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11 January 19
App note-FAMEs analysis method revalidation from helium to hydrogen for GC
FAMEs, analysis, method, revalidation, from, helium, to, hydrogen, for, GC, Article, Ed, Connor
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FAMEs analysis method
revalidation from helium
to hydrogen for GC
from helium to
hydrogen for GC
Ed Connor DR. SC.
GC Product Specialist
Changing carrier gas from helium to
hydrogen does not always present an
opportunity for faster sample analysis.
Method revalidation can be simplified by
keeping the new method as close to the old
method as possible, which will limit changes
to sample selectivity and resolution whilst
maintaining the retention times of analytes.
Fatty acid methyl esters (FAMEs) analysis is
an important method used across a range of
product analyses to characterise fat content.
Analysis of FAMEs is conducted in a wide
range of products of biological origin including
foodstuffs, biodiesels and microbes.
quantification of FAMEs is usually conducted
using gas chromatography with flame ionisation
detection (GC-FID) using a highly polar column.
Many labs use helium carrier gas for their GC
analysis of FAMEs, but as prices continue to
rise, changing carrier supply to hydrogen
supplied by a gas generator is becoming more
attractive to many analysts. When people
consider using hydrogen, faster analysis is
one of the features often cited as a reason for
switching carrier gas from helium. However, if
peaks are poorly resolved, increasing carrier
gas flow rate can have the effect of causing
peak co-elution. One option that can reduce
running costs, in terms of switching to a lowercost carrier gas, and that avoids the need for
major method revalidation is to keep as many
method parameters the same as possible.
Here we look at FAMEs analysis of a
38-component standard mixture (Supelco
F.A.M.E. mixture Catalogue # 18919-1AMP) with
transpalmitelaidic acid methyl ester (purchased
from Matreya, Catalogue # 1148)). Figure 1
shows a section of the total chromatogram
including the poorly resolved peaks 29-33 in
the original method run using helium carrier
gas. This method used a carrier flow rate of 34
cms-1 with a 20 mLmin-1 split flow (Table 1). The
results of this analysis suggest that there is little
opportunity for increasing sample throughput.
However, to see whether a translated method
using hydrogen carrier gas would give similar
results, the method parameters were kept the
same, with only the carrier gas linear velocity
altered as a result of changing the gas from
helium to hydrogen (Table 1). Figure 2 shows
the comparative results of using helium (2A)
and hydrogen (2B) carrier gas. The increase
in linear velocity resulted in a shorter analysis
time, but co-elution of peaks 29-33 meant
that method revalidation was required.
The method was revalidated to match the
linear velocity of hydrogen with that of the
original helium method. This required reduction
of the GC head pressure to reduce the flow
rate of the carrier gas. This had the effect of
reducing peak size because of the reduction of
carrier flow rate combined with the relatively
high split flow. Peak height was improved by
reducing the split flow from 20 mLmin-1 to
5.6 mLmin-1 which increased the amount of
analyte transferred onto the column (Table 1).
After revalidating the method to match the
linear velocity of hydrogen to the linear velocity
of helium in the original method, results
were almost identical to those obtained with
helium (Figure 3). This meant no reduction
in analysis time, but like-for-like results
with an increase in peak height of samples
run using hydrogen carrier gas because of
improved efficiency associated with its use.
Although hydrogen is often cited as a fast
carrier gas, it is not always possible to
speed up analysis because of lack of peak
resolution. Here we have shown that it is
possible to revalidate your method by keeping
the linear velocity of hydrogen the same as
that of helium. This approach will ensure
that compounds elute at the same retention
time despite a change in the carrier gas. By
adopting this approach, you can a column
with the same dimensions and will only need
to make slight adjustments to the GC method.
Ed Connor DR.SC. is a GC Product Specialist,
Peak Scientific, Inchinnan Business Park,
Scotland, UK . Prior to joining Peak in February
of this year, Ed completed his Dr.Sc. at ETH
Zurich in Switzerland using GC-MS to look
at herbivore induced plant volatiles and their
interaction with beneficial insects. He then
joined the University of Zurich where his work
focused primarily on volatile collection methods
and analyses using GC-MS and GC-FID.
Figure 1: Section of the total chromatogram of the 38 component
FAME standard showing poorly resolved peaks 29-33.
Average Linear Velocity
Table 1. HS-GC-FID Operating Conditions
Figure 2: 2A showing a section of the chromatogram produced using
the original helium method including poorly resolved peaks 29-33 Figure 2B showing co-elution of peaks 29-30 and 31-33 when running the
sample using hydrogen carrier gas at a higher linear velocity.
Figure 3: Results of the original method using helium carrier gas (3A)
and results of the revalidated GC method using hydrogen carrier gas
40 °C (2 min hold), 4 °C/min to 250 °C (3 min hold)
FID at 260 °C
Supelco 2380 (30m x 0.25mm x 0.2mm film)
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