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3.4 GAS CHROMATOGRAPHY

         Gas chromatography is a method of chromatographic separation in which the mobile phase is a gas (the carrier gas) and the stationary phase is a solid or liquid coated on a suitable solid support contained in a column. On emerging from the column the carrier gas is passed through a suitable detector.

Apparatus

         The apparatus consists of an injector, a chromatographic column contained in an oven, a detector and data collection devices. The carrier gas flows through the column at a controlled rate or pressure and then through the detector. The chromatography is carried out either at a constant temperature or according to a given temperature programme.

         INJECTORS Sample injection devices range from simple syringes to fully programmable automatic injectors. Direct injections of solutions are the usual mode of injection, unless otherwise prescribed in the monograph. Injection may be carried out either directly at the head of the column using a syringe or an injection valve, or into a vaporization chamber which may be equipped with a stream splitter. The amount of sample that can be injected into a capillary column without overloading is small compared to the amount that can be injected into packed columns and may be less than the smallest amount that can be manipulated satisfactorily by syringe. Capillary columns, therefore, often are used with injectors able to split samples into two fractions, a small one that enters the column and a large one that goes to waste. Such injectors may be used in a splitless mode for analyses of trace or minor components.

         Purge and trap injectors are equipped with a sparging device by which volatile compounds in solution are carried into a low-temperature trap. When sparging is complete, trapped compounds are desorbed into the carrier gas by rapid heating of the temperatureprogrammable trap.

         Headspace injectors are equipped with a thermostatically controlled sample heating chamber. Solid or liquid samples in tightly closed containers are heated in the chamber for a fixed period of time allowing the volatile components in the sample to reach an equilibrium between the nongaseous phase and the gaseous or headspace phase.

         After this equilibrium has been established, the injector automatically introduces a fixed amount of the headspace in the sample container into the gas chromatograph.

         COLUMNS Capillary columns which are usually made of fused silica, are typically 0.2 to 0.53 mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase, which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 μm thick, although nonpolar stationary phases may be up to 5 μm thick.

         Packed columns, made of glass or metal, are 1 to 3 m in length with internal diameters of 2 to 4 mm. Those used for analysis typically are porous polymers or solid supports with liquid phase loadings of about 5 per cent (w/w). High-capacity columns, with liquid phase loadings of about 20 per cent (w/w), are used for large test specimens and for the determination of low molecular weight compounds such as water. The capacity required influences the choice of solid support.

         Supports for analysis of polar compounds on lowcapacity, low-polarity liquid phase columns must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanizing prior to coating with liquid phase. Acid-washed, flux-calcined diatomaceous earth is often used for drug analysis. Support materials are available in various mesh sizes, with 80- to 100-mesh and 100- to 120-mesh being most commonly used with 2- to 4-mm columns.

         Helium or nitrogen is usually employed as the carrier gas for packed columns, whereas commonly used carrier gases for capillary columns are nitrogen, helium and hydrogen. Retention time and the peak efficiency depend on the carrier gas flow rate; retention time is also directly proportional to column length, while resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in ml per minute at atmospheric pressure and room temperature. It is measured at the detector outlet with a flowmeter while the column is at operating temperature. The linear flow rate through a packed column is inversely proportional to the square of the column diameter for a given flow volume. Flow rates of 60 ml per minute in a 4-mm column and 15 ml per minute in a 2-mm column give identical linear flow rates and thus similar retention times. Unless otherwise specified in the monograph, flow rates for packed columns are about 30 to 60 ml per minute. For capillary columns, linear flow velocity is often used instead of flow rate. This is conveniently determined from the length of the column and the retention time of a dilute methane sample, provided a flame-ionization detector is in use. At high operating temperatures there is sufficient vapour pressure to result in a gradual loss of liquid phase, a process called bleeding.

         DETECTORS Flame-ionization detectors are used for most pharmaceutical analyses, with lesser use made of thermal conductivity, electron-capture, nitrogenphosphorous (alkali flame-ionization), mass spectrometric, Fourier transform infrared spectrophotometric detectors, and others, depending on the purpose of the analysis. For quantitative analyses, detectors must have a wide linear dynamic range: the response must be directly proportional to the amount of compound present in the detector over a wide range of concentrations. Flame-ionization detectors have a wide linear range and are sensitive to most organic compounds. Detector response depends on the structure and concentration of the compound and on the flow rates of the combustion, air, makeup, and carrier gases. Unless otherwise specified in individual monographs, flameionization detectors with either helium or nitrogen carrier gas are to be used for packed columns and helium or hydrogen is used for capillary columns.

         DATA COLLECTION DEVICES Modern data stations receive the detector output, calculate peak areas and peak heights, and print chromatograms, complete with run parameters and peak data. Chromatographic data may be stored and reprocessed, with integration and other calculation variables being changed as required. Data stations are used also to program the chromatograph, controlling most operational variables and providing for long periods of unattended operation.

         Data can also be collected for manual measurement on simple recorders or on integrators whose capabilities range from those providing a printout of peak areas and peak heights calculated and data stored for possible reprocessing.

         The design of a particular chromatograph may require modification of the conditions detailed in the monograph. In such a case, the analyst should be satisfied that the modified conditions produce comparable results. If necessary, adjust the flow rate of the carrier gas to improve the quality of the chromatogram or to modify the retention times of the peaks of interest.

Performance

         Criteria for assessing the suitability of the system are described in the “Chromatographic Separation Techniques” (Appendix 3.9). The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given.

Procedure

         Equilibrate the column, the injector and the detector at the temperatures and the gas flow rates specified in the monograph until a stable baseline is achieved. Prepare the test solution(s) and the standard solution(s) as prescribed in the monograph. The solutions must be free from solid particles. Using standard solution determine experimentally suitable instrument settings and volumes of the solutions to be injected to produce an adequate response.

         In applications where an internal standard is used, an injection of sample solution containing only the substance being examined should be made to determine whether any peak is present that will interfere with that of the internal standard. If an interfering peak is present, a suitable correction should be made.

         Inject the selected volumes of the solutions prescribed in the monograph and record the resulting chromatograms. Repeat the determinations to ensure a consistent response. For qualitative analysis, the retention time for a peak in the chromatogram obtained for a test specimen is “the same as,” or “corresponding to” that obtained for a standard preparation under the conditions specified in the individual monograph.

         For quantitative analysis, determine the peak areas or, alternatively, when the symmetry factor is between 0.80 and 1.20, determine the peak heights corresponding to the components of interest. From the values obtained calculate the content of the component or components being determined.

         Assays require quantitative comparison of one chromatogram with another, and lack of control of the specimen size injected is a major source of error. Addition of an internal standard to the test specimen minimizes this error. The ratio of peak response of the components of interest to the internal standard is compared from one chromatogram to another. Where the internal standard is chemically similar to the substance being examined, minor variations in column and detector parameters are controlled also. In some cases,the internal standard may be carried through the assay procedure prior to gas chromatography to control other quantitative aspects of the procedure.

Materials

         Supports, stationary phases and internal standards for gas chromatography are stated in the “Materials for Chromatography” (Appendix 1.7).

         Solvents and reagents used in the preparation of solutions for examination should be of a quality suitable for use in gas chromatography.