20 Gas Chromatography (GSC and GLC), Technique and Sample preparations
Dr. Varinder Kaur
Objectives: To study the basics of gas chromatography and know the following about the following questions
- What is gas chromatography?
- What are the main components of GC?
- Which type of samples are analyzed by GC?
- How is it different from HPLC?
- What are the main applications of GC?
- How is it used in daily life?
1. Description
Gas chromatography (GC) is extensively used in various branches of science and technology. It has played a fundamental role in determining the type and amount of components in a mixture. In this technique, components of a vapourized sample are separated on the basis of their partitioning between mobile phase and stationary phase. In this case, a gas is used as mobile phase, therefore, it is termed as gas chromatography.
Principle
Gas chromatography is based on the principle of partition of a volatile compound between a liquid/solid stationary phase and a gaseous mobile phase in a fixed set of parameters. In gas chromatography, sample is a gas or in vapour phase, eluent is a gas and stationary phase is non-volatile liquid coated of a solid surface or a solid. The analyte in vapor phase distributes between the stationary and mobile phase establishing equilibrium between the two phases.
2. Types of Gas chromatography
On the basis of stationary phase used, gas chromatography is categorized into two types.
1. Gas solid chromatography (GSC)
2. Gas liquid chromatography (GLC)
In gas solid chromatography, stationary phase is solid and equilibrium is established between for the distribution of a component between solid and gas. In this technique, solid stationary phase physically adsorbs analytes leading to retention of the analyte on the column. Thus, applications of GSC are limited because problems like long retention of active or polar molecules and tailing of elution peaks are encountered in this case.
In gas liquid chromatography, a thin layer of a liquid supported over a solid phase plays a role of stationary phase. The components distribute between the gaseous mobile phase and the liquid stationary phase and establish equilibrium. This equilibrium is very rapid and therefore, GLC has been adopted as the most useful method. It is generally termed as Gas Chromatography (GC). GC or GLC is advantageous over GLC as availability of wide range of liquid coatings afford diverse separations and evaluation of wide range of concentrations. It shows good resolution of peaks in a shorter analysis time.
3. Instrumentation of GC
The main components of GC are carrier gas, sample injection port, column, thermostatic oven and detector. A block diagram of GC is given below and the components are discussed in detail.
3.1 Carrier gasThe mobile phase used in gas chromatography (i.e. gas) is called as carrier gas. It delivers the sample to the column and then, to the detector. The carrier gas must be chemically inert. Therefore, the gases like argon, helium, nitrogen and carbon dioxide are the most commonly used carrier gases. In some of the case, hydrogen is also used as carrier gas, however, its use is not preferred because of fire hazards. The gases are available in pressurized tanks, therefore, their release is controlled by pressure regulators. The constant flow of the carrier gas is maintained with the help of a flow meter (inlet flow is generally 10-50 psi).
3.2 Sample injection system
This component is used to apply a sample into the system via injection port. The sample is injected by a microsyringe directly into a flash vapourizer port by piercing a rubber septum. The temperature of the sample port is maintained usually at 50°C (or higher than the boiling point of the least volatile component of the sample). The volume of the sample varies according to the type of column i.e. 0.1 µl to 20 µl in packed column and 10-3 mL in capillary column.
3.3 ColumnColumn is used for the separation of different components of a mixture. It is filled with the stationary phase, so equilibration between stationary phase and mobile phase takes place in the column. The column used in GC should be rigid and able to withstand moderate pressures (up to 50 psi [~145 kpa]). It should be stable at high temperature and should be chemically un-reactive. These are generally made up of stainless steel, flass, fused silica or Teflon. The columns used in GC vary in length from 2-50 m or more and are coiled to fit in a thermostat. In general, two types of columns are used in GC.
1. Packed column
2. Open tubular or capillary column
Packed columns are densely packed with a solid material with a coating of liquid stationary phase. The stationary phase is coated in the form of a thin liquid film on the surface of a finely divided, inert material. The mobile phase comes in contact with the liquid stationary phase and the components undergo equilibrium.
Open tubular columns (capillary columns) are hollow in the centre and possess a narrow opening running down the centre (a capillary) through which the mobile phase travels. The capillary columns are more efficient and faster as compared to packed columns. These are further of two types; wall coated open tubular column (WCOT) and support coated open tubular column (SCOT).
Wall coated open tubular columns are capillary tubes coated with thin layer of stationary phase whereas in support coated open tubular columns, inner surface of capillary is supported with a diatomaceous support, which is coated with liquid stationary phase.
3.4 Column thermostat oven
The column thermostat oven is used to control the column’s temperature. In case if isothermal separation, column is maintained at a constant temperature. The temperature chosen for the separation should be below the boiling point of the lowest boiling solute. In case of temperature programming, the temperature of the column is increased slowly either uniformly or in a series of steps.
3.5 Detection system
The separation of components is detected with the help of a detector. The mobile phase coming out from the column is directly delivered to the detector, where the components are detected on the basis of their physical properties. In the past, numerous detectors have been used with gas chromatography. However, the best detector in GC should have the following characteristics.
3.5.2. Thermal conductivity detector
This detector works on the basis of the fluctuations in the thermal conductivity of carrier gas due to the presence of components in the mobile phase. The presence of small amounts of organic compounds in the carrier gas coming out from the column decreases the conductivity of the gas and thus increases the temperature of the detector. The thermal conductivity device consists of an element composed of fine Pt, Au or W wire, which was heated by supplying a constant electric power. Its temperature varies with the thermal conductivity of the carrier gas. These devices are fixed at two different positions; before the sample injection port and after the column to measure the thermal conductivity of carrier gas before injecting sample and the carrier gas coming out of the column. This arrangement cancels the thermal conductivity of carrier gas itself giving the response due to the components in the sample. Generally, He and H2 gases are used as carrier gases in this detection system because their thermal conductivity is high as compared to organic compounds. This detector is simple to use, responds to both organic and inorganic analytes, non-destructive and work in wide range. However, it suffers due to low sensitivity of the detector.
3.5.3. Electron capture detector
This detector measures the variation in current produced by the ionization of the carrier gas. It utilizes a -emitter (generally Ni-63), which gives rise to the ionization of carrier gas. This produces electrons, which generate a constant flow of current between the electrodes. However, the presence of sample components having highly electronegative functional moieties in the carrier gas coming out form the column interact with the ions and tend to capture the electrons. This reduces the flow of current generating a signal. Generally N2 is used as carrier gas in electron capturing detection. This detector is used for the analysis of samples containing halogens, peroxides, quinones, nitro compounds, pesticides and polychlorinated hydrocarbons. However, it cannot be used with the samples containing amines, alcohols, and hydrocarbons. It is partially non-destructive and highly sensitive detector.
3.5.4 Mass spectrometry
This is the best detector for GC and the combination of two techniques is termed as GC-MS. In this detector, the sample components are detected on the basis of the masses of ions produced from each component. The carrier gas containing sample components enter into the mass spectrometer through an inlet interfaced between the GC (at atmospheric pressure) and MS (low pressure). Then, the sample undergoes ionization in the ionization chamber to form the ions of the components termed as molecular ions. The vacuum pump pulls the ionized and non-ionized species into analyzer where they are analyzed on the basis of their m/z value. The data system gives the separation of components in the form of chromatogram.
4. Working with GC
Three important steps are followed to analyze a sample using GC.
4.1 Sample preparation for GC analysis
Before the GC analysis, some important considerations should be taken into account such as; 1) components of sample to be analyzed by GC must be volatile, 2) the concentration of components should be appropriate for the detection, 3) the sample should not degrade under GC conditions, 4) Before injecting a sample, it should be concentrated to get better results.
Various methods for the concentration of sample are listed below.
1. Liquid–liquid extraction
2. Derivatization
3. Solid-phase microextraction (HS-SPME and DI-SPME)
4. Solid phase extraction
5. Stir-Bar sorptive extraction
4.2 Determination with GC
Before loading the sample into the GC for analysis, gas is set to flow at a constant rate from the cylinder. Then the ample is injected into the injection port, which is carried by the carrier gas into the column. In the column, the components get separated by differential partition in between the mobile gas phase and stationary phase. The partitioned components come out of the column with the carrier gas according to their desorption rate and delivered to the detector for analysis. In the detector, separation of the components is detected and displayed in the computer in the form of chromatogram.The chromatograms are analyzed and quantified, which result in the qualitative and quantitative analysis of the components.
Advantages and disadvantages of GC
5. Factors affecting the separation of components
The separation of components in GC depends upon various factors. Some of the important factors are discussed below.
5.1 Vapor pressure
The vapor pressure is related to the boiling point of the compound. The compounds with lower boiling point (higher vapor pressure) spent less time on the stationary phase and more time in the gaseous phase, therefore, their retention time is comparatively less and they come out fast from the column.
5.2 Polarity of components
The compounds having similar polarity with stationary phase interact possess more retention time because they interact strongly with the stationary phase. Therefore, the retention time of polar molecules on polar stationary phases is more as compared to non-polar stationary phases.
5.3 Column temperature
The choice of column temperature is very important for separation purposes. The higher temperature of column results in the volatilization of all the components and as a result they interact poorly with the stationary phase. This gives poor separation of components with shorter retention times. Therefore, the temperature is selected in such a way that components may interact effectively with the stationary phase and result in good separation.
5.4 Carrier gas flow rate
If the carrier gas flows at a higher rate, the separation would be poor due to less retention time of the component. This is same as above because the components would not be able to interact with the stationary phase effectively.
5.5 Column length
The retention time is proportional to the column length. With the increase in the column length, retention time increases and results in the broadening of the peak as well. The broadening is also observed due to less flow rate and finite rate of mass transfer between phases.
5.6 Amount of material injected
The amount of sample injected effects the shape of the peak. In actual, the shape should be symmetric, however, it may transform into a tailed peak if the sample contains higher concentration of components. Therefore, the separation of components is affected.
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