Table II. Calibration of Recording Dilatometer with Water
Specific
Recorder Reading
1.01207 1.01162 1.01116 1.01072 1.01028 1.00985 1.00943 1 . m 1
5.8 18.9 32.7 45.2 58.1 70.1 81.6 93.9
1.01162 1.01116 1.01072 1.01028 1.00985 1.00943 1. m 1 1.OO861
13.6 27.3 39.8 52.3 64.5 76.1 87.9 99.1
To,C. Volume of W a W
The recorder presently used is a portable one manufactured by Varian b ciatea and requires 10 mv. for full-scale displacement (Model G-11A). Any similar recorder could be used as well. CAUBRATION
Kl.0 49.0 48.0 47.0
46.0 45.0 44.0 43.0
Run 2 49.0 48.0 47.0 46.0 45.0
44.0 43.0 42.0 Data taken from reference (3).
in Figure 3. This circuitry permits accurate adjustment of recorder span to correspond always to a given displacement of the syringe barrel placement. The recorder zero adjustment permits setting the start of a run a t zero on the recorder chart regardless of the actual initial position of the syringe barrel. These two adjustmenta, zero and span, are not completely independent, but no dficulty is encountered in bringing both to the desired settings.
OF INSTRUMENT
Teats were made to check the output of the null balancing system to determine whether it accurately indicated displacement of the syringe barrel. The results are presented in Table I. The figures in column 1 represent the physical displacement of the syringe barrel from an arbitrary zero position. This displacement was measured by a dial indicator graduated in divisions of one thousandth of an inch. Column 2 gives the angular displacement of the potentiometer shaft. Column 3 gives the recorder reading corresponding to these displacements. These data have been fit by least squares to a straight line giving a value of 368.8 divisions per inch displacement. Given the above value for chart divisions per inch of displacement and the diameter of the syringe plunger (0.3368 inch in this instrument), the number of chart divisions per unit change in volume in the reaction chamber is readily calculated. For this ins&ment, the value 253 chart units per cc. was obtained. This method of palibration has proved easier than methods bawd on the volume of the dilatometer
bulb and the known temperature coefficients of specific volumes of various liquids, such aa water. Two examples of the latter method are given in Table 11, however. They serve to illustrate the quality of results which may be expected. The calibration constants calculated by least squares from these two runs were 246 and 250 scale divisions per cc. change in volume. These are in’good agreement with themselves and with the value 253 estimated above, Both runs were made with a dilatometer bulb which contains 116.0 cc. when the barrel of the syringe is in its uppermost position. The instrume‘nt accurately records changes in volume ‘of the dilatometer contents. LITERATURE CITED
(1) Automatic Temperature Control Co., Philadelphia, Pa., Bulletin R-31,“Differential Transformers aa Applied to
the Measurement of Straight Line Motions.” (2) Burnett, G. M., Trans. Faraday Soc.
46,772 (1950). (3) “Handbook of Chemistry and PhyaICE,” Chemical Rubber CO., 38th ed., 19561957 p. 1990. (4) Kelly, d. J., Harris, H. M., J . Am. Ceram. Soc. 39, 344-8 (1956). (5) Schulz, G. V., Harborth, G., Angm. Chem. 59,W (1947).
RECEIVEDfor review March 23, 1959. Accepted June 22,1959.
Rapid Determination of Blood Alcohol by Diffusion Oxidation in High Vacuum H. S. MAHAL Forensic Science laboratory, Bombay 8, India
bA
new simple, quick, and accurate procedure for estimations of blood alcohol has proved very useful in this laboratory. An analysis was done in 5 minutes with 0.5 ml. of blood. Recovery of alcohol was very good from blood having alcohol ranging from 0.01 to 0.50 gram per 100 ml. Apparatus and reagents used are readily available in all chemical laboratories, This procedure was not specific for ethyl alcohol, as other volatile reducing substances-e.g., methyl and propyl alcohol, ether, chloroform, and formaldehyde, if present, interfered with the estimation. Acetone, however, did not interfere, and volatile acids were fixed and did not diffuse while carrying out the test.
1908
ANALYTICAL CHEMlSTRY
Q
UANTITATIVE removal and oxida-
tion of ~1~0401 in blood are carried out in a minute by diffusion oxidation in high vacuum. The quantity of dichromate used in the oxidizing difbate is determined by iodometric titration. Volatile acids and free acetone in blood do not interfere with the test. With the introduction of complete prohibition in the state of Bombay, this laboratory has been concerned with thousands of determinations of alcohol in blood samples for liquor consumption cases each year. Because the courts in these cases depend on the blood alcohol concentration, it became imperative to have a rapid and axurate microtest that could be applied for routine multiple analyses.
Numerous micro- and macromethods have been described for the quantitative determination of alcohol in biological fluids. Most of these methods are based on Widmark’s method (6)which utilizes oxidation of the separated alcohol by acid,dichromate solutions; an alkaline permanganate solution had also been tried as an oxidizing agent (a,but was generally abandoned. Alcohol has been separated from blood by distillation and by aeration or diffusion desiccation a t room or higher temperatures ( I , 2 ) . Some of these methods, no& withstanding their lack in specificity as compared to enzymatic procedures (4), have been generally favored for routine multiple analyses. A special committee of the British Medical Association on the
Figure 1. Apparatus for oxidation in high vacuum
diffusion
recognition of intoxication, following the expert advice of the Royal Institute of Chemistry, London, recommended the use of chemical methods for routine analyses (3). Various procedures suggested for carrying out Widmark's method are tedious and time-consuming. The present procedure separates and oxidizes alcohol simultaneously in vacuum. It takes only a minute to complete the reaction, and the unused chromic acid is titrated in the reaction flask. A blood sample is analyzed in 5 minutes. This procedure was in use for a number of years in this laboratory and has been very eucrP88ful. REAGENTS
Standard potassium dichromate solution, approximately 0". Standard sodium thiosulfate solution, approximately 0.05N. Sodium carbonate, anhydrous. Sulfuric acid, concentrated. Potassium iodide solution, myo, freshly prepared. Starch indicator, lyo, freshly prepared. Pure! glassdistilled water. APPARATUS
The apparatus shown in Figure 1 consists of a round-bottomed 250-ml. flask with a shorbring neck, A , a 7.5 ml. glass bulb with neck 5 cm. long and 6 mm. internal diameter, B, and a three wa!7 stopcock, C. PROCEDURE
Potassium dichromate solution, 2.5 ml., is added to A followed by 10 ml. of concentrated sulfuric acid. A is rolled to wet three fourths of its inner surface with the solutions and is attached t o C.
One gram of anhydrous sodium carbonate is introduced into B followed by 0.5 ml. of blood. A and B are attached to C , so that A can either join B or the high vacuum pump, H , through the usual intervening traps containing concentrated sulfuric acid and strong potassium hydroxide solution on pumice stone pieces. A is first evacuated to about 1 mm. pressure. Stopcock, C , is then so manipulated that A is disconnected from H and cautiously connected to B. A beaker containing boiling water is gradually raised until B dips into it and is kept there for 45 seconds. The hot water is then removed and B disconnected from C. This allows air to rush into A which is given two or three revolving movements and disconnected from C. Then 150 ml. of pure glass-distilled water are gradually added to A and the unused chromic acid determined by iodometric titration. An excess of 5oy0 potassium iodide solution is added to A and the iodine thus released is immediately titrated with standard thiosulfate solution, using freshly prepared starch solution a t the end to determine the exact equivalent point. Each milliliter of 0.1N dichromate consumed corresponds to 1.15 mg. of ethyl alcohol in the blood used for the test. Acetonr, 0.500 gram per 100 ml. of blood, was repeatedly subjected to the diffusion oxidation procedure, but recovery was never more than the equivalent of 0.005 gram per 100 ml., and further dilutions, therrfore, were not tried. RESULTS
The procedure of diffusion oxidation in high vacuum was first tested by analyzing a series of six alcohol-water dilutions. About 300 mg. of anhydrous ethyl alcohol were dissolved in 100 ml. of pure glass-distilled water and accurately diluted with the requisite quantities of distilled water. The dilutions subjected to direct oxidation as well as to diffusion oxidation. Similarly, a blood sample containing about 500 mg. of ethyl alcohol was first prepared and six blood-alcohol concentrations made from it by accurately diluting with thr requisite quantities of normal blood. Recowry of alcohol from aqueous solutions and blood was good (Table I). Blood containing acetone showed practically no recovery of acetone by this procedure. DISCUSSION
Diffusion oxidation in high vacuum provides a rapid and accurate micromethod of determining alcohol in blood.
1.
Recovery of Alcohol by Diffusion Oxidation Alcohol contenta, gam/100 ml.
Table
Dilute Aqueous Solutions Blood Direct ReReOxidationo coveree Added covered" n- 215 o 488 0.486 __. o . 3ix .__ 0.210 0.105 0.052 0.026 0.013 hlean of 0
0.208
0.107 0.050
0.323
0.3% 0.162 0.081
0.024
0.040
0.012
0.020
0.160
0.079 0.038 0.017
three determinations.
Making the sample alkaline with anhydrous sodium carbonate rliminates volatile acids from interfrring with the results. Acetone in blood does not reduce chromic acid solution under similar conditions. Other volatile substancrs such as methyl and propyl alcohol, ether, chloroform, and formaldehyde, however. do interfere with the test. Because the oxidizing solution is titrated in the reaction flask, the procedure is fast and convenient and any loss or contamination of the oxidizing solutions that might occur in transfers to other vessels for titration purposes is obviated. Ordinary precautions necessary in microanalytical methods must be strictly adhered to. All ch(tmica1s used should be of reagent quality standard. All apparatus should be well cleaned with hot chromic acid and finally, 5 to 6 times with distilled water bfore use The atmosphere of the room where this test is carried out should be free from all gases and impurities likely to rffect the oxidizing solution. To prc>vc,nt frothing of blood, which may occur a t times in B while connecting it to A or while d i p ping it into the hot water bath, these o p erations should be carried out cautiously. A loose plug of glass wool may be inserted between B and C t o prrvcfintblood from going to thc stopcock at any time LITERATURE CITED
(1) Chaikelis, A. S., Foersheim, R. D., Am. J . Clzn. Pathol. 16, 180 (1946).
(2) Feldskin, hl., Klendaho;, N. c., Can. J . M e d . l'echnol. 16,48 (1954). (3) Kent-Jones, D. W., Taylor, G., =inalyst 79,121 (1954). (4) Kirk, P. L., Gibor, A , , Parker, K. P., ANAL.CHEM. 30, 1418 (1958). (5) Maclead, I,. I)., J . B i d . C h c n . 181, 323 (1940). ( 6 ) Widmark, E. M. P., Hiochem. %. 13i, 473 (1922); 218, 465 (1930;.
RECEIWDfor review February 18, 1059. Accepted July 6, 1959.
'401. 31, NO. 1 1 , NOVEMBER 1959
0
1ma
