated (in cases where the aldehyde or ketone content is high, the flask is stoppered and shaken vigorously) and then the distillation is carried out as before.

Oxidation-A 150 cc. fat extraction flask, with a 100 cc. beaker serving as the cover, is used for the oxidation. The sample is introduced either by direct distillation, as described above, or by

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Determination of Ethyl Alcohol

pipette from the volumetric flask. In order to determine the size of the aliquot and the strength of the permanganate solution, trial runs may be made. If the aliquot chosen has a volume of 10.0 cc., 15 cc. of water should be added to the extraction flask after the introduction of the sample, for it is important, that the total volume during the oxidation should be 60 to 70 cc. (see Table I). To the sample are now added 10 cc. of 5 N sodium hydroxide and 25.0 cc. of a potassium permanganate solution of appropriate strength. More consistent results are obtained if the permanganate is added with constant rotation. In ordinary work, where moderately large quantities of alcohol are expected, a 0.02 N solution should be used. After further rotation to insure thorough mixing of the contents, the flask is placed in a boiling water bath. At the end of 20 minutes, it is removed to a bath of cold running water. As soon as it is cool, 10 cc. of 10 N sulfuric acid are added. After a few more minutes of cooling, the flask is removed; 0.2 to 0.5 gm. of potassium iodide is added, and the iodine titrated with standard sodium thiosulfate of approximately the same strength as the permanganate. 1 to 2 cc. of starch solution are added when the color of the solution is a pale yellow. The titration is continued until the solution is colorless.

Blank determinations should be made simultaneously. CuZcuhtions-A (cc. of thiosulfate required for the blank) minus

B (cc. of thiosulfate required for the sample) equals C (cc. of thiosulfate equivalent to the potassium permanganate used in the oxidation).

The amount of alcohol present in the solution oxidized can now be calculated. 1.00 cc. of 0.020 N KMn04 = 0.0855 mg. of ethyl alcohol, or 0.00186 mM; 1.00 cc. of 0.010 N KMn04 = 0.0420 mg., or 0.000912 mM; 1.0 cc. of 0.005 N KMn04 = 0.0215 mg. or 0.000467 mM. These factors apply only when C is less than 5 cc. (6 cc. in the case of the 0.02 N KMnOJ. If C is greater than 5 cc., approximate results may be obtained by using factors given in Table I.

If S is the number of cc. of initial sample equivalent to the aliquot taken for oxidation, the following formulae give the amount of alcohol in mg. per cent.

C X 8.55/S = mg. ‘% alcohol, with 0.020 N KMnO, ‘I x 4.20/” = “ % “ “ 0.010 “ “ t‘ x 2.15/“ = ‘1 % “ “ 0.005 (‘ “

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TABLE I Oxidation of Pure Ethyl Alcohol

25.0 or35.0cc. of alcohol solution, 10 cc. of 5~NaOH,25.0 co. ofKMnO4; total volume. 60 or 70 cc. Heated20minutes in a boiling water bath. Water blanks = 25.6 to 25.6 cc. of 0.021 0, 0.010, or 0.005 N thiosulfate.

T - I T 0.02 N KMnO, 0.01 N KMnO4

Alcohol equiva-

lent

CiZO

3.13 5.43 3.0316

5.66 5.10 I.0274

5.08 4.53 I.0230 3.88 3.65 I.0224 3.42 3.30 3.0228

3.25 3.09 3.0215 2.90 2.63 3.0215 2.91 2.69 3.0210

2.24 2.07 3.0208

1.50 1.30 1.0207 3.85 0.63 1.0184*

-7 Alcohol oxidized

ml.

0.796 0.778 0.716 0.700 0.637 0.622 0.557 0.544 0.517 0.466 0.438 0.398 0.389 0.350 0.348 0.311 0.305 0,272 0.233 0.218 0.194 0.174 0.155 0.131 0.117 0.087 0.078 0.0700 0.0697 0.0623 0.0610 0.0545 0.0467 0.0389 0.0311 0.0156

y;i!* ume

60 cc.

~AE.0

8.53 7.89 7.96 7.41 7.30 6.84 6.56 6.21 6.17 5.46 5.12 4.71 4.55

Alcohol e uiva-

9 ent

GO

8.32 7.76 7.65 7.35 7.09 6.72

6.14 5.50

mg. per cc.

0.0933

0.0986 0.0900 0.0945

0.0873 0.0909 0.0849 0.0876 0.0838 0.0853 0.0855 0.0845 0.0855

3.62 0.0859

2.74 0.0850

1.82 0.0852

0.89 0.08761

Alcohol equiva-

lent

cc. CM?%0 I

mg. per cc.

6.64 6.82 6.51 6.32 5.88 5.36 5.08 4.68 4.15 3.68 3.12

3.10

5.24 5.07

1.18 3.68

0.0527 0.0510 0.0478 0.0483 0.0463 0.0435 0.0429 0.0415 0.0419 0.0421 0.0420

1.83 0.0426 1.65 0.0424

1.51

1.31 1.15 0.93

0.0413

0.0416 0.0406 0.0418 *

- - * Not included in any calculations.

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54 Determination of Ethyl Alcohol

Precautions-Inasmuch as the success of the method depends upon the controlled oxidation of a minute quantity of an organic compound (ethyl alcohol), it is absolutely essential that all other oxidizable substances be excluded. In practically every instance those who have described oxidimetric methods have stressed the importance of clean glassware. We too urge that great care be taken to use glassware that is free from dust and grease. Cleaning with chromic-sulfuric acid mixture (with not less than 80 per cent HzSOJ is recommended. It is also important that the distilled water be of good quality. If organic impurities are present in the water, it is impossible to obtain accurate results.

The rubber stopper used to connect the Kjeldahl flask with the condenser may be the source of considerable volatile oxidizable material. The amount is small and fairly constant, however, (so that a correction for this error is provided by the blank) if the still is steamed out each day before a series of distillations is begun.

The reagents for the oxidation should be measured with pipettes, and not burettes. It is well to plug all pipettes with cotton in order to avoid contamination of the solutions with chance droplets of saliva. The lower part of these pipettes should not be allowed to touch the desk, nor should the lower part of these pipettes be touched with the hands, and, if used repeatedly, each pipette should be placed in an upright position in a test-tube.

Xaliva-If a beverage or solution containing alcohol has been taken orally, the mouth should be rinsed well with several changes of water in order to remove the last traces of the liquid. At least 5 minutes should then elapse before a sample of saliva is collected for analysis. A rapid flow of saliva may be induced by chewing a small piece of parafhn. Chewing gum should never be used. The sample should be preserved by the addition of sodium fluoride (0.1 gm. for 10 cc. of saliva). 1 cc. samples are then distilled according to the directions given above. A second distillation is unnecessary (see Table V).

Blood-In the collection of samples, the skin is cleansed with soap and with 0.1 per cent mercuric chloride solution. If 0.2 cc. samples are desired, the skin is pricked as in making a blood count. 10 drops or more are allowed to run into a small test-tube contain- ing a small amount (0.1 to 0.2 per cent) of a mixture of equal parts of powdered sodium fluoride and potassium nxalate. For the

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T. E. Friedemann and R. Klaas 55

collection of larger samples, a dry sterile syringe for venous punc- ture is used. After collection of the blood, the container is tightly stoppered and immediately placed in the refrigerator. The analysis should be undertaken as soon as possible, at least within 24 hours after collection.

In the measurement of 0.2 cc. samples, a pipette of 0.2 cc. capacity, such as is used in the measurement of the Kahn antigen, has been found to give fairly accurate results (see Table III). Blood is drawn up past the mark; the tip is wiped with a towel, and the meniscus adjusted to the mark. The pipette is then allowed to empty slowly into the Kjeldahl flask (time necessary, about 1 minute) ; the last drop is blown out. The distillation from the sodium tungstate and mercuric sulfate is then carried out, with a 150 cc. extraction flask as the receiver.

Whenever larger amounts of blood are available, 1 cc. samples are taken. The pipettes should be allowed to drain slowly. The 100 cc. volumetric flask is used as the receiver, and a suitable aliquot of the distillate taken for oxidation. In the case of blood, as with saliva, a redistillation is not required, because of the presence of only very small amounts of interfering substances.

Urine-The samples are preserved with sodium fluoride (1 gm. per 100 cc.). Since urine often contains rather large amounts of volatile reducing materials, especially in the case of diabetic individuals, the two distillations outlined above are necessary in order to obtain accurate values for alcohol content. 1 cc. samples are generally used, although 5 or 10 cc. may be taken if the amount of alcohol is low.

Culture Media-Sulfuric acid is added to stop the metabolic activities of microorganisms as recommended by Friedemann and Brook (10). 5 or 10 cc. samples are taken for analysis; two distil- lations are required because of the frequent occurrence of aldehydes or ketones as products of metabolism. Samples of the same cul- ture medium in which no microorganisms have grown are run in order to obtain blank values. Certain bacteria produce other alcohols besides ethyl alcohol. Our method is fairly specific for alcohols as a group, but it cannot be used to determine et,hyl alcohol when other alcohols are also present.

Tissues--The sample is frozen in liquid air and crushed accord- ing to the procedure of Graeser, Ginsberg, and Friedemann (11).

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56 Determination of Ethyl Alcohol

A weighed sample of the crushed tissue is introduced directly into the Kjeldahl flask and treated like a sample of blood.

EXPERIMENTAL

O~zZalion of Pure Ethyl Alcohol-Commercial absolute ethyl alcohol was heated with unslaked lime under a reflux condenser for 15 hours. The alcohol was then distilled; only the middle third of the distillate was retained. Solutions of known composi- tion by weight (approximately 15 per cent) were prepared, and these were immediately diluted to form 1 per cent solutions. The latter were kept in the refrigerator and diluted further as needed. The oxidations were carried out as described above, with 25 cc. of the alcohol solution, 10 cc. of 5 N sodium hydroxide, and 25 cc. of potassium permanganate. Oxidations were also carried out

TABLE II

Recovery of Ethyl Alcohol after Distillation

Weight of alcohol distilled

Distillation from HgSOr + NaiWO4

(1)

ma. per cent recovery

1.560 98 0.936 101 0.730 93 0.312 97

Distillation from Heso + Cs(OH)x Two distillations:

(2) (1) followed by (2)

per cent remvery

100 103 98 96

in which the sample (25.0 cc.) was further diluted with 10 cc. of water before the addition of the alkali and permanganate. The study covered a wide range of concentrations of alcohol and three concentrations of potassium permanganate. Typical results are shown in Table I.

With a given permanganate solution, the results are fairly con- stant until the volume of oxidizing agent used exceeds 5 cc. (6 cc. in the case of the 0.02 N KMnOJ. Thus, in the case of the data presented in Table I, the greatest deviations from the average are -1.2 and +0.5 per cent with 0.02 N KMnO+ and -3.3 and +2.1 per cent with 0.01 N KMn04 It may be noted that the values change rapidly when the volume used in the oxidation is greater than 5 (or 6) cc. In no case is the alcohol oxidized completely to carbon dioxide and water; the calculated equivalents are 0.0768,

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T. E. Friedemann and R. Klaas 57

0.0384, and 0.0192 mg., for 0.02, 0.01, and 0.005 N solutions, re- spectively.

We have chosen as the alkali concentration one which permits the oxidation to proceed most nearly to completion. With higher or lower concentrations, the extent of the oxidation rapidly diminishes.

Distillation. E$ect of Various Agents on the Recovery-Alcohol solutions of varying concentrations were distilled according to the procedure outlined above. Typical results are given in Table II. It is evident that the reagents used remove little if any of the alcohol. The average recovery in all determinations shown in Table II is 98.6 per cent. Low recoveries, such as 93 per cent in the single distillation in the second column, are rarely encountered if considerably more than one-half of the solution is distilled.

Recovery of Alcohol from Blood and Urine-l cc. samples of blood were measured into Kjeldahl flasks. To these were added 1 cc. samples of alcohol solutions varying in concentration from 79 to 278 mg. per cent. Distillations were carried out from the mercuric sulfate and sodium tungstate reagents, and the appropriate aliquots of the distillates oxidized. 0.2 cc. samples of blood and alcohol solutions were treated similarly, except that the distillates were collected in extraction flasks. The results are shown in Table III. In the case of the smaller samples, it will be noted that the recovery was somewhat high and variable. Because the error in the measurement of small quantities of liquid may be rather large, the pipettes should be carefully calibrated or the samples weighed whenever a higher degree of accuracy is desired.

To 1 cc. samples of normal urine amounts of alcohol varying from 63 to 156 mg. per cent were added. The recovery here was excellent, just as in the case of blood.

Removal of Interfering Substances-Various methods have been recommended by different workers for the removal of aldehydes and ketones, two classes of substances which are very similar to alcohol in volatility and ease of oxidation. Gorr and Wagner (12) review the literature up to 1925 and then outline a new procedure. The sample is heated under a reflux condenser with a mixture of mercuric chloride and slightly less than the equivalent amount of alkali. Pelgroms (13) merely distils slowly from mercuric oxide. He does not describe his method in detail or state the alkali con-

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58 Determination of Ethyl Alcohol

centration used. We have made a careful study of the use of mercuric oxide and its efficiency in the removal of aldehydes and ketones, and we find that it is a very satisfactory reagent for the purpose. We recommend the use of excess of calcium hydroxide. The alcohol is recovered practically quantitatively (Table IV) at

TABLE III

Recovery of Added Alcohol from Blood and Urine

Blood

T

-

Sample

cc.

1.00 1.00 1.00 1.00 1.00 0.20 0.20 0.20 0.20

-

-

-

T

mkzY 79

119 159 198 278 79

119 159 198

RWW- =Y

per cent

101 99

101 100 103 106 103 101 107

Urine A

Urine B

S8mplE

cc.

1.00 1.00 1.06 1.00 1.00 1.00 1.00 1.00

TABLE IV

Removal of Acetaldehyde and Acetone

The procedure outlined for urine was followed.

-

-

Ethyl alcohol used

ml.

1.49 0.87 0.75 1.49 0.87 0.75 0.29

“z&Y 63 78 91

156 63 78 91

156

-

-

per cent

96 97

102 100 98 99

100 99

Substmce added Ethyl alcohol recovered

fw. nag. per cent

Acetaldehyde 1.1 102 “ 1.1 102 ‘I 1.1 105

Acetone 1.0 99 I‘ 1.0 99 I‘ 1.0 100 ‘I 1.0 100

this alkalinity when mixed with equal or larger quantities of ace- tone or acetaldehyde. At least 99 per cent of the acetone is removed, and acetaldehyde is removed to the extent of 95 to 98 per cent under the conditions specified.

Whenever complex mixtures are dealt with, the first distillation from an acid medium results in the removal of volatile basic

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T. E. Friedemann and R. Klaas

substances, such as the amines, and the second, from the alkaline mercuric oxide, permits the removal of phenols and the lower aliphatic acids, aldehydes, and ketones. The method therefore provides an excellent separation of alcohol from other substances commonly present in biological materials.

Blank Values of Blood, Saliva, and Urine-The quantity of oxidizable material obtained by the distillation, according to our procedure, of 1 cc. of normal blood, saliva, or urine is very small. Thus, in the case of urine, the volume of 0.01 N potassium perman- ganate used in the oxidation of a 25 cc. aliquot of the distillate varies from 0.02 to 0.10 cc. The same values are obtained with diabetic urines. In the case of a diabetic urine, four determina- tions with 1 cc. samples gave an average titration of 0.05 cc., equiv- alent to about 0.8 mg. per cent of ethyl alcohol, whereas the same urine, when distilled without any reagents whatsoever, gave a titration of 3.30 cc., equivalent to 55 mg. per cent of alcohol. With saliva and blood, just as with urine, the blank values are very small. Hence, in ordinary work it is unnecessary to apply corrections for the oxidizable material present in the samples.

Normal Alcohol Content of Blood, Saliva, and Urine-That ethyl alcohol is normally present in blood and tissues has been demon- strated conclusively by Gettler, Niederl, and Benedetti-Pichler (14). These workers isolated the alcohol, identified it by means of its physical properties, and prepared characteristic derivatives. Using their quantitative microethoxy method, which is specific for alcohols, they have obtained the following average values for the normal alcohol content of blood: human, 4 mg. per cent; dog, 1.3 mg. per cent.3 Their results are the lowest thus far published.

Our method gives the results summarized in the last column of Table V. It is to be noted that there is a fairly close agreement among the values obtained for the three body fluids, blood, urine, and saliva, despite the fact that the content of volatile materials varies widely, being greatest in urine.

The data in the third column of Table V have been included in order to show the necessity for two distillations in the case of urine. Single distillations gave high and variable results. It may also be

3 A summary of determinations by previous workers of the normal alcohol content of tissues and body fluids is given by Gettler, Niederl, and Bene- detti-Pichler.

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60 Determination of Ethyl Alcohol

noted that in certain instances the quantities of reagents used were not sufficient for the volume of urine taken. Thus, the 20 cc. sample of diabetic urine, after two distillations, yielded 1.38 mg. per cent of oxidizable material calculated as ethyl alcohol, whereas approximately one-half as much was obtained from the 10 and 5 cc. samples. Again, normal Urines U and V yielded 8.9 and 6.0

TABLE V

Normal Alcohol Content of Urine, Blood, and Saliva

With the exception of blood (see below) the quantity of reagents used in these analyses was that recommended for 1 cc. of sample.

Description of sample

Urine A, normal

“ B, “ “ R, diabetic*

“ U, normal “ v ‘I

Blood A: normal?

‘I B, “ t

Saliva A, normal “ B, “

cc.

20 20 20 10

5 1

10 10 10 10

10 10 10 10 10

Treatment

Distillation from W30, + NazWO

(1)

mg. per cent

2.0 4.4

17.3 8.9 6.0

0.38 0.36

mg. pa cent 0.77 0.50 1.38 0.53 0.74 0.8 0.92 0.62 0.10 0.15 0.17 0.40 0.40 0.32 0.31

* This sample contained much glucose and gave a strong reaction with ferric chloride (acetoacetic acid).

t The first distillation was carried out with 10 cc. of acid HgSO, and 40 cc. of Na*WO, in a total volume of 160 cc.

mg. per cent, respectively, after the first distillation; after two distillations, they gave 0.92 and 0.62 mg. per cent. With an excess of mercuric sulfate (5 gm. of HgSOb added to the mercury- tungstate mixture), practically the same results (not shown in Table V) were obtained after one distillation as with the two; viz., 0.81 and 0.96 mg. per cent.

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T. E. Friedemann and R. Klaas 61

SUMMARY

A method for the determination of ethyl alcohol in biological materials has been outlined. The alcohol is separated from other constituents by distillation after addition of reagents which remove many of the commonly occurring volatile substances. The distil- late, or an aliquot thereof, is oxidized in a water bath with alkaline permanganate for a period of 20 minutes. After the mixture has been cooled, it is acidified, and the excess permanganate deter- mined iodometrically. The necessity for cleanliness of glassware and purity of water has been emphasized. The high degree of reliability of the method is indicated by the following criteria: (a) practically quantitative recovery of alcohol from pure solu- tions, (b) practically quantitative recovery of alcohol when added to blood and urine, (c) excellent removal of many of the common volatile substances which occur in biological materials, and finally (d) obtaining a low and consistent value, approximately 0.4 mg. per cent, for the normal alcohol content of blood, saliva, and urine.

We wish to thank Dr. H. Necheles of the Michael Reese Hospital for advice and cooperation given in the course of this study.

BIBLIOGRAPHY

1. See Harger, R. N., J. Lab. and Clin. Med., 20, 746 (1935), for a brief review of the literature.

2. Widmark, E. M. P., Biochem. Z., 131,471 (1922). 3. Nicloux, M., Bull. Sot. chim. biol., 13, 857 (1931). Nicloux, M., Le

Breton, E., and Dontcheff, A., Bull. Sot. chim. biol., 16,1314 (1934). 4. Chapman, E. T., and Smith, M. H., J. Chem. Sot., 20, 301 (1867). 5. Morris, H. E., Chem. Rev., 10, 465 (1932). 6. Evans, W. L., and Day, J. E., J. Am. Chem. Sot., 38, 375 (1916); 41,

1267 (1919). 7. Barendrecht, H. P., Z. anal. Chem., 53,167 (1913). 8. Friedemann, T. E., and Ritchie, E. B., Proc. Sot. Exp. BioZ. and Med.,

30, 451 (1933). 9. Friedemann, T. E., and Graeser, J. B., J. BioZ. Chem., 100,292 (1933).

10. Friedemann, T. E., and Brook, T., unpublished work. 11. Graeser, J. B., Ginsberg, J. E., and Friedemann, T. E., J. BioZ. Chem.,

104,149 (1934). 12. Gorr, G., and Wagner, J., Biochem. Z., 161,488 (1925). 13. Pelgroms, J. D., Natuurw. Tidjschr., 14, 44 (1932). 14. Gettler, A. O., Niederl, J. B., and Benedetti-Pichler, A. A., Mikro-

chemie, 11,167 (1932).

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Theodore E. Friedemann and Rosalind KlaasALCOHOL

THE DETERMINATION OF ETHYL

1936, 115:47-61.J. Biol. Chem. 

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