CHAPTER 10: TOXICOLOGY: BLOOD ALCOHOL

Toxicology: Blood Alcohol

Drinking and driving and the crime of operating a vehicle under the influence of drugs or
alcohol (OVI) are common and costly to society. During the past decades in the United States,
OVI laws and greater awareness have reduced the number of accidents and deaths due to alcohol,
but the problem persists. Alcohol taken into the body is rapidly absorbed, circulated to all parts
of the body, including the brain, by the circulatory system, metabolized in the stomach and liver,
and finally excreted in the urine. A small portion of the imbibed ethanol also leaves the body
unchanged in the person’s breath. Alcohol acts a depressant, reducing inhibitions as well as
motor control. All U.S. states have laws for adult drivers that restrict the blood alcohol
concentration to 0.080 g ethanol per 100 mL of blood or less.

Testing for ethanol for legal purposes is performed on breath samples or blood/serum
samples. Breath alcohol content (BrAC) is measured by infrared spectrophotometry using a
specifically designed, automated instrument, such as the Intoxilyzer, that captures expired breath
and measures the transmittance of the gaseous sample at several frequencies of light
corresponding to ethanol, water vapor, acetone, and other expected vapors. The Intoxilyzer is
found in many police laboratories and provides a reasonably accurate measure of blood ethanol,
though BrAC must be converted mathematically to blood alcohol content (BAC) before the
determination of the guilt or innocence to an OVI charge can be made.

The BAC can be measured directly by headspace gas chromatography of a blood sample.
The blood sample is place in a vial that is sealed and heated, and substances vaporize to the
extent allowed by Henry’s Law. Fortunately, few compounds have sufficient volatility to become
significant interferences in the measurement of ethanol and the added internal standard. A small
volume of the gas above the solution, the so-called headspace, is removed and injected into a gas
chromatograph, fitted with a specialty column for alcohols and a flame ionization detector.
Ethanol and internal standard are measured, and the amount of ethanol in the sample is
quantified by comparison to an internal standards calibration plot. The gas chromatographic
method for blood ethanol is selective and sensitive and the method to which all others are
compared.

References on Blood Alcohol and Toxicology in Forensic Science

Stripp, R. “Forensic and Clinical Issues in Alcohol Analysis” in Forensic Chemistry Handbook, Koblinsky,
L.F. and Levine L., Editors, John Wiley & Sons, Hoboken, NJ, 2012, Chapter 12.

“Forensic Toxicology” in Criminalistics: An Introduction to Forensic Science, Saferstein, Richard ; 10th

edition; Prentice Hall: Upper Saddle River, NJ, 2011; Chapter 9.

Thompson, R.Q. “The thermodynamics of drunk driving”, J. Chem. Ed. 1997, 74, 532-536.

Logan, Barry K. "Analysis of alcohol and other volatiles." Ian Tebbett, ed. Gas Chromatography in
Forensic Science. Ellis Horwood: New York, 1992) pp. 87-108.

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FORENSIC ANALYTICAL CHEMISTRY LABORATORY MANUAL

Technique for Quantifying Ethanol in Blood

Headspace Gas Chromatography with Flame Ionization Detector

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CHAPTER 10: TOXICOLOGY: BLOOD ALCOHOL

Ethanol in Blood by Headspace Gas Chromatography

Goal
To determine the concentration of ethanol in a blood sample

Method of Analysis
Headspace sampling followed by gas chromatography using a flame ionization detector;
internal standard calibration.

Available Standards and Samples

Pure ethanol and pure t-butanol (or n-propanol), serving as an internal standard, are
available in the laboratory. Prepare a 0.20 w/v% internal standard solution for use by weighing
the compound and diluting with water.
Prepare an ethanol stock solution by weighing ethanol into a volumetric flask and
diluting to the mark with water. Choose a convenient concentration for the stock solution. Next
prepare, by volumetric dilution of the stock, aqueous ethanol working standards with
concentrations that cover the range expected for sober and impaired drivers. Make fresh working
standards daily.
Store both stock standard solutions in soft-walled bottles, filled up to the top, squeezed to
eliminate air space, tightly capped, and stored at 4 oC (refrigerator). The solutions should be
stable for at least two weeks.
[INSTRUCTOR NOTE: We prepared a stock ethanol solution of 5.0 w/v% and a stock internal
standard solution of 0.20%. Ethanol working standards were prepared by volumetric dilution in
pure water.]

Blood (synthetic) is available in the laboratory, stored in the refrigerator. Blood samples
(synthetic) collected from several persons are available for testing and stored in the refrigerator.
Allow the blanks and samples to warm to room temperature prior to use.

[INSTRUCTOR NOTE: Human blood is over 90 w/v% water with dissolved proteins (~7%) and
salts making up the remainder. Synthetic blood is prepared by mixing the white of one large
chicken egg (about 6 g of albumin protein) with 100 mL of water. Stir gently for 10 min, allow
undissolved solids to settle, and pour off about 90 mL into two 50-mL plastic centrifuge tubes.
Centrifuge the mixture to remove any remaining solids and retain the supernatant as “blood”. Add
a tiny amount of solid dye (e.g. phenol red) to color, if desired. Do not add dyes in solution, such
as food coloring, because these solutions often contain ethanol or other alcohols. Store the
synthetic blood in the refrigerator for up to two weeks.
Prepare the samples by adding 0.01 g to 0.15 g ethanol to synthetic blood in a 100-mL
volumetric flask. For instance, a person found guilty of OVI might have a blood sample containing
0.10 g ethanol per 100 mL of blood. Store the blood samples in the refrigerator for up to two
weeks. Eliminate headspace in stored solutions as much as possible.]

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Headspace Sampling
Prepare 100 ml of a 2 M aqueous solution of potassium carbonate as ionic strength
adjuster. Store the solution at room temperature.

[INSTRUCTOR NOTE: The increase in ionic strength resulted in higher extraction efficiency and
higher signals for ethanol and the internal standard. 28 g of K2CO3 are dissolved in 75 mL water
and then diluted to exactly 100 mL to produce a 2 M solution.]

To a 10-mL headspace vial add (in order) 5.00 mL of aqueous standard or sample, 1.00
mL of internal standard solution, and 1.00 mL of ionic strength adjuster. Immediately seal the
vial with a septum cap, and heat the vial to 80 oC. Heat the syringe that you will use to sample
the headspace as well. After 30 min, sample the headspace according to your instructor’s
directions and manually inject 1.0 mL of the gas into the gas chromatograph. Make sure that the
needle does not extend into the liquid phase during sampling.

[INSTRUCTOR NOTE: Headspace sampling is most often accomplished by means of an

automated headspace sampler unit paired with the gas chromatograph. After gas-liquid
equilibrium is reached in the sample vial, carrier gas is used to pressurize the vial and to force an
aliquot of the headspace into the autoinjector. A known and reproducible volume of headspace is
injected. If an automated headspace sampler is available, use it instead of these directions for
manual sampling and injection.
The temperature and time for manual headspace sampling were determined experimentally
to provide good sensitivity and precision in a reasonable amount of time. Signals for ethanol and
internal standard increase with time and then level off around 30 min. Likewise, signals increase
o
with temperature. The chosen temperature, 80 C, is about the highest most people can stand in
handling the syringe (same temperature as the headspace vial) without resorting to more than
thin cotton gloves.
A 22-gauge syringe needle with luer-lock connector, cut off to give the proper length and a
blunt end, is fitted into a one-holed, #5, rubber stopper. See photos below. The needle/stopper
assembly placed on top of the headspace vial punctures the septum without coring and acts as a
spacer to ensure that the headspace, not the liquid phase, is sampled. In sampling the gas
phase, pressurize the vial with 2 mL of air from a 5-mL luer lock syringe, wait 5 seconds for
mixing, draw up the syringe to the 1.5-mL mark, and then wait 20 seconds for the syringe to
completely fill with headspace gas. Remove the needle/stopper assembly from the vial,
disconnect the syringe, add a syringe needle appropriate for the gas chromatograph, and depress
the plunger to the 1-mL mark. Immediately inject the gas sample into the gas chromatograph.]

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CHAPTER 10: TOXICOLOGY: BLOOD ALCOHOL

Gas Chromatography
Follow instructions to set up the gas chromatograph for use. Ensure that the appropriate
column is installed as well as an unpacked split injection liner. The injection mode is split
injection with a 20:1 split and injection port temperature of 200 oC. The oven is programmed to
begin at 40 oC, hold for 5 min, and increase to 120 oC at 20 oC/min. The total run time is about 11
min, including time for the oven to cool down prior to the next injection.

[INSTRUCTOR NOTE: We used an Agilent 7890A gas chromatograph with flame ionization
detector and BAC Plus 1 column (30 m x 0.32 mm x 1.8 µm) from Restek installed. BAC columns
from other manufacturers or even a standard WAX column may also suffice. For such volatile
polar analytes, a thick, polar stationary phase coating and low oven temperature are required for
o
adequate retention and separation. The oven program finishes at 120 C so that any water vapor
transferred to the column is eluted during each run. The flow rate of hydrogen carrier gas through
the column was 1.6 mL/min.]

Data Interpretation
Record the retention times and peak areas for analyte and internal standard for each
blank, standard, and sample analyzed. Create an internal standards calibration line for ethanol.
Determine the concentration of ethanol in the original blood sample. Answer the question of
whether or not the sample tests positive for OVI in your jurisdiction.

Points to Ponder
Blanks, precision, activity of non-ionic solutes, timing of sampling and analysis

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Results and Discussion

Ethanol elutes prior to t-butanol in the chromatogram (Figure 10-1). Peak

assignments can be made because the ethanol peak size varies with ethanol
concentration, while the t-butanol peak is much more constant in size. No other peaks
should be observed, though impurities from the reagents, water, and column may
occasionally appear.

Figure 10-1. Chromatogram of ethanol (0.07 g/dL) and t-butanol (internal standard) at
40 oC on BAC Plus 1 column (30 m x 0.32 mm x 1.8 µm).

The internal standards calibration plot of peak area ethanol / peak area internal
standard versus ethanol concentration is very linear from zero to 0.30 g/dL. The y-
intercept is consistently slightly negative. The statistics of our calibration line are:
Area ratio = 8.74 ± 0.12 (g/dL ethanol) – 0.024 ± 0.013; correlation coeff. = 0.9996.

Three separate analyses of synthetic blood for ethanol gave the following results.
Accuracy is quite acceptable.

Known conc. (g/dL) 0.050 0.100 0.200

Found conc. (g/dL) 0.053 ± 0.009 0.110 ± 0.009 0.186 ± 0.008
mean ± sd

Six repeated measurements of a synthetic blood sample with an ethanol

concentration of 0.080 g/dL gave peak area ratios with a relative standard deviation of
6%. Precision is good for a method that involves manual sampling.

158 Robert Q. Thompson, Oberlin College