UNIT G1.3
Identification and Quantitation of Aroma
Compounds
After isolation of the volatiles using different extraction and concentration procedures
(
UNIT G1.2
) the samples are injected into a gas chromatograph (GC) for separation of
individual compounds. Identification is based on their Kovats retention indices on two
columns of different polarity, as well as comparison with mass spectroscopy (MS) spectra
of the corresponding reference compounds. Quantification is then performed using one
or several compounds as internal standards, which naturally do not occur in the sample,
added to the sample before extraction, if possible. Depending on the type and availability
of these standards, quantitation is preferably performed by gas chromatography-flame
ionization detection (GC-FID; see Basic Protocol) or gas chromatography-mass spec-
trometry (GC-MS) using isotope dilution assays (IDAs; see Alternate Protocol). Other
methods for quantification based on external standards are less accurate, and those based
on multiple standard addition at different concentrations are only rarely needed.
BASIC
PROTOCOL
IDENTIFICATION (GC-FID; GC-MS) AND QUANTIFICATION OF
VOLATILES (GC-FID)
Volatiles are separated by GC and their elution time monitored relative to a series of
n
-alkanes that are injected under identical conditions. The most common GC detector is
the flame ionization detector (FID), which is also widely used in flavor research. The
detector responds to all organic compounds that burn or ionize in its flame, and is
characterized by a large dynamic range (Buffington and Wilson, 1987). The temperature
program chosen for the separation of compounds is dominated by mutually exclusive
parameters like speed, resolution, and capacity. For separation of highly volatile odorants,
sub-ambient GC-start temperatures are recommended. The steps described in this unit for
the identification are limited to compounds that are listed in common data banks.
Analytical methods to identify a compound that has never been reported before are not
discussed, and the reader is referred to experts in natural products chemistry. Unambigu-
ous identification requires that an authentic standard has been shown to have the same
chromatographic, spectral, and olfactory properties. Anything less is tentative; however,
like other problems in analytical biochemistry the biological activity (i.e., olfaction) is a
powerful indication of the identity of a standard and an analyte especially when combined
with chromatography.
Materials
Aroma samples
Solution of 10 to 100
µg/ml n-alkanes (C5 to C25) in diethylether (freshly
distilled)
Reference compound
Internal standard compound
Gas chromatograph with FID and sniffing port (Hewlett Packard)
DB5 and DBWAX capillary columns (e.g., 30-m
× 0.25-mm; f = 0.25 µm; J&W
Scientific)
Gas chromatograph with MS detector (with EI mode and optional CI mode;
Hewlett Packard)
Additional reagents and equipment for extraction methods (see
UNIT G1.2
)
Contributed by Christian Milo
Current Protocols in Food Analytical Chemistry (2001) G1.3.1-G1.3.12
Copyright © 2001 by John Wiley & Sons, Inc.
G1.3.1
Smell Chemicals