The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. In the packed columns, the liquid phase is deposited on a finely divided, inert solid support, such as diatomaceous earth, porous polymer, or graphitized carbon, which is packed into a column that is typically 2 to 4 mm in internal diameter and 1 to 3 m in length. In capillary columns, which contain no packing, the liquid phase is deposited on the inner surface of the column and may be chemically bonded to it. In gas-solid chromatography, the solid phase is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated with a liquid phase.
When a vaporized compound is introduced into the carrier gas and carried into the column, it is partitioned between the gas and stationary phases by a dynamic countercurrent distribution process. The compound is carried down the column by the carrier gas, retarded to a greater or lesser extent by sorption and desorption on the stationary phase. The elution of the compound is characterized by the partition ratio, k¢, a dimensionless quantity also called the capacity factor (see Glossary of Symbols for the definition of symbols). It is equivalent to the ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unretained compound. The value of the capacity factor depends on the chemical nature of the compound, the nature, amount, and surface area of the liquid phase, the column temperature, and the gas flow rate. Under a specified set of experimental conditions, a characteristic capacity factor exists for every compound. Separation by gas chromatography occurs only if the compounds concerned have different capacity factors.
Apparatus—
A gas chromatograph consists of a carrier gas source, an injection port, column, detector, and recording device. The injection port, column, and detector are temperature-controlled. The typical carrier gas is helium, nitrogen, or hydrogen, depending on the column and detector in use. The gas is supplied from a high-pressure cylinder or high-purity gas generator and passes through suitable pressure-reducing valves and a flow meter to the injection port and column. Compounds to be chromatographed, either in solution or as gases, are injected into the gas stream at the injection port. Depending upon the configuration of the apparatus, the test mixture may be injected directly into the column or be vaporized in the injection port and mixed into the flowing carrier gas prior to entering the column.
Once in the column, compounds in the test mixture are separated by virtue of differences in their capacity factors, which in turn depend upon vapor pressure and degree of interaction with the stationary phase. The capacity factor, which governs resolution, retention times, and column efficiencies of components of the test mixture, is also temperature-dependent. The use of temperature-programmable column ovens takes advantage of this dependence to achieve efficient separation of compounds differing widely in vapor pressure.
As resolved compounds emerge separately from the column, they pass through a differential detector, which responds to the amount of each compound present. The type of detector to be used depends upon the nature of the compounds to be analyzed and is specified in the individual monograph. Detectors are heated to prevent condensation of the eluting compounds.
Detector output is recorded as a function of time, producing a chromatogram, which consists of a series of peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlap. The elution time is a characteristic of an individual compound; and the instrument response, measured as peak area or peak height, is a function of the amount present.
Injectors—Sample injection devices range from simple syringes to fully programmable automatic injectors. 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 temperature-programmable 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. A list of liquid phases in current use is given in the section Chromatographic Reagents.
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% (w/w). High-capacity columns, with liquid phase loadings of about 20% (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 low-capacity, 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. Supports and liquid phases are listed in the section Chromatographic Reagents.
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 individual 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 vapor 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, nitrogen-phosphorus, and mass spectrometric detectors. 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, flame-ionization detectors with either helium or nitrogen carrier gas are to be used for packed columns, and helium or hydrogen is used for capillary columns.
The thermal conductivity detector employs a heated wire placed in the carrier gas stream. When an analyte enters the detector with the carrier gas, the difference in thermal conductivity of the gas stream (carrier and sample components) relative to that of a reference flow of carrier gas alone is measured. In general, the thermal conductivity detector responds uniformly to volatile compounds regardless of structure; however, it is considerably less sensitive than the flame-ionization detector.
The alkali flame-ionization detector, sometimes called an NP or nitrogen-phosphorus detector, contains a thermionic source, such as an alkali-metal salt or a glass element containing rubidium or other metal, that results in the efficient ionization of organic nitrogen and phosphorus compounds. It is a selective detector that shows little response to hydrocarbons.
The electron-capture detector contains a radioactive source of ionizing radiation. It exhibits an extremely high response to compounds containing halogens and nitro groups but little response to hydrocarbons. The sensitivity increases with the number and atomic weight of the halogen atoms.
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 to those providing chromatograms with peak areas and peak heights calculated and data stored for possible reprocessing.
Procedure—
Packed and capillary columns must be conditioned before use until the baseline and other characteristics are stable. This may be done by operation at a temperature above that called for by the method or by repeated injections of the compound or mixture to be chromatographed. The column or packing material supplier provides instructions for the recommended conditioning procedure. In the case of thermally stable methyl- and phenyl-substituted polysiloxanes, a special sequence increases inertness and efficiency; maintain the column at a temperature of 250
for 1 hour, with helium flowing, to remove oxygen and solvents. Stop the flow of helium, heat at about 340
for 4 hours, then reduce the heating to a temperature of 250
, and condition with helium flowing until stable.
Most drugs are reactive polar molecules. Successful chromatography may require conversion of the drug to a less polar and more volatile derivative by treatment of reactive groups with appropriate reagents. Silylating agents are widely used for this purpose and are readily available.
Assays require quantitative comparison of one chromatogram with another. A major source of error is irreproducibility in the amount of sample injected, notably when manual injections are made with a syringe. The effects of variability can be minimized by addition of an internal standard, a noninterfering compound present at the same concentration in test and standard solutions. The ratio of peak response of the analyte to that of the internal standard is compared from one chromatogram to another. Where the internal standard is chemically similar to the substance being determined, there is also compensation for minor variations in column and detector characteristics. In some cases, the internal standard may be carried through the sample preparation procedure prior to gas chromatography to control other quantitative aspects of the assay. Automatic injectors greatly improve the reproducibility of sample injections and reduce the need for internal standards.
Many monographs require that system suitability requirements be met before samples are analyzed (see System Suitability and Interpretation of Chromatograms).
