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Research with thin-layer and column chromatography showed that separations are much more effective when the stationary phase is a very thin layer on the surface of very small and very uniform spherical beads. However, resistance to flow of the mobile phase is very much higher, and in order to get a useful flow of a liquid mobile phase, e. g., 1 - 3 milliliters/minute, pressures of around 15 Mpa (about 2,000 psi) must be applied to the mobile phase. It is possible to apply such pressure from a cylinder of compressed gas, but most systems use a reciprocating piston pump or diaphragm pump with some means of damping the pressure fluctuations from the piston. The sample is usually dissolved in the mobile phase before injection. Columns are typically 4.6 mm ID (6 mm OD) stainless steel tubing 250 mm long. A typical packing will have octadecylsilyl (C18-Si-) (ODS) groups bonded to 5 µm silica beads. The packing is held inside the column by “frits”, discs with pores about 0.5 µm in diameter.
Liquid-liquid chromatography began with samples dissolved in organic solvents and a stationary phase of water adsorbed on particles or fibers of the solid support. More generally, the stationary phase was more “polar” than the mobile phase. That is the so-called "normal phase" chromatography. But stationary phases such as ODS have been particularly useful for separating samples dissolved in water (and most HPLC is now done with bonded phases). Liquid chromatography with the stationary phase less polar than the mobile phase is called “reverse phase”, but is now the common situation. The mobile phase is very often not just water but a mixture of water with methanol (CH3OH) or acetonitrile (CH3CN). “Solvent programming”, a stepwise or continuous change (gradient elution) of the mobile phase composition, is used to speed up separations, like temperature programming in gas chromatography.
“Chiral”columns have been developed relatively recently to separate optical isomers. This separation is important because many pharmaceuticals are active in only one chiral form. For instance, natural Vitamin E is D-a-tocopherol, while half of synthetic Vitamin E is the less active L- isomer.
Liquid chromatography (LC) is a specific technique in which the mobile phase is a liquid. In this type, the stationary phase consists of a finely powdered solid adsorbent packed into a thin metal column.
The mobile phase consists of an eluting solvent forced through the column by a high-pressure pump. The mixture of molecules to be analyzed is injected into the column. A detector monitors this process.
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