THE INTERFEROMETRIC DETERMINATION OF ALCOHOL IN BLOOD

BY JOSEPH C. BOCK

(From the Department of Physiological Chemistry, Marquette University Medical School, Milwaukee)

(Received for publication, July 20, 1931)

INTRODUCTION

A critical study of the methods for the determination of ethanol in biological fluids and tissues brings the realization of the fact that the very large majority of these procedures is not sufficiently accurate to allow the determination of minute amounts nor the estimation of small variations when larger amounts occur.

With the exception of one method by Widmark (l), all proce- dures remove the alcohol by distillation. The material is diluted with water and the alcohol is distilled in VUCUO, with steam or under normal pressure.

The alcohol in the distillate is determined by various processes. A large number of these consist of modifications of Nicloux’s (2) method. All of the methods in this group are based on the oxi- dation with bichromate. The reagents used in these methods are not stable and must be restandardized frequently. The color changes, which determine the end-point, are very difficult to ob- serve, the exactness of the procedure being based on the subjective judgement of the observer, to the extent that errors of 10 drops or more may occur. In one of the best of the oxidation methods by Gettler and Tiber (3), the authors themselves admit consistently low results and apply a correction factor.

Fischer and Schmidt (4) convert the alcohol into ethyl nitrite and acetic acid. The nitrite is aerated with a stream of carbon dioxide into a potassium iodide solution acidified with hydrochloric acid. The method is not accurate, is time-consuming, and neces- sitates the use of complicated apparatus.

In the methods of Bugarsky (5), Stolz (6), and Spechter (7), 645

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646 Interferometric Alcohol Determination

bromine is used as an oxidizing agent, the alcohol being converted to acetic acid with the formation of hydrobromic acid. The neces- sary excess of bromine must be removed by heating, a procedure which results in appreciable losses of hydrobromic acid.

The alcohol in the distillate can be determined by the pycnom- eter as described by Kuhn (8) and Vollmering (9). The pro- cedures as well as the vaporimeter method of Nungesser (10) necessitate several distillations and are of doubtful value where small amounts of alcohol are concerned.

The immersion refractometer is not sufficiently accurate when used with very dilute alcohol solutions.

Although the study of the literature undertaken for this work is not claimed to be exhaustive, we have found only one attempt to remove the proteins before distillation. This method of Maignon (ll), however, is rather complicated and inaccurate. In the present work we remove the blood proteins with a single reagent, distil in a very simple distilling apparatus, and determine the alcohol by means of an interferometer. This instrument has been used for this purpose by Kionka (12). He distils the alcohol from the whole blood in uucuo, using a very complicated, costly, and fragile apparatus, which shortcomings the present method attempts to overcome.

Interjerometric Measurements

Before describing our procedure it is deemed necessary to give a very brief discussion of interferometric measurements. A de- tailed description of the instrument and its many uses can be obtained from the makers, Carl Zeiss, Jena, Germany. The prin- ciples of physics involved in its construction are described by the inventor, Lowe (13, 14).

The Lowe-Zeiss interferometer for liquids (and gasses) is an instrument which measures the di$erence of the refractive indices of two liquids or solutions. Two coherent pencils of light, coming from a small lamp, traverse the two media under examination. If both liquids are of exactly the same density the observer sees through the eyepiece of the instrument two horizontal fields with series of vertical bands, the interference bands. The fields are traversed by two black median bars bordered on each side by colored bands. The two pairs of black bands, the minima of the

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J. C. Bock 647

first order, coincide only if both liquids are alike. If there is the slightest difference in their refractive indices, the two black bands of the upper field move from their median position, to the right or left. By turning the compensator with the micrometer screw, the bands of the upper field are made to coincide with those of the lower and immovable comparison spectrum. The amount of rotation can be read from the scales attached to the micrometer

,

FIG. 1 FIG. 2 FIG. 1. Micrometer screw showing the two scales to be considered in the

interferometric measurements. The circular or drum scale has 100 di- visions: the vertical scale 30.

FIG. 2. The gold-plated metal chamber, consisting of two cells closed with glass at each end, in which the liquids to be compared are contained.

screw. As seen in Fig. 1 there are two scales to be considered. The circular or drum scale has 100 divisions, the vertical scale has 30. One full turn of the drum moves the circular scale 1 division upward on the vertical scale, therefore 1 division of the latter is equivalent to 100 drum scale divisions. The reading shown in Fig. 1 is 174.5.

The liquids to be compared are contained in a gold-plated metal chamber, consisting of two cells closed with glass on each end (Fig. 2). The inside measurements, in the optical axis, vary; 5

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648 Interferometric Alcohol Determination

mm., 10 mm., 20 mm., 40 mm., and 80 mm. chambers being available. For the present work the two largest sizes were used. If both cells are filled with the same liquid the reading for the sallze chamber will be the same. To illustrate:

40 Mm. Chamber

Both cells water.. . . . . . . . . . . . . .drum reading, 18 “ “ 0.1 per cent alcohol. . . . . “ “ 18 I‘ “ 0.01 “ “ “ . . . . . . . . . ‘( “ 18

With the 80 mm. chamber with the same three liquids three readingg of 33 are obtained.

When beginning a series of determinations one must always de- t,ermine the zero value of the chamber aa illustrated above. For our purpose water is indicated as comparison fluid. The tempera- ture must be the same throughout the whole set of observations. After the zero value of the chamber has been ascertained, the comparison fluid (water) is left in one cell, preferably the right one. The other cell is emptied by means of a pipette. The tip of the pipette should be protected with a short piece of small rub- ber tubing, to prevent scratching. The cell is then dried with rolls of filter paper and finally with a wad of absorbent cotton. Soft linen, commonly used in cleaning optical instruments, should be avoided, because it may contain traces of starch which will introduce a serious error. After the cell is dry, it is filled with the alcohol solution and the chamber put in the interferometer. After the temperatures of the cells are equal (3 to 5 minutes) the position of the bands is observed, the micrometer screw is turned until the bands coincide, and the scales are read. The reading is in- terpreted by means of a previously determined curve (Figs. 3 and 4). The curve is obtained by plotting the scale reading of solutions of known strength against the percentage of these solutions. It is beat to plot two or three curves, depending on the kind of work. We have one for concentrations from 0 to 0.01 per cent, the second to 0.1 per cent; a third can be carried as high as the expected results demand. Instead of a curve, interpolation may be used. If, for instance, a known alcohol dilution of 0.02 per cent reads 79 against water in an 80 mm. chamber and another of 0.04 per cent reads 119, a scale reading of 99 would indicate an alcohol concen-

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J. C. Bock 649

tration of 0.03 per cent. From the above figures it can be easily seen that 1 scale division is equivalent to 0.0005 per cent alcohol when the 80 mm. chamber is used.

55 53 240

220 51

200 49

180 41

160 45

140 45

120 41

100

39 ii 5l 70

60

35 :: 33 30 0 Essxsg+s:%g ddddddoddo

FIQ. 3 FM. 4 FIQ. 3. Standardization curve; 0.001 to 0.01 per cent

Fro. 4. Standardization curve; 0.01 to 0.1 per cent

Distilling Apparatus

The purpose of the first set of experiments was to determine to what degree the recovery of very small amounts of alcohol could be accomplished by a simple distillation process.

The distilling flask used is a 50 ml., round bottom, Pyrex flask. The connection leading from the flask to the condenser is made from a 5 ml. pipette having a tube of comparatively large diameter. This tube must be of the same dimension as the condenser tube,

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650 Interferometric Alcohol Determination

i.e. 6 to 7 mm. or $ inch outside diameter. The two tubes must touch very closely and are held together by a heavy walled rubber tube. It was found that the condenser tube had to be fairly short, which fact caused a comparatively small condensing surface. In order to get satisfactory recoveries the distillation had to be ex- ceedingly slow. This difficulty was finally overcome by using a metal condenser tube. Either silver’ or copper’ tubing may be used, although the use of silver is indicated when the highest ac- curacy is desired. The length of tube, before bending, is 48 cm. The metal tubing must first be washed with dilute nitric acid and then rinsed with water several times. After steaming the tube, it is washed again two or three times with distilled water. It is advisable to keep the metal condenser tube closed or filled with water when not in use. If the condenser has not been used for more than 24 hours, it is best not only to rinse the tube but to clean it further by distilling about 20 to 30 ml. of water through it. These precautions will not appear superfluous when one considers that the interferometer is capable of measuring refraction dif- ferences of liquids within 2 units of the seventh decimal.

The receiver, usually a 25 ml: volumetric flask, contains a small (1 to 2 ml.) amount of water and is placed so that the condenser tube touches the water at the bottom of the flask, forming a seal. As, the distillate accumulates, the receiver is gradually lowered, so that the end of the condenser tube is only a short (3 to 5 mm.) distance under the surface of the liquid. The lowering of the flask is easily done by using a wedge-shaped support. The small flame of the micro burner should be shielded as a further precaution against back suction.

Recovery of Alcohol by Distillation

The effectiveness of the distillation procedure was tested in the following manner. A series of alcohol dilutions was made from pure ethanol, certified burettes and flasks being used. The solu- tions were further checked by means of an immersion refractometer whenever the dilution permitted. 25 ml. quantities were measured into the distilling flask and distilled, a 25 ml. volumetric flask being

1 The silver tubing can be obtained from the American Platinum Works, Newark, New Jersey. The copper tubing, 2 inch outside diameter, can be bought from any automobile supply store.

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J. C. Bock 651

used as a receiver. The process was continued until about 20 ml. had been collected. The condenser is rinsed into the flask with small amounts of water, the condenser tube being then above the liquid level. The flask is made to volume and the contents thoroughly mixed. The distillates are analyzed with the inter- ferometer. In this series of experiments we did not use water as comparison fluid, nor did we make use of the plotted curves. We use the original alcohol solutions instead; that is to say, one portion was distilled, the other serves as comparison liquid. It has been mentioned above, that if both cells of the interferometer

TABLE I Recovery of Alcohol from Solutions of Known Strength

Alcohol Sale reading

Left cell

per ten

Stock solution ...... .O. 5 Distillate ........... .O. 5

“ ............ 0.5

Stock solution ...... .0.5 Distillate ........... .O. 5

Stock solution. ..... .0.05 Distillate ........... .O. 05

Stock solution ...... .O. 01 Distillate .......... .O. 01

-

. _ t

-

Right cell

per cen1

Stock solution ..... .O. 5 “ “ ..... .0.5 “ “ ...... 0.5

I‘ “ ..... .O.l ‘I “ ..... .O.l

“ “ .... ..o.o 5 “ “ .... ..o.o 5

“ ‘I ..... .O.Ol I‘ “ .... ..o.o 1

40 mm. 80 mm. chamber chamber

17 16 17

17 17

18 19

18 17

33 33 34

34 33

35 35

34 33

chamber are filled with the same liquid the reading is the zero value. We established this value by using the various alcohol solutions in both cells, and then reading the distillate from each dilution against the original (stock) solution. This procedure furnishes the most accurate check on the method. The instru- ment can be read accurately within 1 or 2 scale divisions or, in the present case, within limits of 0.0005 to 0.001 per cent ethanol by volume. Table I gives some of the results. From the table it will be seen that all the readings are within 1 scale division of the zero value and no appreciable loss of alcohol has occurred in the distillation process.

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652 Interferometric Alcohol Determination

Reagent

Almost every method for the determination of alcohol in blood uses distillation without removing the proteins. The disadvan- tages of such a procedure are obvious. We found that the blood proteins may be removed and the filtrate used for distillation without any appreciable loss of alcohol. A number of protein precipitants were considered and tried. Those containing volatile material are ipso facto excluded. Others did not yield enough filtrate for the determination or called f or dilutions too great for our purpose.

A phosphomolybdic acid reagent was found satisfactory. It gives a coarse precipitate and ample filtrate. The composition is as follows: 12 gm. of phosphomolybdic acid and 10 gm. of sodium sulfate (crystals) are dissolved in about 600 ml. of water. 9 ml. of concentrated sulfuric acid are added and the solution is then boiled for approximately 15 minutes. After cooling make to 1 liter.

Procedure

10 ml. of blood are run slowly into a 50 ml., glsss+&oppered volumetric flask, containing about 35 ml. of the phosphomolybdic acid reagent. The flask is gently agitated (not shaken) to mix the contents and iilled to the mark with reagent. Mix well, let stand about 10 minutes, and transfer to a 50 ml. centrifuge tube. This tube has a slightly constricted neck and is closed with a rubber cap.” The tube is centrifugated for approximately 10 minutes at high speed. The supernatant liquid is poured through a small coarse iilter to free it of the small particles of precipitate which adhere frequently to the upper part of the tube.

25 ml. of filtrate are transferred to the distilling flask and dis- tilled at a moderate rate of sp,eed until about 20 ml. have been re- ceived in a 25 ml. volumetric flask. The flask is made to volume, the contents are well mixed, and the alcohol is determined in the interferometer.

Tests for Losses in Manipulation

In order to determine if any measurable losses of alcohol- will occur with the procedure described above we performed the fol-

* International Equipment Company, Boston, Massachusetts, cata- logue; tube No. 5‘20, cap No. 584.

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lowing experiments. Instead of using blood we used alcohol solu- tions of known strength. 10 ml. samples of these solutions were diluted withreagent to 50 ml. and centrifugated in the capped tubes for the same length of time as the blood samples. They were then run through a filter and distilled. In the regular analysis of blood we use water as comparison fluid in the interferometer chamber. In this set of experiments we used the same alcohol dilutions as

TABLE II

Recovery of Added Alcohol

Sample No. Alcohol added Alcohol recovered Dil%X‘XXe

per cent per cent per cent 0.04 0.0385 -0.0015 0.04 0.0395 -0.0005 0.04 0.0400 0.0000

0.06 0.0605 +0.0005 0.06 0.0585 -0.0015 0.06 0.0600 0.0000 0.06 0.0595 -0.0005

0.02 0.0205 +0.0005 0.02 0.0190 -0.0010 0.02 0.0205 +0.0005

0.1 0.0990 -0.0010 0.1 O.looo 0.0000 0.1 0.0985 -0.0015

0.2 0.1985 -0.0015 0.2 0.2000 0.0000 0.2 0.1995 -0.0005

comparison fluid which we used for the manipulations described above. If we dilute 10 ml. of 0.2 per cent alcohol to 50 ml. in the above procedure we obtain a 0.04 per cent solution. This solution, after centrifugation, filtration, and distillation was compared in the interferometer with a 0.04 per cent alcohol which in turn was obtained by diluting 10 ml. of the same 0.2 per cent alcohol to 50 ml. Several series of triplicate determinations were made with alcohol concentrations varying from 0.01 to 0.2 per cent.. The greatest

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654 Interferometric Alcohol Determination

variation from the theoretical reading, the zero value, was 4 scale divisions, but 93 per cent of the results showed readings within +1 or - 1 of the zero value, 2 scale divisions being equivalent to 0.001 per cent of ethanol when the 80 mm. chamber was used.

Recovery of Alcohol Added to Blood

The next problem was to determine whether or not a definite amount of alcohol added to the blood could be successfully re- covered. The alcohol contents of bloods were first, determined. Accurately measured amounts of alcohol were then added to por- tions of the same blood and the samples analyzed as described be- fore. From Table II it will be seen that the losses are very small. The results given in Table II are representative of the findings from a large number of deberminations.

Interfering Substances

The presence of volatile substances other than ethanol has to be considered. Acetone, acetaldchyde, lact.ic acid, and glycerol have been reported in human blood. Our distillates were examined for these substances.

Acetone and Acetaldehyde-Traces of these substances were found to be present in less than 3 per cent of the cases studied. The distillates showing posit,ive resuhs were not read in the interferometer.

Glycerol--Dilute solutions of glycerol were distilled in the same manner as t.he blood filtrates. The results were negative.

Lactic Acid-A 0.1 per cent solution of lactic acid subjected to the usual distillation procedure gave distillates which read within the limits of error of the method.

The present work has confined itself to bloods of normal individ- uals. It was found that the blood of adults, taken before break- fast, about 12 hours after the last meal, fluctuates from 0.0015 per cent to 0.0113 per cent, the majority of cases varying only from 0.003 to 0.005 per cent ethanol.

A study of the alcohol concentrat.ion of pathological bloods and of tissues is in progress.

SUMMARY

A rapid and accurate method for the determination of alcohol in normal blood is presented.

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J. C. Bock 655

BIBLIOGRAPHY

1. Widmark, E. M., Biochem. Z., 131, 473 (1922). 2. Nicloux, M., Compl. rend. Sot. biol., 9,341 (1906). 3. Gettler, A. O., and Tiber; A., Arch. Path. and Lab. Med., 3,75 (1927). 4. Fischer, W. hf., and Schmidt, A., Ber. them. Ges., 67, 693 (1924); 69,

079 (1926). 5. Bugaraky, A., Math. u. Naturwissenach. Ber. Ungarn, 23, 35 (1905). 6. Stolz, Inaugural dissertation, Giessen (1914). 7. Spechter, T., Inaugural dieeertation, Geiseen (1917). 8. Kiihn, H., Inaugural dissertation, Giessen (1912). 9. Vollmering, J., Inaugural dissertation, Gieeeen (1911).

10. Nungeaser, W., Inaugural dissertation, Giessen (1913). 11. Maignon, F., Compt. rend. Acad., 140, 1063 (1905). 12. Kionka, Pharmakologische Beitriige zur Alkoholfrage, Jena, pt. 1 (1927). 13. L8we, F., Physik. Z., 11, 1047 (1910). 14. Lijwe, F., 2. Znstrumentenk., 39, 321 (1910).

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Joseph C. BockBLOOD

DETERMINATION OF ALCOHOL IN THE INTERFEROMETRIC

1931, 93:645-655.J. Biol. Chem. 

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