Preparative Liquid Chromatography

Liquid chromatography (Hostettman et al, 1986) in its many forms is a separation technique based on the polarity of the analytes and their partition between the mobile and stationary phases, and is therefore complementary to fractional distillation, which separates materials by their boiling point. The usual sequence for fractionating an essential oil or extract is to distil it first and then apply liquid chromatography to the distillation fractions as a further fractionation procedure, rather than as an analytical tool. The selectivity of the technique is achieved by choosing a stationary phase, usually from the various activities of silica gel, and varying the polarity of the mobile phase, the solvent, by mixing a non-polar component (such as hexane or pentane) with different amounts of a more polar component (such as diethyl ether, ethyl acetate or chloroform).

In the simplest form of liquid chromatography, the solvent is applied to the top of the column by gravity from a reservoir, a slow but reproducible process. Greater speed and resolution can be achieved, even with relatively large columns containing up to a few kilograms of silica, by using ‘flash chromatography’, in which air or nitrogen under pressure is used to force the solvent through the column. This is a convenient and reasonably reproducible method for quickly separating fractions of very different polarities. For greater resolution and reproducibility on a smaller scale, the solvent can be pumped through the column at a continuous and controlled rate, a technique variously called medium-pressure chromatography or preparative high-perfor­mance chromatography (Prep HPLC; Verzele and Dewaele, 1986), according to the equipment used. Detectors are not used with gravity or flash systems, but a non-destructive detector, such as a UV spectrophotometric detector or a refractive index detector (relative refractometer), is used with Prep HPLC to monitor the solvent stream and allow accurate collection of fractions as they elute from the column.

Solvent gradients can be used to improve resolution. A simple, step­wise gradient involves sequentially using solvents of increasing polarity and can be applied to any type of column chromatography. Contin­uous solvent gradients can be generated by modern HPLC pumps which mix solvents of different polarities to increase gradually the polarity of the mobile phase. Computer-controlled systems equipped with an autosampler and automatic fraction collector are available and can be programmed to repeat a separation many times and bulk the fractions from each separation. This allows larger amounts of sample to be processed, while achieving the resolution of a small-scale separa­tion.

Analytical HPLC necessarily includes a detector on the outlet from the column, which responds to the presence of analytes in the solvent stream. Narrow-bore columns and fine particle sizes are used to achieve the best possible resolution. Although analytical HPLC is used in the fragrance industry to investigate the non-volatile fractions of essential oils and product bases, for example, it is much more widely used in other industries (such as the pharmaceutical industry, for which it is the main research tool).

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