APPLICATIONS OF GAS CHROMATOGRAPHY

IN ANALYSIS OF FLAVOURS

1. INTRODUCTION TO GAS CHROMATOGRAPHY

Gas chromatography is a method of separating the components of mixtures of
gases and vapors; it is widely applied in analytical chemistry and is finding an
increasingly useful place in physiological laboratories. The method is more
versatile, more accurate, simpler to operate and less dependent upon technical skill
than many of the old-established ways of identifying and measuring gases
contained in biological fluids. Thus, a chromatographic system suitably designed
will identify and measure oxygen, carbon dioxide and nitrous oxide in less than
fifteen minutes and just as readily, gas chromatography can be employed to
measure levels of volatile anesthetics and alcohol in blood and tissues.

The simplest concept of a chromatograph is to consider it as a molecular filter

which selectively retards the passage of molecules according to certain physical
characteristics such as shape, size, weight and boiling point. A mixture of gases, in
the course of its passage through the chromatograph, will divide into its
components and emerge from the distal end of the column in regular order, to be
measured by a detector whose output may be recorded graphically or
electronically.

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 2. A SUCCESSFUL TECHNIQUE

Flavors and fragrances are mixtures of volatiles representative of matrices mainly

in the food or cosmetic fields that can be sampled with different but specific
approaches and/or techniques such as headspace, distillation or extraction. Flavors
and fragrances are in general complex but homogeneous mixtures of volatiles
requiring high efficiency techniques for their analysis such as gas chromatography
(GC) as such or combined with mass spectrometry (GC-MS). Flavor and fragrance
analysis has radically changed over the last 15–20 years, by the introduction of
new strategies for sample preparation, analysis and data elaboration, and to the

development of automatic systems. GC is an appropriate technique for the

separation and characterization of volatiles in different food matrices.

3. ANALYTICAL METHODS

The primary technique used in flavor analysis is gas chromatography, with and
without mass spectrometry and olfactometry. The GC is one of popular instrument
used in the world. Majority of analyses of flavor in foods are done by GC but
liquid chromatography also plays a significant role in flavor analysis, particularly
for sugars, capsaicin, protein fragments (umami), and other compounds that are not

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amenable to GC because they are nonvolatile, thermally labile, or too polar to be
separated by GC.

3.1: MASS SPECTROMETRY

Passing the effluent from the GC into a mass spectrometer allows identification
and quantification of the chemical components detected by the GC. The three
most-used types of MS are quadrupole, ion-trap, and TOF. The first two typically

provide 10 spectra/sec. The fastest GC-MS on the market, provides 500

spectra/sec.

3.2: OLFACTOMETRY

One of the challenges ahead is trace composition analysis, particularly to identify

compounds that are present at very low concentrations but are very odor active,
providing subtle nuances of odor or off-odor. They may be present at levels that
can’t be detected instrumentally but can be detected by the nose. In olfactometry,
regions of the gas flow from the gas chromatograph can be split, sent to a mass
spectrometer and to a sniff port, where the effluent can be sniffed to identify the
particular odor.

3.3: DECONVOLUTION

One of the problems with GC is that although we get very good sensitivity and
lower detection limits, we might also get coelution—overlapping—of peaks.
Separation of the peaks can be accomplished through the use of deconvolution
software consisting of algorithms that provide precise, rapid compound
identification and quantification. The algorithms identify target compounds present
even in complex mixtures.

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 4. EXTRACTION METHODS

Some compounds such as essential oils (orange, lemon, etc.) can be directly
injected into the GC and analyzed, but once the compounds are formulated into a
product, they need to be extracted before injection. There are two primary methods
of extraction: solvent based and solventless.

1. Solvent-based extraction is widely used, but has a major shortcoming in that

the solvent is also being injected into the GC and might obscure the peaks of
interest.
2. Among the most widely used solventless techniques, are solid phase
microextraction (SPME); solid phase dynamic extraction (SPDE); and stir
bar sorptive extraction (SBSE). SPME uses an absorbent coating on fiber
within a syringe that is exposed in the headspace over a sample in a vial.
After equilibration, the compounds concentrated on the SPME fiber are
thermally desorbed into a GC. SPDE is similar to SPME, except that the
coating is on the inside of a syringe needle and the syringe must be pumped
repeatedly to draw sample across the phase.
5. APPLICATIONS

Gas chromatography (GC) is used widely in applications involving food analysis.

Typical applications pertain to the quantitative and/or qualitative analysis of food
composition, natural products, food additives, flavor and aroma component. GC is
rarely used in bulk compositional assays, it is the primary tool for analysis of fatty
acids, sterols, alcohols, oils, aroma profiles, and off-flavors, and in other food-
composition applications. GC is also the method of choice for analysis of any
volatile component in food.

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Flavor compounds in foods may, however, be at concentrations too low to be
accurately detected by GC; concentration of volatiles may, therefore, be required
prior to GC operation.

5.1: TWO-DIMENSIONAL GAS CHROMATOGRAPHY

Comprehensive two-dimensional gas chromatography (GC × GC) is a technique

utilizing two columns of different selectivity connected in series by the modulation
device. The modulator cuts slice from the first-dimension column effluent and re-
injects them to the secondary column. Due to the difference in column polarity,
each simple compound is subjected to two independent separation mechanisms.
Compared to one-dimensional GC, this technique brings dramatically increased
peak capacity, improved peak resolution, and up to an order of magnitude increase
in compounds’ detectability. In contrary to heart-cutting variety, in GC × GC all
effluent from the primary column passes through the secondary column,
maximizing sample resolution throughout the entire analysis.

In GC × GC, two basic orthogonality rules should be kept:

 Independence of separation mechanisms, i.e., the two columns should

possess of different selectivity.
 Preserving of the first-dimension separation.

5.2: SIMULTANEOUS DISTILLATION EXTRACTION (SDE)

Simultaneous distillation extraction (SDE) is one of the most important

extraction methods and more popular among all the known methods for rice
aroma chemical analysis. It is a solvent extraction technique. SDE allows
simultaneous extraction, concentration and isolation of flavor constituents within a
short time due to continuously recycling the two immiscible solvents. Only a small

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amount of solvent is used for extraction so the volatile compounds can be easily
concentrated more than tenfold.

5.3: SOLVENT-ASSISTED FLAVOR EVAPORATION (SAFE)

Solvent-assisted flavor evaporation (SAFE) is considered to be the best overall

method to produce a “clean” aroma extract to avoid the loss of labile aroma
compounds or the formation of thermally generated artifacts during gas
chromatographic (GC) analysis. However, SAFE is both time consuming and labor
intensive, especially when applied repeatedly for quantitation by stable isotope
dilution analysis. In the SAFE procedure, odor-active components are distilled
under high vacuum, thus, the extraction bias during SAFE is based on volatility
rather than polarity, like in the case of solvent extraction. SAFE seemed to be the
most useful technique, due to the low extraction temperature and ability to extract
the most compounds.

5.4: HEAD-SPACE SOLID PHASE MICROEXTRACTION

(HS-SPME)

Different methods such as purge and trap, static headspace, liquid-liquid, solid
phase extraction, and solid phase microextraction are used for extraction and
concentration of volatile compounds. Among various separation and concentration
techniques, head space solid phase microextraction (HS-SPME) using a fused-
silica fibre combined with gas chromatography-mass spectrometry (GC-MS) has
gained increasing attention for the extraction and analysis of volatile, semi-volatile,
polar and non-polar compounds in foods such as vegetables, legumes, beverages
and dairy products. In comparison with conventional extraction techniques, HS-
SPME is a solvent-free, less expensive, fast, and simple technique and involves the
adsorption of volatile compounds onto an adsorbent fibre. In fibre-SPME, adsorption

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is based on the equilibrium partitioning of the analytes between the solid-phase of the
SPME fibre, liquid or solid sample matrix. Upon heating, adsorbed analytes are
desorbed onto a GC column and analyzed by gas chromatography. SPME is a
partition-based extraction technique in which the profile of extracted compounds is
highly dependent on fiber coating used.

5.5: GAS CHROMATOGRAPHY-OLFACTOMETRY-MASS

SPECTROMETRY (GC-O-MS)

Gas chromatography-olfactometry-mass spectrometry (GC-O-MS) is a

combination of gas chromatography-olfactometry (GC-O) and gas
chromatography–mass spectrometry (GC–MS). GC-O-MS technique is a
powerful tool to study food flavors and it has been widely applied for aroma
and flavor analysis of various food items. Gas chromatography-olfactometry
(GC-O) has been used almost since the introduction of gas chromatography, as the
human nose is the most appropriate detector to monitor the presence of an odorant
in the effluent of a gas chromatograph. It is worth mentioning that the human nose
is able to detect the odoriferous compound at the amount of 10−17 g, while
detectors commonly used in gas chromatography required at least 10−13 g.

In combination with different technologies, GC-O-MS can be applied to solve

many flavor problems in the food industry such as quick mapping of aroma-
active compounds, identification of key aroma-active compounds, cluster
analysis based on the aroma-active compounds, relationship between
odorants and sensory properties, and clarification of formation mechanism of
important odorants.

5.6: GC-IMS (ION MOBILITY SPECTROMETRY) However, the

technique is time-intensive, involving aroma extract dilution analysis (AEDA), to
identify aromatic constituents present in food. Consequently, it cannot be used to

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characterize the volatile constituents of food rapidly. GC-IMS (Ion Mobility
Spectrometry) represents a new gas-phase separation detection methodology which
harnesses the high separation ability of gas chromatography, together with the fast
response of IMS. Gas chromatography–ion mobility spectrometry (GC–IMS) is a
powerful technique for the separation and sensitive detection of volatile organic
compounds. It has a fast response, high sensitivity, easy operation, and low cost.

6. DETECTING AROMAS

The key aroma components of food can also be determined by GC-IMS. The levels
of polyamines and monoamines can be Quantified using the technique – these
serve as an indicator of food freshness as these products are formed by the
degradation of amino acids.

With regards to off flavour, GC IMS can be used cat to quantify product of lipid
oxidation which produce undesirable flavours in food (and undetectable by human
smell). In the context of fermentation, the technique can quantify characteristic
flavours to enable fermentation to be terminated at the correct time point; this is
especially useful in the production of beer, as well as cheese ripening.

Volatile metabolites such as those produced by lactic acid bacteria can be

identified by GC-IMS. As a result, the technique can be used to select strains of
bacteria the influence cheese flavour.

7. MECHANISM

A gas chromatograph is made up of a narrow flow-through tube, known as the

column, through which the sample passes in a gas stream (the carrier gas) at
different rates depending on their various chemical and physical properties and

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their interaction with a specific column lining or filling, called the 'stationary
phase'. As the chemicals exit the end of the column, they are detected and
identified electronically. The function of the stationary phase in the column is to
separate different components, causing each one to exit the column at a different
time. Other parameters that can be used to alter the order or time of retention are
the carrier gas flow rate, column length and the temperature.

In GC, the mobile phase or carrier phase is an inert gas such as helium and the
stationary phase is a very thin layer of liquid or polymer on an inert solid support
inside a column. The volatile analytes interact with the walls of the column, and
are eluted based on the temperature of the column at specific retention time. The
eluted compounds are identified with detectors. Flame ionization and mass
spectrometry are the most commonly used detectors for flavor analysis.

8. ANALYSIS

In a GC analysis, a known volume of gaseous or liquid analyte is injected through

a rubber disk and into a hot, temperature controlled, port attached to the
column. As the carrier gas transports the analyte molecules through the column,
there is adsorption of the analyte molecules either onto the column walls or onto
packing materials (stationary phase) in the column to give separation. Since each
type of molecule has a different rate of progression, the various components of the
analyte mixture are separated as they progress along the column and reach the end
of the column at different times (retention time). A detector is used to monitor the
time at which each component reaches the outlet and ultimately the amount of that
component can be determined. Generally, substances are identified (qualitatively)
by the order in which they elute from the column and by the retention time of the
analyte in the column.

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As a flavor or food sample passes through a heated capillary column, the chemical
components are evaporated and separated, producing peaks according to the time it
takes to reach a detector, such as a flame ionization detector, the most common
type. There are two basic types: comprehensive GCxGC and heart-cutting GC.

3. In comprehensive GCxGC, he said, the effluent from one GC column is

injected into a second column, increasing resolving power and resulting in a
very complicated analysis, suitable for detailed sample characterization and
component class determinations. This technique typically is best done with a
fast time-of-flight (TOF) mass spectrometer.
4. In heart-cutting GC, a small region of the gas flow from one column
containing specific peaks of interest is diverted by a pressure controller
called a dean’s switch to a second column and analyzed. Heart cutting GC
can be coupled to olfactometry and is gaining in popularity.
9. CONCLUSION

Gas chromatograph, as a common instrument in most analytical laboratories

worldwide, can be successfully applied in authentication and fraud detection
procedures of various food and beverage products. It can be concluded that
utilization of a GC device in further development of authentication methodologies
could provide us with meaningful results, thus representing a significant
contribution to this emerging field in the future.

REFERENCES

1. Deibler, K. D., Acree, T. E., & Lavin, E. H. (1999). Solid phase microextraction application in gas
chromatography/olfactometry dilution analysis. Journal of Agricultural and Food Chemistry, Vol. 47, No.
4, pp. 1616–1618, ISSN 0021-8561.
2. Song H, Liu J. GC-O-MS technique and its applications in food flavor analysis. Food Res Int. 2018
Dec;114:187-198. doi: 10.1016/j.foodres.2018.07.037. Epub 2018 Aug 3. PMID: 30361015.
3. Anandram Venkatasubramanian, Vincent T. K. Sauer, Swapan K. Roy, Mike Xia, David S. Wishart,
and Wayne K. Hiebert . Nano-Optomechanical Systems for Gas Chromatography. Nano
Letters 2016, 16 (11) , 6975-6981. https://doi.org/10.1021/acs.nanolett.6b03066

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