Appendix III H. Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) is a method of chromatographic separation in which the mobile phase is a fluid in a supercritical or a subcritical state. The stationary phase, contained in a column, consists of either finely divided solid particles, such as a silica or porous graphite, a chemically modified stationary phase, as used in liquid chromatography, or, for capillary columns, a cross-linked liquid film evenly coated on the walls of the column.

SFC is based on mechanisms of adsorption or mass distribution.

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

Pumping systems are required to deliver the mobile phase at a constant flow rate. Pressure fluctuations are to be minimised, e.g. by passing the pressurised solvent through a pulse-damping device. Tubing and connections are capable of withstanding the pressures developed by the pumping system.

Microprocessor controlled systems are capable of accurately delivering a mobile phase in either constant or varying conditions, according to a defined programme. In the case of gradient elution, pumping systems which deliver solvent(s) from several reservoirs are available and solvent mixing can be achieved on either the low or high-pressure side of the pump(s).

Injection may be carried out directly at the head of the column using a valve.

Stationary phases are contained in columns which have been described in the chapters on Liquid chromatography (2.2.29) (packed columns) and Gas chromatography (2.2.28) (capillary columns). A capillary column has a maximum internal diameter (Ø) of 100 µm.

Usually the mobile phase is carbon-dioxide which may contain a polar modifier such as methanol, 2-propanol or acetonitrile. The composition, pressure (density), temperature and flow rate of the prescribed mobile phase may either be constant throughout the whole chromatographic procedure (isocratic, isodense, isothermic elution) or may vary according to a defined programme (gradient elution of the modifier, pressure (density), temperature or flow rate).

Ultraviolet/visible (UV/Vis) spectrophotometers and flame ionisation detectors are the most commonly employed detectors. Light scattering detectors, infrared absorption spectrophotometers, thermal conductivity detectors or other special detectors may be used.

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.