In the Laboratory
Was the Driver Drunk? An Instrumental Methods Experiment
for the Determination of Blood Alcohol Content
Jennifer L. Zabzdyr and Sheri J. Lillard*
Department of Chemistry, University of California, Riverside, Riverside, CA 92521; *sheri.lillard@ucr.edu
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 pro-
cedures 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 mean-
ingless number.
Experiments of a forensic nature can be easy to incor-
porate into existing laboratory formats using available instru-
ments. 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
JChemEd.chem.wisc.edu • Vol. 78 No. 9 September 2001 • Journal of Chemical Education
W
routinely accepted technique for such a determination is gas
chromatography (GC) (12–15). Since the experiment was de-
signed 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 quan-
titate 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/
1225
 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 add-
ing 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.
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% 10000

Carbowax 1500 on Chromosorb W 60–80 mesh support.

A helium mobile phase (20–40 mL/min) is used. Hydrogen 8000
y = 5988.6x − 95.857
(30 mL/min) and compressed air (240 mL/min) are detector R 2 = .9952
Peak Area

gases. Injection, column, and detector temperatures are 175, 6000

80, and 200 °C, respectively. Headspace analysis is performed

by withdrawing and injecting exactly 500 µL of vapor from the 4000

vials using a 1-mL Hamilton Gastight syringe. The syringe

is flushed with air between runs to prevent cross-contamination. 2000

Hazards 0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

There are no significant hazards associated with this 0.8

experiment. We recommend the use of latex gloves and
eye protection.
0.6 y = 0.4044x + 0.0007
R 2 = .9998
Area Ratio

Results
0.4

Qualitative Analysis
A representative chromatogram obtained by the students 0.2

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, 0.0
respectively. Owing to this variation, the relative retention 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

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-pro-
panol internal standard) was 0.637 ± 0.008 (n = 26), based
on student data.
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.

1226 Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu


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 typi-
cally 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:
serum ethanol concn (g ethanol/100 mL serum)
BAC =
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 per-
cent 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
JChemEd.chem.wisc.edu • Vol. 78 No. 9 September 2001 • Journal of Chemical Education
In the Laboratory
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 com-
mercial 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.
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