Gas Chromatography

International Journal of Agriculture Innovations and Research Volume 6, Issue 1, ISSN (Online)
2319-1473 M anuscript Processing Details (dd/mm/yyyy) : Received : 10/07/2017 | Accepted on : 17/07/2017 |
Gas Chromatography: Principles,

Published : 26/07/2017
Advantages and Applications in Food Analysis

Wedad Q. AL-Bukhaiti1*, Anwar Noman1, 2 Aseela Saeed Qasim3, Ammar AL-Farga4 1State key
Laboratory of Food Science and Technology & School of Food Science and Technology, Jiangnan University, 1800 Lihu,
Avenue, Wuxi 214122, P.R. China. 2Department of Agricultural Engineering, Faculty of Agriculture, University of Sanaà,
Sanaà, Yemen 3 Department of Chemistry, Faculty of Education, University of Sanaà, Sanaà, Yemen 4 School of Food and
Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China *Corresponding

author: Tel: +86 15251516277,
E-mail address: wedadqasim@yahoo.com
Abstract – Gas chromatography (GC) is a common Chromatographic separation methods are without
kind of chromatography used as a piece of analytical any doubt the most frequently employed analytical
science for segregating and investigating exacerbates that techniques for compositional analysis [1]. Gas
can be vaporized without disintegration. Regular chromatography is a unique and versatile technique. In
employments of GC are trying the immaculateness of a its initial stages of development it was applied to the
particular substance, or separating of the distinctive
analysis of gases and vapors from very volatile
segments of a blend. Development of the analytical
components. Gas chromatography is the analytical
methods for identification, purity evaluation and
technique used for product identification (under very
quantification of drugs and food has received a great deal
controlled conditions) and must be directly coupled to a
of attention in the field of separation science. This review
describes GC method development and validation in mass spectrometer when information other than a
general way. A general and very simple approach for the comparative fingerprint (program) is required, such as
GC method development for the separation of compounds positive identification of peaks on the chromatogram [2].
was discussed. Knowledge of the physiochemical The basic principal of gas chromatography is that greater
properties of the primary compound is of utmost the affinity of the compound for the stationary phase,
importance prior to the any GC method development. more the compound will be retained by the column and
Several steps are being considered for GC method longer it will be before it is eluted and detected. Thus the
development like column section (stationary phase and heart of the gas chromatograph is the column in which
dimensions: column id, length, and film thickness), carrier separation of the component takes place, and to this
gas selection (Nitrogen, Helium, flow rate), temperature must be added the source and control of the carrier gas
programing (Initial temperature, initial hold, ramp rate, flow through the column, a mean of sample introduction
final temperature, and final hold), injector selection, and a means of detection of
Injector temperature, detector selection and detectorthe components as they elute from the end of the
temperature. Optimized method is also need to be
column. Since temperature will influence the volatility of
validated with various parameters (e.g. specificity,
the analytes, the column is placed in a thermostatically
precision, accuracy, detection limit, linearity, etc.).
controlled oven [3]. Faster gas chromatographic
Keywords – Gas Chromatography, Development, separation is a generally beneficial option. Since the
Column, Detectors, Application, Validation. decrease time of analysis results in the increased
sample. Reduction of analysis time can be achieved by
I. INTRODUCTION changing column parameters: shorter length, smaller
column inner diameter, thinner film of stationary phase,
or operational parameters: faster temperature program
rate, isothermal analysis. Different carrier gas, higher the compounds are adequately thermally steady and
carrier gas flow rate or a combination of both approaches reasonably volatile [5].
can be applied [4]. Chromatography (GC) 50 years ago,
GC has been used to help determine food composition, II. PRINCIPLES OF GAS CHROMATOGRAPHY
discover our nutritional needs, improve food quality, and
introduce novel foods. Furthermore, GC has been the The basis of the separation is a retardation of the
only adequate approach to measure many of the organic ndividual components as they are moved through a long
contaminants that occur at trace concentrations in column by a carrier gas, usually helium or nitrogen. The
complex food and environmental samples. GC has been column consists of a steel or glass tube filled with an
instrumental in helping humans realize that we must use nert packing material such as glass or ceramic beads
caution with agricultural and industrial chemicals to avoid (see Figure 1). In gas-liquid chromatography (GLC),
health and environmental damage. these are coated with an in volatile liquid, so that the
Gas Chromatography (GC) is a normally utilized surface area of the liquid in contact with the gas is large.
analytic technique as a part of numerous research and For some applications, the packing may be a solid
industrial research facilities for quality control and in without any liquid coating; it is then called gas-solid
addition identification and quantitation of components in chromatography (GSC), but this is less widely used than
a mixture. GC is likewise utilized technique as a part of GLC.
numerous environmental and forensic labs since it takes The sample is injected into the carrier gas stream.
into consideration the detection of very little quantities. As it moves through the column with the carrier gas, the
An expansive variety of tests can be analysed the until
Copyright © 2017 IJAIR, All right reserved
123
International Journal of Agriculture Innovations and
Research Volume 6, Issue 1, ISSN (Online) 2319-1473
them is needed. Two types of detector are commonly
used: thermal conductivity and flame ionization.
molecules of each substance present in the sample willThermal conductivity detectors (TCDs) rely on changes
distribute between the gas and the liquid. Individualn the thermal conductivity of the gas leaving the column.
molecules will constantly move between the gas and theThe pure helium carrier gas passes over a hot
liquid in a dynamic equilibrium. While a molecule is in thetungsten-rhenium filament, causing it to cool, since
gas phase it will pass along the column, while it remainshelium has a very high thermal conductivity. When a
dissolved in the liquid it will be stationary. The morechemical substance emerges with the carrier gas, cooling
volatile a substance, the greater proportion of time itswill be less, and the temperature of the filament will rise.
molecules will be moving in the carrier gas, and so theAs with most metals, its electrical resistance increases
sooner it will emerge from the column. In this way eachwith temperature and this can be measured and
substance will become separated within the column andrecorded. Flame ionization detection (FID) is more
emerge separated by time at the end. frequently encountered in food applications, since many
The time taken from injection to emergence iscompounds under investigation are organic (containing
known as the retention time (Rt), and is characteristic forcarbon), and FID is around a thousand times more
each substance under any given set of conditions. Itsensitive than thermal conductivity detection for organics.
depends on the volatility of the substance, as well as theThe gas emerging from the column is burned with a
temperature of the column and its length and diameter.hydrogen and air mixture. This forms ions, which conduct
Many substances have an inconveniently long retentionan electric current which can be amplified and recorded
time at room temperature, and this is overcome byon a chart recorder. Although the number of ions formed
heating the column in an oven. Having separated then this way is small, perhaps only 0.0001 per cent of the
components in the column so that they emergetotal carbon atoms present in the sample, the proportion
individually, some method of detecting and measuringproduced is always constant. This means that the total
signal recorded on the chart recorder is proportional to the amount of the chemical substance present [6].
Fig. 1. Schematic Diagram of Gas

Chromatography
GC can be utilized [2]. Gas
III. APPLICATIONS chromatography (GC) has been an indispensable
analytical technique in the application of fatty acid
Gas chromatography (GC) is used widely indeterminations in oilseed plant breeding, biosynthesis
applications involving food analysis. Typical applicationsand human metabolism. As well as the characterization
pertain to the quantitative and/or qualitative analysis ofof complex mixtures of geometric isomers when
food composition, natural products, food additives, flavorcombined with other chromatographic separations and
and aroma components, a variety of transformationspectroscopic identification [13]. Plant breeders uses GC
products, and contaminants, such as pesticides,as a more precise and rapid technique for studying the
fumigants, environmental pollutants, natural toxins,variation and inheritance of fatty acids in oilseed crops
veterinary drugs, and packaging materials [7]. Andsuch as rapeseed [14]. flaxseed [15], and safflower [16].
particular food applications involving GC, such as
carbohydrates and amino acids [8]. Lipids and IV. ADVANTAGES OF GC PERFORMANCE
accompanying lipophilic compounds [9, 10]. flavors and
aroma [11, 12]. Optimum qualitative and quantitative GC analysis
GC can be used for the direct separation and of complex mixtures presupposes: (1) good resolution, as
analysis of gaseous samples, liquid solutions, andshown by sharp and symmetric peaks; (2) high
volatile solids. If the sample to be analyzed isrepeatability and reproducibility of retention times; (3)
non-volatile, the techniques of derivatization or pyrolysishigh precision

124
through volatility, polarity or concentration; (4) minimum
thermal and catalytic decomposition of sensitive sample
and accuracy in quantitation based on peak area components [17]. The use of fused-silica capillary
measurements, i.e. no discrimination of components columns with improved surface inertness, thermal
stability and resolution [18]. Best fulfils most of these improperly used column can often generate confusing,
requirements. In capillary GC the peak resolution, inadequate, and poor separations which can lead to
expressed in terms of column efficiency, separation and results that are invalid or complex to interpret. There are
retention factors [19]. Is primarily affected by the polarity over 10, 000 compounds that can be analysed by GC
of the stationary phase, column length, internal diameter and over 400 GC capillary columns [26]. The initial
and film thickness [20, 21, 22]. A variety of columns with choices of column and supporting instrumentation have a
different properties are available. In addition, fused-silica profound influence on the possibilities for, and ultimate
columns are highly applicable in practical work due to results of, separation
their flexibility and simplicity in handling and easyoptimization. Manipulation of column parameters
connection to GC and mass spectrometers [23]. In order(stationary phase, inner diameter, length, and film
to improve the sensitivity of the GC analyses it is alsothickness) gives chromatographers control over column
important to test the effect of carrier gas flow rate, as wellefficiency, resolution and speed of analysis. [2]. In gas
as gas flows in the FID, in order to reduce the noise fromchromatography, the elution order of analytes is
the hydrogen flame [24]. The carrier gas, usuallygoverned by several factors such as latent vapour
hydrogen or helium, and its purity can also affect thepressures, solubilities in stationary phase, and
resolution [20]. propensities for molecular interaction in the stationary
phase. All of these effects change with temperature and
V. DEVELOPMENT OF G C METHOD their concerted effect ultimately determine the equilibrium
distribution of solute molecules between the mobile and
Methods are developed for new products when no stationary phase [28]. Selection of Column:
official methods are available. Alternate method for A column is of course, the starting and central
existing (Non- Pharmacopoeial) products are to reduce piece of a chromatograph. An appropriately selected
the cost and time for better precision and ruggedness. column can produce a good chromatographic separation
When alternate method proposed is intended to replace which provides an accurate and reliable analysis. An
the existing procedure comparative laboratory data improperly used column can often generate confusion,
including merit/demerits are made available [25]. Severalinadequate, and poor separations which can lead to
steps are being considered for GC method development results that are invalid or complex to interpret. There are
like column selection (stationary phase and dimensions: over 10, 000 compounds that can be analysed by GC
column id, length, and film thickness), carrier gas and over 400 GC capillary columns. It is a challenge for a
selection (Nitrogen, Helium, flow rate), temperature column manufacturer to give detailed column selection
programing (Initial temperature, initial hold, ramp rate, guidelines to meet such a wide variety of applications. An
final temperature, and final hold), injector temperature optimized chromatographic separation begins with the
and detector temperature [26]. Steps involved in Method column. The selection of the proper capillary column for
development [27]: - Understanding the Physicochemical any application should be based on four significant
properties of sample. - Selection of chromatographic factors which are: stationary phase, column internal
conditions. - Developing the approach of analysis. - diameter, film thickness, and column length. The
Sample preparation. - Method optimization. - Method differences in the chemical and physical properties of
validation. injected organic compounds and their interactions with
stationary phase are the basis of separation process.
VI. SELECTION OF CHROMATOGRAPHIC When strength of the analyte-phase interactions differs
CONDITIONS significantly for two compounds, one is retained longer
than the other. How long they are retained in the column
(retention time) is a measure of these analyte-phase
1. Column Selection
interactions. Changing the chemical features of the
A column is of course, the starting and centralstationary phase alters its physical properties. Two
piece of a chromatograph. An appropriately selectedcompounds that co-elute (do not separate) on a
column can produce a good chromatographic separationparticular stationary phase might separate on another
which provides an accurate and reliable analysis. An
phase of different chemistry [26]. 1-1 Selection of capillary column that is practical should be selected. Note
Column Internal Diameter (i.d.) High Efficiency that very narrow bore columns, such as 0.10 or 0.18 mm
Column id plays two contradicting roles in i.d., may require specialized equipment, such as GC with
a pressure regulator that allows higher column head
separation. The efficiency of a capillary column,
measured in plates (n) or plates per meter (n/m), pressure [29]. 1-2 Sample Injection Port
increases as the i.d. of the column decreases. This is For optimum column potency, the sample mustn't
one of the basic principles behind fast GC but decreased be large, and may be introduced onto the column as a
sample loading capacities. When a column is overloaded "plug" of vapor - slow injection of enormous samples
with sample, the plate number is decreased greatly. If the causes band broadening and loss of resolution. The
sample to be analysed contains many analytes, or has foremost common injection methodology is wherever a
analytes that elute closely together, the most narrow i.d. micro syringe is

125
used for many analyses when using helium as a carrier
gas. When using hydrogen as the carrier gas, try an
employed to inject sample through a rubber septum into initial average linear velocity of 60 cm/sec. If better
a flash vaporizer port at the top of the column. The resolution is desired, reduce the velocity to not less than
temperature of the sample port is sometimes concerning 50 cm/sec; however, the analysis time will be increased.
50oc beyond the boiling purpose of the smallest amount If a shorter analysis time is desired, increase the velocity
volatile element of the sample [30-32]. 1-3 Column to 70-80 cm/sec; be aware of potential resolution losses
Temperature at these higher linear velocities. Average linear velocities
of 60-70 cm/sec are used for many analyses when using
For precise work, column temperature should be
hydrogen as carrier gas. [35]. The choice of gas to be
controlled to at intervals tenths of a degree. The optimum
used as mobile phase in gas chromatography is
column temperature is dependent upon the boiling
influenced by the following requirements and
purpose of the sample. As a rule of thumb [33], a
considerations:
temperature slightly on top of the typical boiling purpose
Inertness, Dryness, Freedom from oxygen, Safety,
of the sample ends up in associate extraction time of two
- half-hour. Least temperatures offer sensible resolution, Cost and Availability [3]. 3 . Optimization of Column
however increase extraction times [34]. 2 . Selection of Oven Temperature Program.
Carrier Gas: Finding the Best Carrier Gas Average The column resides in an oven, and temperature,
Linear Velocity D etermining the best average linear which greatly affects the effectiveness of the
velocity is fairly easy and only involves a small amount of chromatographic separation, is an extremely important
trial and error. Hydrogen provides the best resolution in factor used in controlling GC. In many cases, isothermal
shortest amount of time. Helium provides similar is not the most effective temperature mode for sample
resolution, but at a longer analysis time. Nitrogen is not separation; in such cases, a temperature program can be
recommended for use with capillary columns due to the used. Most GC temperature program have initial
extremely long analysis times. When using helium as the temperature, a ramp (degree increase per minute) and a
carrier gas, try an initial average linear velocity of 30 final temperature. Using a linear temperature program as
cm/sec. If better resolution is desired, reduce the velocity a starting point if previous analysis information is not
to not less than 25 cm/sec; however, the analysis time available to use as a guide, the first program
will be increased. If a shorter analysis time is desired, development step is to try a simple, linear temperature
increase the velocity to 35-40 cm/sec; be aware of program. To improve the resolution of earlier eluting
potential resolution losses at these higher linear peaks, decrease the initial temperature or increase the
velocities. Average linear velocities of 30-35 cm/sec are initial hold time. Decreasing the initial temperature
usually results in the largest resolution improvement, but on-column injections. Of these, split and splitless
analysis times are substantially increased. The resolution injections are the most commonly used techniques. Split
of peaks eluting in the middle of the chromatogram can Injector was selected for analysis of sample with high
be altered by change in ramp rate. If there is excessive concentration levels. In the split injection mode, only a
peak resolution, the ramp rate can be increased to fraction of the vaporized sample is transferred onto the
reduce resolution and the analysis time. If there is head of the column. The remainder of the vaporized
insufficient resolution, decrease the ramp rate, but there sample is removed from the injection port via the split
will be an increase in the analysis time. Better resolution vent line. Split injections should be used only when
of later eluting peaks often occurs when decreasing the sample concentrations are high enough to allow a portion
ramp rate. Another option to alter resolution of peaks in of the sample to be discarded during the injection
the middle of a chromatogram is to use a mid-ramp hold. process, while still maintaining a sufficient concentration
A mid ramp hold is a several minute isothermal portion of analytes at the detector to produce a signal [35, 36]. 5.
somewhere during a temperature ramp. Stop the Selection of Stationary Phase
temperature program shortly after last peak has eluted When selecting a column, first determine the
from the column. If column’s isothermal temperature limit
samples characteristics to match the columns stationary
is reached and peaks are still eluting, a final hold time is
phase. Stationary phases are in general divided into 3
necessary. Only use a final hold time if the temperature
categories first non-polar second mid-polar and third
limit is reached [26, 35]. 4. Optimization of Injector Polar. Stationary phases are further categorized by
Type, Temperature & Injection Volume Siloxane (non-polar and mid-polar) and Polyethylene
Introduction of the sample into GC system is a glycol (PEG or polar). G1, G2 and G38 (100% Methyl
critical step in separation. The reproducibility of the Polysiloxane) does not undergo hydrogen bonding
amount of sample injected is important to ensure the interactions. The change in the elution order of Hexanol
reproducibility of results. A sample can be injected and Phenol with G14, G15, G16, G20, G39 and G47
manually into the system or by using an auto sampler (Polyethylene glycol) is a combination of dipole and
system. A major errors in GC is poor injection technique. hydrogen bonding interaction [26]. 6. Optimization of
The injector temperature for separation. The temperature Detector Type & Detector Temperature
of injector is used to rapidly vaporize the liquid sample A variety of detector is commercially available to
into gaseous phase that can be carried to the column for
be used with GC, each having its own limitations and
separation. In capillary and micro packed gas
advantages. The most commonly used detector in GC is
chromatography (GC), there are four primary techniques
flame ionization detector. Detector temperatures and the
for vaporizing a sample and transferring it onto the inlet
relative flow rates
of the analytical column: split, splitless, direct, and
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International Journal of Agriculture Innovations and Research Volume 6, Issue 1, ISSN (Online)
2319-1473
of carrier gas, hydrogen and air into the detector are the key operating parameters [26]. A series of
standards is defined for evaluation of detector parameters such as drift, noise, sensitivity, linear range,
dynamic range etc. The variation in detector response with flow rate depends on whether the detector is
concentration or mass flow dependent. For concentration dependent detectors (e.g., thermal conductivity
detector, photo ionisation detector) a decrease in the flow-rate does not affect the peak height, which
remains approximately constant. However the peak width, and consequently the peak area, increase. In
contrast, for
mass flow detection systems (e.g., flame ionization detection, flame photometric detection, nitrogen
phosphorus detection) the response is inversely proportional to the retention time [37]. 7. Detectors
There are several detectors which may be employed in gas activity. Totally different detectors can offer
differing kinds of property. The response of a mass flow dependent detector is unaffected by make-up
gas. As shown in the table (1) [38-43].
Table: 1 Types of detectors which may be employed in gas activity Detector Type Support gases Selectivity
Detectab
ility
Dynamic range Flame ionization (FID)
Mass flow Hydrogen and
air
Most organic cpds. 100 pg 107
Thermal conductivity (TCD)
Concentration Reference Universal 1 ng 107
Electron capture (ECD)
Concentration Make-up Halides, nitrates, nitriles, peroxides,
anhydrides, organometallics
50 fg 105
Nitrogen- phosphorus Mass flow Hydrogen and
air
Nitrogen, phosphorus 10 pg 106
Flame photometric (FPD)
Mass flow Hydrogen and air possibly oxygen
Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium
100 pg 103
Photo- ionization (PID)
Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics
2 pg 107
The effluent from the column is mixed with gas and air, and lit. Organic compounds burning within the
flame manufacture ions and electrons which might conduct electricity through the flame [44]. An outsized
electrical potential is applied at the burner tip, and a collector conductor is found higher than the flame
[45]. The present ensuing from the transformation of any organic compounds is measured [46].
VII. CONCLUSION
Gas chromatography is an important analytical technique for qualitative and quantitative analysis in a wide
range of application areas. It is fast, provides a high peak capacity, is sensitive and allows combination
with a wide range of selective detection methods including mass spectrometry. However, the application
area of GC is limited because the molecules to be analysed have to be thermally stable and sufficiently
volatile. Numerous molecules do not meet these requirements and hence are not amenable to direct GC
analysis. Recent research has resulted in better chromatographic columns and methods for sample
preparation that enable a significant expansion of the molecular application range of GC. The strategies
exploited include conversion of (macro) molecules into smaller species and approaches to reduce the
polarity of molecules.
Abbreviations m m millimeter fg femtogram ng nanogram pg pictogram
Concentration Make-up
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International Journal of Agriculture Innovations and Research Volume 6, Issue 1, ISSN (Online)

2319-1473 M anuscript Processing Details (dd/mm/yyyy) : Received : 10/07/2017 | Accepted on : 17/07/2017 |