Appendix III B. Gas Chromatography

Gas chromatography (GC) is a chromatographic separation technique based on the difference in the distribution of species between two non-miscible phases in which the mobile phase is a carrier gas moving through or passing the stationary phase contained in a column. It is applicable to substances or their derivatives which are volatilised under the temperatures employed.

GC is based on mechanisms of adsorption, mass distribution or size exclusion.

The apparatus consists of an injector, a chromatographic column contained in an oven, a detector and a data acquisition system (or an integrator or a chart recorder). 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.

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 vaporisation chamber which may be equipped with a stream splitter.

Injections of vapour phase may be effected by static or dynamic head-space injection systems.

Dynamic head-space (purge and trap) injection systems include a sparging device by which volatile substances in solution are swept into an absorbent column maintained at a low temperature. Retained substances are then desorbed into the mobile phase by rapid heating of the absorbent column.

Static head-space injection systems include a thermostatically controlled sample heating chamber in which closed vials containing solid or liquid samples are placed for a fixed period of time to allow the volatile components of the sample to reach equilibrium between the non-gaseous phase and the vapour phase. After equilibrium has been established, a predetermined amount of the head-space of the vial is flushed into the gas chromatograph.

Stationary phases are contained in columns which may be:

• — a capillary column of fused-silica whose wall is coated with the stationary phase,

• — a column packed with inert particles impregnated with the stationary phase,

• — a column packed with solid stationary phase.

Capillary columns are 0.1 mm to 0.53 mm in internal diameter (Ø) and 5 m to 60 m in length. The liquid or stationary phase, which may be chemically bonded to the inner surface, is a film 0.1 µm to 5.0 µm thick.

Packed columns, made of glass or metal, are usually 1 m to 3 m in length with an internal diameter (Ø) of 2 mm to 4 mm. Stationary phases usually consist of porous polymers or solid supports impregnated with liquid phase.

Supports for analysis of polar compounds on columns packed with low-capacity, low-polarity stationary phase must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanising prior to coating with liquid phase. Acid-washed, flux-calcinated diatomaceous earth is often used. Materials are available in various particle sizes, the most commonly used particles are in the ranges of 150 µm to 180 µm and 125 µm to 150 µm.

Retention time and peak efficiency depend on the carrier gas flow rate; retention time is directly proportional to column length and resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in millilitres per minute at atmospheric pressure and room temperature. Flow rate is measured at the detector outlet, either with a calibrated mechanical device or with a bubble tube, while the column is at operating temperature. The linear velocity of the carrier gas through a packed column is inversely proportional to the square root of the internal diameter of the column for a given flow volume. Flow rates of 60 mL/min in a 4 mm internal diameter column and 15 mL/min in a 2 mm internal diameter column, give identical linear velocities and thus similar retention times.

Helium or nitrogen are usually employed as the carrier gas for packed columns, whereas commonly used carrier gases for capillary columns are nitrogen, helium and hydrogen.

Flame-ionisation detectors are usually employed but additional detectors which may be used include: electron-capture, nitrogen-phosphorus, mass spectrometric, thermal conductivity, Fourier transform infrared spectrophotometric, and others, depending on the purpose of the analysis.

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 reference solution(s) as prescribed. The solutions must be free from solid particles.

Criteria for assessing the suitability of the system are described in the chapter on Chromatographic separation techniques (2.2.46). The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given in this chapter.

Static head-space gas chromatography is a technique particularly suitable for separating and determining volatile compounds present in solid or liquid samples. The method is based on the analysis of the vapour phase in equilibrium with the solid or liquid phase.

The apparatus consists of a gas chromatograph provided with a device for introducing the sample that may be connected to a module that automatically controls the pressure and the temperature. If necessary, a device for eliminating solvents can be added.

The sample to be analysed is introduced into a container fitted with a suitable stopper and a valve-system which permits the passage of the carrier gas. The container is placed in a thermostatically controlled chamber at a temperature set according to the substance to be examined.

The sample is held at this temperature long enough to allow equilibrium to be established between the solid or liquid phase and the vapour phase.

The carrier gas is introduced into the container and, after the prescribed time, a suitable valve is opened so that the gas expands towards the chromatographic column taking the volatilised compounds with it.

Instead of using a chromatograph specifically equipped for the introduction of samples, it is also possible to use airtight syringes and a conventional chromatograph. Equilibration is then carried out in a separate chamber and the vapour phase is carried onto the column, taking the precautions necessary to avoid any changes in the equilibrium.

Using the reference preparations, determine suitable instrument settings to produce an adequate response.

Separately introduce into identical containers the preparation to be examined and each of the reference preparations, as prescribed in the monograph, avoiding contact between the sampling device and the samples.

Close the containers hermetically and place in the thermostatically controlled chamber set to the temperature and pressure prescribed in the monograph; after equilibration, carry out the chromatography under the prescribed conditions.

Add to a set of identical suitable containers equal volumes of the preparation to be examined. Add to all but one of the containers, suitable quantities of a reference preparation containing a known concentration of the substance to be determined so as to produce a series of preparations containing steadily increasing concentrations of the substance.

Close the containers hermetically and place in the thermostatically controlled chamber set to the temperature and pressure prescribed in the monograph; after equilibration, carry out the chromatography under the prescribed conditions.

Calculate the linear equation of the graph using a least-squares fit, and derive from it the concentration of the substance to be determined in the preparation to be examined.

Alternatively, plot on a graph the mean of readings against the added quantity of the substance to be determined. Extrapolate the line joining the points on the graph until it meets the concentration axis. The distance between this point and the intersection of the axes represents the concentration of the substance to be determined in the preparation to be examined.

If prescribed, the successive withdrawal method is fully described in the monograph.

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.

Unless otherwise stated in the monograph, use nitrogen as the carrier gas and a flame ionisation detector. Occasionally reference is made to on-column injection, in which case the sample is injected directly on to the packing material without the use of an inlet heater. When non-volatile material is to be injected on to the column, a suitable interchangeable pre-column may be used.

Solvents and reagents used in the preparation of solutions for examination should be of a quality suitable for use in gas chromatography. A wide range of chemical substances is used as stationary phases, including polyethylene glycols, high-molecular weight esters and amides, hydrocarbons, silicone gums and fluids (polysiloxanes often substituted with methyl, phenyl, nitrilo, vinyl or fluoroalkyl groups or mixtures of these) and microporous cross-linked polyaromatic beads. A suitable stationary phase, its concentration and the nature and grade of a suitable solid support are stated in the monograph. The column should be conditioned in accordance with the manufacturer's instructions. In most cases reference is made to a particular commercial brand that has been found to be suitable for the purpose, but such statements do not imply that a different but equivalent commercial brand may not be used.

Reagents used as internal standards should not contain any impurity that would produce a peak likely to interfere in the determination described in the monograph.

Where no injection volume is specified in the monograph, the analyst should select an appropriate volume for their specific application. The volume chosen is dependent on the response of the analyte, the detector used, the efficiency of the column and the overall performance of the chromatographic system. Where a volume is not indicated, 1 µL is usually appropriate; however this should be checked for suitability under the local operating conditions.

Reference may be made to a secondary peak. A secondary peak is a peak in the chromatogram other than the principal peak and any peaks due to internal standard, solvent and derivatising agents. Peaks identified as being due to the counter-ion and/or other excipients including preservatives in the material being examined may also be excluded.