2. Principal Chemical and Analytical Methods Used in Reverse Engineering
2.4 Chromatography
2.4.3 Gas Chromatography (GC)
Gas chromatography is a process by which a mixture of samples is separated into its constituents by a moving gas phase passing over a stationary sorbent, as shown in Figure 2.19. Gas chromatography is divided into two major categories: gas-liquid chro- matography (GLC) with which separation occurs by partitioning a sample between a mobile gas phase and a thin layer of non-volatile liquid coated on an inert support, and gas-solid chromatography (GSC) that employs a solid of large surface area as the station- ary phase.
A gas chromatograph consists of several basic modules joined together to:
1. Give a constant flow of mobile phase, which is carrier gas
2. Allow the introduction of sample vapors into the flowing mobile phase 3. Have the sufficient length of a stationary phase
4. Maintain the column at the appropriate temperature
5. Detect the sample components as they elute from the column
6. Provide a readable signal proportional in magnitude to the amount of each component
Flow controller
Sample injector
Waste
Detector
Column oven Column
Carrier gas FIGURE 2.19
Typical instrumental diagram of gas chromatography.
2.4.3.1 Carrier Gas
The mobile phase used is either helium, nitrogen, hydrogen, or argon—the selection of gas depends on factors such as availability, purity required, consumption, and the type of detector employed. Helium gas is preferred when thermal conductivity detectors are employed because of its high thermal conductivity relative to that of the vapors of most organic compounds. The operating efficiency of the apparatus is highly dependent on the maintenance of a constant flow of carrier gas.
2.4.3.2 Sample Injection
Various applicators are available for introducing the sample, but the major applicator is a hypodermic syringe, a self-sealing silicone rubber septum, as common applications involve liquid samples that are injected onto a glass liner within a metal block. The tem- perature of the sample port is kept high so that the liquid is rapidly vaporized but with- out decomposing or fractionating the sample. Generally, the sample port temperature is around the boiling point of the least volatile component.
Many samples are, however, unsuitable for direct injection into a gas chromatograph because of their polarity, low volatility, or thermal instability.
2.4.3.3 The Column
Two basic types of columns are generally used: the packed column and the open tubular or capillary column. The actual separation of sample components is affected in the column where the nature of the solid support, type and amount of liquid phase, method of pack- ing, length, and temperature are important factors in obtaining the desired resolution.
The internal column diameter should be at least eight times the diameter of the support particles. The essential requirements for an ideal support are:
1. It must be chemically inert to solutes passing through the column.
2. It must have a large surface area to volume ratio.
3. It must have a low resistance to gas flow.
4. It should be capable of being uniformly wetted.
5. It should have good mechanical strength.
Stationary phase is the part of the column packing which is directly responsible for the separation of the solute mixture; therefore, it is important to make the correct choice of phase. The essential requirements of a stationary phase are as follows:
1. It must have low vapor pressure. The maximum allowable pressure is governed by the sensitivity of the detector being used.
2. It should be thermally stable at the operating temperature used. Any instability will lead to an excessive background signal and high noise level.
3. It should be a good solvent for the solute under investigation or the components will elute too early. It must also have low viscosity or the solutes will remain too long in the liquid phase.
4. It must be chemically inert to the solute, column material, etc.
The temperature range of the stationary phase must also be considered. Every phase has a maximum recommended temperature (MRT) which is defined as the temperature at which the phase has a vapor pressure of 0.1 mm of mercury. Columns should not be used at temperatures above the MRT.
These columns require conditioning because the liquids used as stationary phases are frequently mixtures of polymers which have varied molecular weights. Due to this spread of molecular weight, such polymers exert a considerable vapor pressure even at low temperature. The finite vapor pressure of these liquids results in a small amount of stationary phase being swept by the carrier gas into the detector. This removal of column stationary phase by the carrier gas is called column bleed. If a highly sensitive detector like a flame ionization detector is used, a large background current will be generated due to the column liquid burning in the flame. By conditioning the column, low molecular weight impurities and residual solvents are removed. Conditioning of the column can be achieved by heating the column overnight at 20 to 30°C below the MRT of the stationary phase with a low flow rate of carrier gas passing through the column.
It is important to disconnect the column from the detector during conditioning to avoid the risk of contamination.
Capillary columns may be prepared from stainless steel or glass, but these have been almost completely replaced by fused silica. Fused silica is an inert material that is extremely flexible, robust, and has a low thermal mass; it is thus ideal for a capillary col- umn. Capillary columns have an internal diameter of 1 mm or less.
The main disadvantage with the capillary column is the sample capacity of the column, which depends on film thickness. With thin-film and narrow-bore columns, maximum column efficiencies are achieved but the sample capacity is low. With wide-bore, thick-film columns, the sample capacity is increased but at the cost of column efficiency.
A large selection of columns is therefore available for gas chromatography. Packed col- umns are relatively cheap, simple to use, and do not require specialized injectors. However, the separating power of packed columns is limited. Capillary columns have a high separa- tion capability but are more expensive and require a higher level of competence.
2.4.3.4 Detectors
A detector located at the exit of the separation column detects the presence of the indi- vidual components as they leave the column. The choice of detector will depend on factors such as the concentration level to be measured and the nature of the separated compo- nents. Detectors usually translate the column’s separation process into an electrical signal to be used for qualitative and quantitative measurements by means of recorders, electronic integrators, etc. Some of the important properties of a detector in gas chromatography are:
• Sensitivity
• Linearity
• Stability
• Universal or selective response
The following are different detectors in gas chromatography:
• Thermal conductivity detector (TCD)
• Flame ionization detector (FID)
• Thermionic emission detector (TED)
• Electron capture detector (ECD)
• Flame photometric detector (FPD)
• Photoionization detector (PID) 2.4.3.4.1 Thermal Conductivity Detectors
Detectors in this group, including hot wire and thermistor types, are designed to continu- ously measure the thermal conductivity of column effluent. The principle of operation is simple. The carrier gas is passed into a cell, the walls of which are maintained at a constant temperature, with the use of an electrical filament or thermistor. When a constant current is applied to the hot element, the rate of production of heat is constant. This heat must be dissipated. Such dissipation is largely conduction of heat through the carrier gas. The appearance of vapor in the gas changes its thermal conductivity so that different tempera- ture gradients are necessary to maintain the required rate of dissipation, thus causing the hot element to change temperature.
The sensitivity of the detector is affected by the current through the wires, the relative thermal conductivity of the carrier gas and the sample compartments, the carrier gas flow rate, and the relative temperatures of the cell block and the hot elements. The heavier the carrier gas and the lower the thermal conductivity, the less is the sensitivity.
The advantages of thermal conductivity detectors are that they are non-destructive (i.e., the sample can be collected for further investigation if required), and they will respond to all compounds eluting from the column. Their disadvantages are that an expensive ther- mostated oven is required to house the detectors, and compared with the various ioniza- tion detectors, they are relatively insensitive.
2.4.3.4.2 Flame Ionization Detector
The flame ionization detector is the most popular detector in use today. This popularity is due to its high sensitivity, the wide sample concentration range over which its response is linear, and its robust design. It is a mass sensitive detector—that is, its response is propor- tional to the total mass of compound entering the detector per unit time.
A good flame ionization detector should have the following characteristics:
• The change in current through the detector should be as large as possible for a given mass of sample (i.e., it should have high sensitivity).
• The change in current through the detector should be proportional to the mass of sample over as wide a range of sample sizes as possible (i.e., it should have a wide linear dynamic range).
• The signal generated for the same masses of different compounds should be equal (i.e., the relative response factor should be unity).
• The signals generated when no sample is passing through the detector should be minimal (i.e., low noise level).
• The detector should not respond to changes in the carrier gas flow rate, and because of their small dead volume, they should have a fast response speed.
The advantages of a flame ionization detector are high sensitivity, fast response, and wide dynamic range. In addition, it is insensitive to changes in temperature, is robust, and can be dismantled easily for cleaning. The disadvantages are that it is somewhat specific in
response (i.e., the detector will not respond to inorganic compounds including the per- manent gases, and organic molecules containing a single carbon atom which is part of a carbonyl group, e.g., formic acid) and that it is a destructive detector in that the sample cannot be collected for further study.
2.4.3.4.3 Electron Capture Detector
The electron capture detector (ECD) was the first truly selective detector for gas chroma- tography. The operation of the ECD is based on the ability of certain substances to react with free electrons. Most ionization detectors are based on measurement of the increase in current, which occurs when a more readily ionized molecule appears in the gas stream.
Compared with the FID, however, the ECD is more specialized and tends to be chosen for its selectivity which can simplify chromatograms. The ECD requires careful attention to obtain reliable results. Cleanliness is essential, and the carrier gases must be pure and dry.
Other detectors that have not been mentioned here are also available for chromato- graphic analysis.
2.4.3.5 Carrier Gas Flow
During an isothermal run, control of the carrier gas flow can be maintained by setting up a constant pressure drop across the column and maintaining the pressure drop by means of a pressure controller. During a temperature programmed run, as the column is heated, the molecular interactions in the carrier gas increase, causing the viscosity to increase and the flow rate to decrease. The flow rate drop during a temperature program can have two effects: the column efficiency may deteriorate rapidly and labile components on the column may be lost. The problem can be overcome by including a flow controller in the carrier gas line. The flow controller increases the pressure drop across the column in proportion to any decrease in flow rate, consequently maintaining a constant flow rate throughout the column regardless of temperature.
2.4.3.6 Injector System
A gas chromatographic system is closed off from the external atmosphere so that any sample introduction must be achieved while maintaining the internal gas pressure. One of the easiest means of doing this is to use a rubber septum that can be pierced by the needle of a micro-syringe, although some injectors employ pneumatically operated seals as an alternative. Some of the common injectors are:
• Packed column injector
• Split/splitless injector
• On-column capillary injector
• Programmable temperature vaporizer 2.4.3.6.1 Split/Splitless Injector
This is a very versatile injection system able to cope with wide concentrations from trace amounts upward. In the split mode the evaporated sample is homogeneously mixed with the carrier gas, and then the mixture is split into two portions, the smaller of which is directed onto the column. This allows concentrated samples to be chromatographed with- out overloading the capillary column. The split ratio is defined as the ratio of inlet flow to
column flow. The major part of the split sample is vented to the atmosphere using a needle valve to vary the split ratio.
Some samples contain trace quantities to be analyzed and thus require the whole sample to go onto the column to ensure adequate sensitivity. The splitless mode was developed to allow this feature. This splitless mode is ideal for low concentrations of components.
2.4.3.7 Sample Handling Techniques
Different types of samples are analyzed by gas chromatography. Depending on sample characteristics, the handling of the sample plays an important role in gas chromatography.
Some of the sample handling techniques are:
• Gas sampling
• Pyrolysis
• Headspace analysis 2.4.3.7.1 Gas Sampling Gas sampling requires:
• Constant temperature (high enough to prevent any condensation of the compo- nents in the mixture)
• Constant pressure
• Gas-tight seals on sample transfer lines and the sampling device
• Fixed amount of sample injected into the GC 2.4.3.7.2 Pyrolysis
Pyrolysis is a technique applied mainly to solid samples, usually polymers. The pyroly- sis system involves the thermal decomposition of the sample—the degradation products enter the gas chromatograph where they are separated and identified.
There are two types of pyrolyzer in general use:
• Filament type: this system is capable of operating in the range of 500 to 1000°C.
• Curie point: in this case, the sample is coated onto the probe.
2.4.3.7.3 Headspace Analysis
In headspace analysis, a sample of the vapor produced by a liquid or solid material is obtained and introduced into the gas chromatograph. The sample is placed in a sealed con- tainer and thermostated for a period sufficient to establish equilibrium in the headspace above the sample. When the headspace sample is injected, a chromatogram is obtained with peaks corresponding to the components.