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 0.2to 0.53mm in internal diameter and 5to 60m in length.The liquid or stationary phase,which is sometimes chemically bonded to the inner surface,is 0.1to 1.0µm thick,although nonpolar stationary phases may be up to 5µm thick.Alist of liquid phases in current use is given in the section Chromatographic Reagents.

Packed columns,made of glass or metal,are 1to 3m in length with internal diameters of 2to 4mm.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 mLper 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 60mLper minute in a 4-mm column and 15mLper 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 30to 60mLper 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 NPor 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.

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.Amajor 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).