In the Laboratory
Was the Driver Drunk? An Instrumental Methods Experiment for the Determination of Blood Alcohol Content
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Jennifer L. Zabzdyr and Sheri J. Lillard* Department of Chemistry, University of California, Riverside, Riverside, CA 92521; *
[email protected] Rationale The combination of forensic science and instrumental analysis is a very effective approach for teaching analytical chemistry (1). A major goal in revitalizing our instrumental methods laboratory is to add forensic-based experiments to spark student motivation and enthusiasm for the course material. A course on forensic analytical chemistry has been taught by Brewer et al. at the University of South Carolina with the similar goal of increasing student participation in the chemistry lecture (2). We want the students to understand the implications of an incorrect determination (a suspect could go to jail), thereby motivating correct analytical procedures and careful interpretation of the data. Our students seemed to enjoy the added responsibility of using their measurements as the basis for important forensic-based conclusions instead of simply calculating a possibly meaningless number. Experiments of a forensic nature can be easy to incorporate into existing laboratory formats using available instruments. These experiments can be performed at all levels, as evidenced by an introductory experiment in the identification of arson accelerants for general chemistry students (3). In our instrumental methods laboratory we have added forensic experiments based on topics included in L. J. Kaplan’s course for non-science majors, Chemistry and Crime: From Sherlock Holmes to Modern Forensic Science (4, 5). Because our course is taught on the quarter system (ca. 27 lectures, 50 min each), time does not permit us to cover some of the interesting topics taught by others (e.g., court testimony) that are not directly related to chemical analysis. The specific objective of this new experiment, designed for a 4-hour laboratory period, is to perform qualitative and quantitative analysis of blood alcohol content (BAC). BAC is typically determined through analysis of breath (6 ) or blood (7, 8). The Breathalyzer, in particular, is a common method used in student experiments for estimation of BAC (9–11). In our experiment students measure the alcohol level in a simulated serum sample and convert the value to BAC. The
routinely accepted technique for such a determination is gas chromatography (GC) (12–15). Since the experiment was designed around existing laboratory equipment, the students will use GC with flame ionization detection (FID) for analysis. FID has the additional advantage that water vapor does not interfere with it (16 ). Qualitative analysis is accomplished by comparing the retention time of ethanol in a standard with the retention times of peaks that occur in the sample. Both external and internal calibration methods are used to quantitate the ethanol content in a (simulated) serum sample from a suspected drunk driver. The significance of this new experiment is to teach students that experiments are performed for a reason and that careful laboratory technique is extremely important. When placed in the context of a drunk-driving scenario with emphasis that an innocent person could go to jail (or a guilty person could be set free) if sloppy experiments are performed, these obvious statements become clearer. Experimental Procedure
Reagents Ethanol and n-propanol were obtained from Fisher Scientific (Fairlawn, NJ) and bovine serum was obtained from Sigma (St. Louis, MO). Deionized water (resistance ≥18 MΩ) was obtained from a Milli-Q system (Bedford, MA). Preparation of Stock Solutions A stock solution of ethanol (10 mg/mL in deionized water) and two stock solutions of n-propanol (1:50 dilutions in both deionized water and 10 mg/mL ethanol) were prepared for student use. Preparation of Standards Five ethanol solutions (0.2, 0.5, 0.8, 1.0, and 1.5 mg/mL) were prepared in 100-mL volumetric flasks using 10 mg/mL ethanol. Ten milliliters of the n-propanol/water stock solution (internal standard) was added to each flask before diluting to the mark with water. Tenfold dilutions of the n-propanol/
JChemEd.chem.wisc.edu • Vol. 78 No. 9 September 2001 • Journal of Chemical Education
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In the Laboratory
water and n-propanol/ethanol stock solutions were also prepared. Fifteen milliliters was transferred immediately to a 20-mL vial and sealed with a screw-top septum cap. To facilitate transfer of volatile components into the vapor phase, each vial was allowed to equilibrate at room temperature for at least 15 min before the sample was withdrawn.
Preparation of Serum Unknowns The teaching assistant prepared serum unknowns by adding 850 to 990 µL of the 10 mg/mL ethanol solution to 10-mL volumetric flasks and diluting to the mark with bovine serum, giving BAC values ranging from 0.0746 to 0.0868. The students prepared serum unknowns for analysis by adding 1.0 mL of n-propanol/water stock solution to a 10-mL volumetric flask and diluting to the mark with the serum unknown. After careful mixing, exactly 7.5 mL of serum unknown was transferred from the volumetric flask to a 10-mL vial and sealed with a screw-top septum cap. A serum blank (i.e., without ethanol) was prepared by adding 7.5 mL of bovine serum to a 10-mL vial and sealing it with a screw-top septum cap. Both the unknown and the blank were allowed to equilibrate in the vials at room temperature for at least 15 min prior to analysis. Figure 1. Chromatogram of 1.5 mg/mL ethanol standard.
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Equipment A Shimadzu GC-14APsF gas chromatograph/flame ionization detector is used in this experiment. Chromatograms are plotted with a chart recorder. The column is a 2-meter × 1 ⁄8-in. stainless steel column with a stationary phase of 10% Carbowax 1500 on Chromosorb W 60–80 mesh support. A helium mobile phase (20–40 mL/min) is used. Hydrogen (30 mL/min) and compressed air (240 mL/min) are detector gases. Injection, column, and detector temperatures are 175, 80, and 200 °C, respectively. Headspace analysis is performed by withdrawing and injecting exactly 500 µL of vapor from the vials using a 1-mL Hamilton Gastight syringe. The syringe is flushed with air between runs to prevent cross-contamination.
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There are no significant hazards associated with this experiment. We recommend the use of latex gloves and eye protection.
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y = 0.4044x + 0.0007 R 2 = .9998
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Qualitative Analysis A representative chromatogram obtained by the students is shown in Figure 1. Typical retention times for n-propanol and ethanol ranged from 0.60 to 0.75 min and 0.38 to 0.48 min, respectively. Owing to this variation, the relative retention time (RRT) method was used for positive identification. The RRT of an unknown peak is the ratio of the retention time of the unknown peak to the retention time of an internal standard. The average RRT value of ethanol (with the n-propanol internal standard) was 0.637 ± 0.008 (n = 26), based on student data.
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y = 5988.6x − 95.857 R 2 = .9952
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Ethanol Concentration / (mg mL᎑1) Figure 2. Representative calibration curves used to quantitate ethanol in serum samples: Top: External standardization using ethanol peak area. Bottom: Internal standardization. Area ratio refers to ethanol peak area divided by n-propanol peak area. Two injections were performed for each standard.
Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu
In the Laboratory
Quantitation Ethanol is quantitated in the serum unknown using both external and internal standardization methods. Examples of external and internal calibration curves (using 0.2 to 1.5 mg/ mL ethanol standards) are shown in Figure 2. When students performed experiments carefully, both calibration curves typically yielded R 2 > .98. Determination of BAC BAC is defined as grams of ethanol per 100 mL of blood. Since dealing with blood presents serious safety issues, especially in an undergraduate teaching laboratory, bovine serum is used. Alcohol does not partition equally between red blood cells and blood serum owing to differences in water content (17). Therefore, to determine BAC using serum instead of blood, a conversion factor (17) is required: BAC =
serum ethanol concn (g ethanol/100 mL serum) 1.14
Compiled student data show that this experiment provides an accurate method for quantitation of alcohol in serum samples. Using external standardization, the differences between actual BAC values and student values ranged from 0.48 to 50.33%, averaging 15.13 ± 13.57% (n = 31). Nineteen percent of the students were within 5% of the actual BAC and 50% were within 10% of the BAC. The results from internal standardization are even better. Differences between actual and student BAC values ranged from 0.44 to 40.68%, averaging 9.61 ± 9.37%. Using this method of quantitation, 33% of the students came within 5% of the actual value and 53% came within 10% of the actual value. Conclusions Gas chromatography is a common method in commercial and forensic laboratories for determination of BAC in suspected drunk drivers. Our experiment is designed for upper-division chemistry students and serves several purposes. The students gain experience using headspace GC as an analytical tool for qualitative and quantitative analysis. The experiment also gives students an opportunity to compare external and internal methods of standardization. Although experiments that illustrate such methodology are common, the novelty in this experiment is that it goes one step further. It shows how these techniques are used outside the classroom in real laboratories and in a situation that is relevant to the students, especially those who consume alcoholic beverages. Students also learn that poor laboratory technique could have penalties far worse than bad grades. The serum unknowns are designed to have BAC values that bracket the California
legal limit of 0.080 by a relatively small margin. If they were real samples, even a seemingly small error by a technician could have severe consequences. This is impressed upon the students, many of whom may soon be working in a commercial laboratory. To achieve accurate results, students must be meticulous in their laboratory practices. Careful technique can result in experimental BAC values within 5% of the actual values; sloppy sample preparation or analysis is evidenced by BAC values that deviate as much as 50% from the actual values. Overall, students found the experiment interesting and a vast improvement over other GC experiments they had performed in the past. W
Supplemental Material
Notes for the instructor and detailed instructions for students are available in this issue of JCE Online. Literature Cited 1. Gaensslen, R. E.; Kubic, T. A.; Deslo, P. J.; Lee, H. C. J. Chem. Educ. 1985, 62, 1058–1060. 2. Brewer, W. E.; Lambert, S. J.; Morgan, S. L.; Goode, S. R. Teaching Forensic Analytical Chemistry; ChemConf ’98, OnLine Conference on Chemical Education 1998; http:// www.inform.umd.edu/EdRes/Topic/Chemistry/ChemConference/ ChemConf98/forensic/ChemConf.htm (accessed May 2001). 3. Elderd, D. M.; Kildahl, N. K.; Berka, L. H. J. Chem. Educ. 1996, 73, 675–677. 4. Kaplan, L. J. http://www.williams.edu/Chemistry/lkaplan/ 113des.html (accessed May 2001). 5. Kaplan, L. J. Crime Lab. Digest 1992, 19, 107–132. 6. Labianca, D. A. J. Chem. Educ. 1990, 67, 259–261. 7. Berkebile, J. M. J. Chem. Educ. 1954, 31, 380–382. 8. Labianca, D. A. J. Chem. Educ. 1992, 69, 628–632. 9. Vitz, E.; Chan, H. J. Chem. Educ. 1995, 72, 920–925. 10. Timmer, W. C. J. Chem. Educ. 1986, 63, 897–898. 11. Anderson, J. M. J. Chem. Educ. 1990, 67, 263. 12. CG Labs, Inc.: Concord, NH; http://www.cglabs.com/ cgl_pro.htm (accessed May 2001). 13. Macchia, T.; Mancinelli, R.; Gentili, S.; Lugaresi, E. C.; Raponi, A.; Taggi, F. J. Anal. Toxicol. 1995, 19, 241–246. 14. Schuberth, J. J. Chromatogr. Sci. 1996, 34, 314–319. 15. Baselt, R. C.; Cravey, R. H. Disposition of Toxic Drugs and Chemicals in Man, 3rd ed; Year Book Medical Publishers: Chicago, 1989; pp 322–326. 16. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed; Harcourt Brace: Philadelphia, PA, 1998; p 707. 17. Payne, J. P.; Hill, D. W.; Wood, D. G. L. Nature 1968, 217, 963–964.
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