national Gas Chromatography Symposiuni of Instrument Society of America,” p. 83, Michigan State
University, June 1961. Harsnape J. N., Whyman, B. H. F., ANAL. HEM. 32, 302 (1960). (5) Durrett, L. R., Simmons, M. C., Dvoretzky, I., Division of Petroleum Chemistry, 139th Meeting, ACS, St. Louis, Mo., March 1961. (4) Desty, D. H.,
(6) Eggertsen, F. T., Groennings, S., ANAL.CREM.30,20 (1958). (7) Groennings, S., ASTM Bull. No. 227, 64 (January 1958) [TP 30). ( 8 ) Jan&, J., J . Chromatog. 3,308 (1960). (9) Knight, H. S., ANAL.CHEM.30, 9 (1958). (10) Martin, R. L., Ibid., 32, 336 (19601. (11) Martin, R. L., Winters, J. C., Zbid., 31, 1954 (1959). (12) Porter, P. E., Deal, C. H., Stross,
F. H., J. Am. Chem. SOC.78, 2999
(1956). (13) Simmons, M. C., Richardson, D. B., Dvorypky, I., in “Gaa Chromatography 1960, R. P. W. Scott, ed., p. 211, Butterwortha, London, 1960. RECEIVED for review March 19, 1962. Accepted June 20, 1962. Division of +a1 tical Chemistry, Sym osium Honoring {. Zechmeister, 141st heeting, ACS, Washington, D. C., March 1963.
Gas Chromatographic Determination of Ethyl Alcohol in Blood for Medicolegal Purposes Separation of Other Volatiles from Blood or Aqueous Solution KENNETH
D. PARKER, CHARLES R.
FONTAN, JOHN L. YEE, and PAUL L. KIRK
School o f Criminology, University of California, Berkeley, Calif.
b A gas chromatographic method is described which, applicable to the medicolegal determination of the ethyl alcohol content of blood and aqueous solutions, offers advantages of rapid analysis, improved accuracy, simplicity, and specificity. Retention data for 56 volatiles indicate the resolution of the castor wax column used and illustrate its general utility for presumptive identification of volatile materials. Seven minutes were required to prepare and quantitate ethyl alcohol in a sample. Use of ethyl acetate as an internal standard obviated the necessity of precise measurement of blood samples. Standard blood and water samples with 0.023 to 0.1 80yoalcohol were analyzed with a precision of 2% and an accuracy of about 4%.
C
n E M I c h L and enzymic methods for the determination of ethyl alcohol in blood have been reviewed and evaluated by Lundquist ( 4 ) . Gas chromatographic methods have been described (1-3, 6),and their routine use has been attcmpted with varying degrees of success. The major problem has been the difficulty of dealing adequately with the high proportion of water in blood. In the use of both the thermalconductivity and the argon detectors, water lengthened the time required for a given analysis unless special chromatographic techniques (8) or special sample preparation ( 1 ) was employed to eliminate water. Chundela and Janak ( 2 ) employed 1-butanol and 2-butanone as internal standards, which avoided the necessity of accurate measurement of injected samples. Many liquid phases, operating parameters, injection methods, and chro-
1234
ANALYTICAL CHEMISTRY
matographic and sample preparation techniques were tested before selecting the described method. The hydrogen flame detector, castor wax column, and direct injection of prepared samples have advantages of rapid analysis, improved accuracy, simplicity, and specificity for routine determinations of ethyl alcohol in urine and blood samples. EXPERIMENTAL
Apparatus and Reagents. The HyFi gas chromatograph (Wilkens Instrument and Research Co., Walnut Creek, Calif.) with hydrogen flame ionization detector, Aerograph Model 600, and the Leeds & Northrup Speedomax H, 0- to 1-mv. recorder, Model S, equipped with the Disc chart integrator Model 207, were employed. The Hamilton microsyringe of 1 4 capacity, Model 7001N, was used to inject blood and aqueous solutions. The chromatographic column was a stainless steel tube inch in o.d., 0.093 inch in i.d., 10 feet in length. It was pack’ed with 60- to 80-mesh Chromosorb W, acid-washed, coated with castor wax 40% by weight. The column was preconditioned a t 190’ C. for about 8 hours. The operating conditions were: injector temperature 170’ C., oven temperature 120’ f 1’ C., flow rate of carrier gas (nitrogen) 13.6 ml. per minute, flow rate of hydrogen 22 ml. per minute, attenuation X8, volume injected 1 pl. The blood, ethyl alcohol, ethyl acetate, and water used to prepare the stock standards were free of impurities when examined by the gas chromatograph a t high sensitivity. Aqueous ethyl alcohol standards were prepared by diluting 5 ml. of C.P. ethyl alcohol, 95 to 98%, to loo0 ml. with water. The ethyl alcohol
concentration of this standard was 0.360 f 0.006% (w./v.) determined by direct oxidation by acid dichromate. Volumetric dilutions of portions of this standard were made, providing the other aqueous ethyl alcohol standards used : 0.180, 0.135, 0,090, 0.068, 0.045, and 0.023% ethyl alcohol. Blood ethyl alcohol standards were made by diluting 5.00 ml. of the appropriate standard to 10.0 f 0.02 ml. with blood. The synthetic blood ethyl alcohol samples contained 0.180, 0.090, 0.068, 0.045, and 0.023’% ethyl alcohol. Ethyl acetate standards were prepared by diluting 15.0 ml. of C.P. ethyl acetate to 2000 ml., making the 0.676% ethyl acetate standard from which, by volumetric dilution, the other aqueous standards-0.338, 0.169, 0.127, 0.085, and 0.043% ethyl acetate (w./v.)-were prepared. Only the 0.16970 ethyl acetate standard was used in the method, however. The other ethyl acetate standards were used only to investigate the linearity of chromatographic response. Procedure. Blood and water, standard and unknown samples, were prepared serially by an identical procedure. The same ethyl acetate standard, serum bottles of the same size, and the same two pipets were used. Pipets, bottles, and liquids were a t room temperature. Using the volumetric pipet, exactly 1.00 ml. of 0.16970 ethyl acetate standard was serially delivered into each clean, dry, and labeled serum bottle and stoppered until the sample containing the ethyl alcohol was delivered. The sample, 1.00 ml. of blood or water, was then placed in the bottle, and the pipet waa washed with the mixed contents of the bottle. The stoppered and mixed prepared samples, kept a t room temperature, were used within 4 hours, and discarded. Although ethyl acetate hydrolyzes a t room temperature and a t the pH of blood,
--
100-
!
90-
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Y
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I I
80'-
-z
70-
80-
70-
60-
0
:: 50- 400)
a
0)
0
0 0
Figure 1. sample
1
2
3
4
mlnutar
5
6
7
8
1
9
I
2
3
5
4
minut a s
6
7
8
9:;
-t
Figure 2. Chromatogram of 1 pl. of aqueous mixture
Chromatogram of 1 pl. of prepared blood
Approximately 0.1 % each of C, D, E, A. Peak resulted from water C. Methanol D. Acetone
1 .OO mi. each of ethyl alcohol (0.065qb) and ethyl acetate 10.1 69%)
F,
G, and I
E.
more than 4 hours is always required for the production of detectable amounts of ethyl alcohol, and no problem was encountered from this source. About 1 pl. of the prepared sample was injected into the gas chromatograph. The retention time and corresponding recorder responses were measured for each peak appearing on the chart. The ratio of the responses (ethyl alcohol to ethyl acetate) was calculated for quantitation of ethyl alcohol. Quantitation by both peak height and area was utilized and compared. The use of blood in the syringe made
Table 1.
Ethylakohd Isopropyl alcohol G. Ethyl acetote I. n-Propyl alcohol 1. impurity in n-propyl alcohol
F.
necessary special care in rinsing-i.e., three water rinses and one with the next sample being analyzed. Routinely, 60 to 80 injections of prepared blood samples were analyzed before cleaning of the injector chamber was necessary. After cooling and opening the bore of the injector tube, dried blood solids were removed by a wire brush, a pipe cleaner, and water. Before recoupling the column and nut,
the injector was heated and the carrier gas turned on to dry the injector tube quickly. The geometry of the injectorsplitter assembly of the later HyFi, Model A-600-B, is different, so that use for this ap lication would require a simple modi cation. Calculations. The following equation was employed to quantitate ethyl alcohol from the observed ratio of the chromatographic responses (ethyl alcohol t o ethyl acetate) obtained from the injection of the prepared sample.
Precision and Accuracy of Method in Comparison of Determination of Ethyl Alcohol in Water and Blood
k'= response of ethyl alcohol X response of ethyl acetate volume ethyl acetate volume ethyl alcohol X 7cethyl acetate ethyl alcohol
Ethyl alcohol content, % (g./lOO ml.). Determined by Direct Determination by Oxidation and Dilution Peak height 0.180 f 0 003 In water 0.179 f 0.001 In blood 0.179 f 0.002 In water 0.087f 0.001 0.090 In blood 0.091 f 0.002 In water 0.071 f 0.001 0,068 In blood 0.069 f 0.001 In water 0.044 f 0.001 0,045 0.044 f 0.OOO In blood 0.023 f 0.001 In water 0.023 In blood 0.022 f O.Oo0 Water average, K A Water average, K H 2.036 Std. deviation 0.054 Blood average, K A Blood average, K H 2.034 Std. deviation 0.039 0 Mean and range of triplicate determinations.
Response Ratios Peak area 0.176 f 0.003 0.180 f 0.004 0.087 f 0.001 0.088 f 0.001 0.070 f 0.OOO 0.070f 0.001 0.045 f 0.OOO 0.046 f 0.001 0.023 f 0.001 0.022 f 0.OOO
1.694
0.050
1.700 0.049
r:
\q.here volume and per cent refer to the iiiitial volume and per cent used in the preparation of the sample. K is the ratio of peak responses (height, K H , or area, K A ) ,ethyl alcohol to ethyl acetate, a t equal weights. Linear responses (peak height and area) were observed for both compounds when 0.1 to 3.0 rg. of injected compounds were plotted on the abscissa against the corresponding responses. Thus, K was derived as the ratio of the slopes of the corresponding response curves (ethyl alcohol to ethyl acetate). VOL 34, NO. 10, SEPTEMBER 1962
1235
Table II. Relative Retention Time of Compounds Studied
Compound Acetaldehyde n-Pentane Ethyl ether Water Methanol n-Hexane Propionaldehyde Acrolein Acetone Isopropyl ether Methyl acetate Methylene chloride Ethyl alcohol Acetonitrile Acrylonitrile Isopropyl alcohol n-Butylamine lert-Butyl alcohol
Relative Retention Time 0.49 0.54 0.59 0.59 0.74 0.81 0.82 0.83 0.87 0.89 0.90 0.98 1.00” 1.05 1.13 1.15 1.15 1.22
Compound Ethyl acetate n-Heptane Cyclohexane %Butanone Methyl iodide Trichloroethane Propionitrile Ethyl iodide Diethylamine Carbon tetrachloride Chloroform n-Propyl alcohol Allyl alcohol Allyl ether Benzene Acetylene dichloride sec-Butyl alcohol Ethylene trichloride
Relative Retention Time 1.30 1.41 1.45 1.47 1.54 1.58 1.59 1.60 1.62 1.67 1.78 1.80 1.88 1.96 1.98 2.04 2.04 2.09
Compound Ethylene dichloride Isobutyl alcohol n-Butyronitrile Dioxane Paraldehyde n-Butyl alcohol BEthylbutyraldehyde Toluene n-Butyl acetate Amyl alcohol n-Butyl ether Ethylene chlorohydrin Pyndine Chlorobenzene Isoamyl alcohol Formic acid Acetic acid Lactic acid Formaldehyde Carbon disulfide
Relative Retention Time 2.11 2.6I 2.70 3.32 3.39 3.46 3.59 3.62 4.30 4.58 4.74 5.22 6.02 6.94 7.38 b b b
I, b
Ethyl alcohol retention time 4.2 minuta. 0.2 pl. of compound injected and no response observed.
As the standard and the unknown samples, containing ethyl alcohol, are prepared identically, the same ethyl acetate standard and pipets being used, and as quantitation is achieved by comparison of response ratios, the per cent ethyl acetate and the pipet, volumes used become independent. RESULTS AND DISCUSSION
Figure 1 is a chromatogram resulting from 1 p l . of prepared blood sample containing 1.0 ml. each of ethyl alcohol (O.OG5%) and ethyl acetate (0.169QJc). The water response, A , in part due to the emerging water which cools the detector flame, was typical of the results obtained from water, urine, or blood injections. The chromatograph was a t X8 sensitivity, which was used without atteuuntion for samples ranging from 0.050 to 0.30070 ethyl alcohol. The ethyl alcohol, E , and ethyl acetate, G, added internal standard, emerged in less than 7 minutes, completing a determination. Either n-butyl or tert-butyl alcohol may serve as internal standard. The objection to them is primarily their possible occurrence in alcoholic beverages, and the fact that n-butyl alcohol has a longer retention time than is desirable. Separation of 1 pl. of aqueous mixture containing approximately 0.1% each of seven compounds is illustrated in Figure 2. Although acetone, D, was not completely separated from ethyl alcohol, E, a t this concentration ratio and a t the operating parameters of the method, a lower oven temperature, while causing greater separation of the 1236
ANALYTICAL CHEMISTRY
two, also extended the time required for an ethyl alcohol determination. Table I compares results of determinations of the ethyl alcohol content of the prepared blood and water standards by the chromatographic method. The K values (KHand K A ) were calculated according to the equation given above, and the average K values for water and blood, respectively, were used in the same equation to calculate thc ethyl alcohol content of the samples. The standard deviation for each average K value is given. From these results it appears that the method has equal applicability to blood and water samples with a precision of 270. As an error of 2y0 might appear in K , the accurarv in the ethyl alcohol determination should be within 4%. As seen from the precision of the ethyl alcohol determinations and standard deviations of the K values, the precision was slightly better, with the equipment used in this study, when the height rather than the area wm used for the calculations. At values of greater than 0.3% ethyl alcohol in blood, the gas chromstographic value may be as much as 2% higher than that obtained by the oxidative method, as contrasted with the agreement a t lower values. This has been explained by some as being a function of the ingestion of alcohol as compared with addition of the ethyl alcohol to the standard. It would appear more probable that the small difference is due to the inadequacy of the oxidative chemical determination with its riunierous possibilities of error, rather than to the gas chromatographic procedure in which n linear response is normally observed.
Table I1 lists retention data for 56 compounds relative to the retention time (4.2 minutes) of ethyl alcohol. These compounds were studied to investigate the specificity for ethyl alcohol and to estimate the general utility of the method for presumptive identification of other volatiles. Methylene chloride, ethyl alcohol, and acetonitrile had nearly identical retention times, being the only compounds studied which limit the specificity of the method for ethyl alcohol. Other compounds whose retention times were found to be close to that of ethyl alcohol, and which were not completely separated from it, were, however, separated sufficiently for their peaks to be observed. LITERATURE CITED
(1) Cadman, W. J., Johns, Theron, “Gas Chromatogra hic Determination of
Ethanol a n 8 Other Volatile8 from Blood,” unpublished data, 9th Annual Pittaburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1958. (2) Chundela, B., Janak, J., J . Forensic Med. 7, 153 (1960). (3) Fox, J. E., Proc. SOC.Exptl. Med. 97, 236 (1958). (4) Lundquist, Frank, “Methods of Biochemical Analysis,” Vol. 7, p. 217, Interscience, New York, 1959. ( 5 ) Salo, Tapio, 2. Lebensm. Unterswh. Forsch. 115, 54 (1961).
RECEIVEDfor review March 26, 1962. Accepted June 11, 1962. Work supported by grant from the National Institutes of Health, U. S. Public Health Service [EF 21(C3)], and from the Committee on Research, University of California. Spring meeting, California Association of Criminalista,’San Diego, Calif., May 1962.
