For euch in jection the ratio of ethanol peak height to propanol peak height was calculated and a graph of the peak heights ratio against ethanol concentration was drawn.

The ethanol concentration corresponding to the peak heights ratio for the test sample was read off the graph giving a concentration of 12.1 gl '.

Choice of column materials

HPLC techniques were initially developed as liquid-liquid chromatographic methods and difficulties in maintaining the stationary phase were resolved by chemically bonding it to the particulate support. Subsequently a whole range of column materials have been developed that enable the basic HPLC instrumentation to be used for the major chromatographic techniques.

In selecting a column material for the separation of a specific substance, it is necessary to decide which physical characteristic of the molecule may be useful (Table 3.5). The initial major consideration for small molecules is usually the polarity of the molecule. A choice can then be made from ion-exchange chromatography for ionic species, adsorption chromatography for molecules showing moderate degrees of polarity and partition chromatography, which can be applicable to most molecules. For large molecules, gel permeation chromatography should be considered, using non-compressible gels which are effective at the pressures used.

The bonding of cyclodextrins to silica has provided a range of media known as chiral stationary phases (CSPs), which are capable of

Table 3.5 Chromatographic media

Name of medium Surface group Chromatographic applications

Adsorption Partisil

LiChrosorb Si60 Nucleosil LiChrosorb Alox Spherisorb A






Steroids Alcohols Organic acids Vitamins Pesticides

Partition (normal phase) Partisil PAC LiChrosorb NIL Nucleosil N02 LiChrosorb Diol

Cyano-amino Amino Nitrite Hydroxyl


Food preservatives

Partition (reverse phase) Partisil ODS LiChrosorb RPB LiChrosorb RP18 LiChrosorb RP2

Octadecylsilane Octyl Octadecyl Silane

Barbiturates Esters, ethers Aromatics Steroids

Ion-exchange Partisil SCX

LiChrosorb KAT Partisil SAX Nucleosil N(CH3)2

Sulphonate Sulphonate

Quaternary ammonium Dimethyl amine

Amino acids, nitrogenous bases Nucleosides Nucleotides Organic acids

Gel permeation LiChrospher Si Macrogel Sephacryl S

Rigid porous silica Semi-rigid polystyrene Dextran-acrylamide

Synthetic polymers separating some diastereoisomers. Cyclodextrins have rigid, highly defined structures and are able to form inclusion complexes with a range of compounds. The structure of the cyclodextrin is often such that one stereoisomer can fit easily into the molecule but the other isomer cannot. The latter isomer is therefore eluted from the column first. Such CSPs provide an alternative method to the production of FLEC derivatives for separating stereoisomers (Figure 3.12). Usually the method is only appropriate for compounds that contain an aromatic group near the chi-ral centre but development of new chiral media is continually extending the range of applications.

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