Blood Alcohol Analysis Utilizing Headspace Autosampling and Dual-column GC Confirmation (GC)

FIELD
APPLICATION
REPORT
Gas Chromatography
Blood Alcohol Analysis
Utilizing Headspace
Autosampling and
Dual-column GC
Confirmation
Introduction
Testing for the presence and subsequent
quantitation of ethanol in blood, breath, and
urine are the highest volume tests performed in
forensic laboratories. In addition to ethanol, the
detection of several other significant alcohols and
their metabolites is necessary. Gas chromatographic
(GC) assays provide the greatest amount of flexibility and specificity in analyzing for these
volatile compounds. Direct injection of biological samples into GC columns has been
used in the past as the method of sample introduction. This typically leads to column
contamination and decreased performance. The incorporation of headspace sampling into
the method prevents the buildup of non-volatile contamination at the head of the column
and helps to maintain consistent performance and extend column lifetime.
Analysis time and resolution are two critical factors when
developing a GC assay for ethanol. Analysis time for each
sample should be as short as possible, while still maintaining
baseline resolution for all analytes. Isothermal operation is the
preferred method of analysis because it eliminates the cooldown period between temperature programmed runs. Overall
analysis time can be reduced in isothermal analysis by raising
the temperature of the analysis or by increasing carrier gas flow
rate. However, in attempting to shorten the analysis time either
by increasing the flow rate or by raising the temperature, many
traditional capillary column stationary phases fail to provide
adequate resolution of all of the components commonly tested
during blood alcohol analysis. PerkinElmer supplies the Elite
BAC-1 and BAC-2, two novel capillary column stationary phases
to meet all of these requirements.
Experimental
The goal of the method developed here was to use headspace
sampling and dual column GC analysis to provide blood alcohol
confirmation in less than three minutes.
The instrumentation requirements for this type of analysis can
be satisfied by selecting from a wide variety of PerkinElmer
products. Any current TurboMatrix™ headspace autosampler (as
well as previous models such as the HS40 or HS110) can be used
for sample introduction. Similarly, the GC requirements can be
fulfilled using the Clarus™ 500 as well as previous models such
as the AutoSystem XL or AutoSystem (as long as the system
incorporates dual FID detectors). Data acquisition and data
processing requirements can easily be addressed using SoftLINK™
and TotalChrom™ software (or Series 600 link box
and TurboChrom® software).
Instrument Parameters
Dual column analysis using a two-hole ferrule. 1.0 mL
headspace sample of a blood alcohol mix.
Oven temperature:
40 °C isothermal
Inj. temperature:
200 °C
Carrier gas:He
Sample equilibration
temperature:
70 °C
Sample equilibration
time:
15 min.
Vial pressure:
30 psi
Vial pressurization time: 0.15 min.
Vial sampling time:
0.01 min.
Transfer line:
0.32 mm ID FS
Hydroguard™ tubing
Transfer line temperature: 200 °C
Injection port sleeve:
2 mm ID
Split flow:
20 mL-per-minute
2
Dual column analysis using a two-hole
ferrule. 1.0 mL headspace sample of
a blood alcohol mix.
Oven temperature: 40 °C isothermal
Inj. temperature: 200 °C
Carrier gas: He
Sample equilibration temperature: 70 °C
Sample equilibration time: 15 min.
Vial pressure: 30 psi
Vial pressurization time: 0.15 min.
Vial sampling time: 0.01 min.
Transfer line: 0.32 mm ID FS
Hydroguard™ tubing
Transfer line temperature: 200 °C
Injection port sleeve: 2 mm ID
Split flow: 20 mL-per-minute
30 m, 0.32 mm ID, 1.8 µm
Elite BAC-1
(part # N9316579)
30 m, 0.32 mm ID, 1.2 µm
Elite BAC-2
(part # N9316577)
1. meth­a­nol
2.acetaldehyde
3.ethanol
4.isopropanol
5.acetone
6.n-propanol
Conc.
w/v
0.1%
0.2%
0.2%
0.1%
0.01%
0.1%
Figure 1. Achieve baseline resolution of all blood alcohol components in less than
three minutes using the PerkinElmer headspace autosampler, gas chromatograph
and Elite BAC-1 and Elite BAC-2 columns.
The TurboMatrix headspace products use a balanced pressure
sampling procedure to transport the sample to the GC. This type
of sampling works better with columns that require higher head
pressure, so 0.32 mm ID analytical columns were chosen for this
application because of their higher operating pressure. Optimal
performance of these columns during headspace analysis
depends on GC/headspace system set up. Band broadening can
occur if there is excess dead volume in the sample flow path
between the sample valve and the head of the column. Low
volume inlet liners or interfaces in the injection port should be
used to reduce the amount of excess volume at the exit end
of the transfer line. A 2 mm ID zero dilution liner was used in
this analysis to reduce dead volume and maintain narrow peak
widths. High carrier gas flow rates through the transfer line
can also be used to maintain narrow sample bandwidths and
speed up sample transfer to the head of the column. A flow
of 40 mL-per-minute was used to optimize the analysis on the
headspace autosampler.
Simulated blood alcohol samples were prepared and analyzed
using a modification of a procedure published by Christmore
et al.1 n-Propanol was used as the internal standard and was
prepared at a concentration of 0.03 g/dL in 1.0 M ammonium
sulfate as a diluent. Five milliliters of diluent was added to 1 mL
of sample in a 20 mL headspace vial.
Product Listing
Elite BAC-1 GC Columns
Length (m)
Elite BAC-2 GC Columns
ID (mm)
df (µm)
Part No.
Length (m)
ID (mm)
df (µm)
Part No.
30
0.53 3.0N9316578
30
0.53 2.0N9316576
30
0.32 1.8N9316579
30
0.32 1.2N9316577
Deactivated Fused Silica Transfer Lines
Length (m)
ID (mm)
OD (mm)
Splitless Liners
Part No.
Description
Part No.
5
0.18 0.34N9301354
Quartz Liner (2 mm)
N6121002
5
0.25 0.37N9301356
Glass Liner (2 mm)
N6101372
5
0.32 0.45N9301357
Deactivated Liner (2 mm, packed with wool)
N6121021
5
0.53 0.69N9301358
Headspace Autosampler Vials & Seals
Dual-Column Analysis Replacement Ferrules – Two-hole
Ferrule
i.d. (mm)
Nut Size
(in)
Column Graphite Graphite/
i.d. (mm)
Vespel®
0.4
1/16
0.18-0.25 N/A
04972392
(1-pk)
Description
Part No.
22 mL clear vial, rounded bottom (100-pk)
B0104236
Headspace vial cap w/PTFE/Silicone septa (1000-pk)
B0104242
0.5 1/16 0.32
N9306001 N9306000
(10-pk)(10-pk)
0.5 1/8 0.32
09903395 (10-pk)
N/A
Results
Conclusion
We demonstrated here that a PerkinElmer headspace
autosampler coupled with dual-column GC confirmational
analysis provided excellent accuracy and precision in the
analysis of blood alcohol. Calibration curves were constructed
using concentrations ranging from 0.01% to 0.5% ethanol.
Correlation coefficients above 0.999 were easily obtained for
all compounds. Response factor repeatability was less than
+1% standard deviation while analyzing six samples at a
concentration of 0.2% ethanol. Based on our experimentation,
a system detection limit of approximately 0.001% ethanol
should be achievable while maintaining a minimum signal-tonoise ratio of 10.
Successful blood alcohol analysis can be performed in less
than three minutes per sample using a PerkinElmer headspace
autosampler and gas chromatograph equipped with Elite BAC-1
and BAC-2 GC columns and dual FID detectors.
References
1. D. S. Christmore, R. C. Kelly, and L. A. Doshier, Journal
of Forensic Science, Volume 29, No. 4, October 1984,
pp.1038-1044.
The data presented in this Field Application Report are not guaranteed. Actual performance and results are dependent upon the exact methodology used and laboratory conditions.
This data should only be used to demonstrate the applicability of an instrument for a particular analysis and is not intended to serve as a guarantee of performance.
Reprinted with permission of Restek Corporation.
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