By Donald A Wellings
A distillation of over two decades of the author's functional adventure of this approach, this booklet intentionally steers away from complicated theoretical facets and concentrates extra at the 'black artwork' parts of the recommendations concerned. hence is a e-book for common use within the laboratory, specifically of curiosity to these within the pharmaceutical, agrochemical and biotechnology industries and to somebody inquisitive about the research and purification of substances, small molecules, proteins and DNA.
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Extra info for A Practical Handbook of Preparative HPLC
Sample text
Organic acids are often added to minimize any cation exchange effects arising from acidic silanol groups on the silica surface. Modes of Chromatographic Separation 37 As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds.
A brief survey suggests that there are over five hundred different reversed phase materials commercially available. However, the eventual scale of operation may limit the number of suitable stationary phases to less than ten. Reversed phase media are designed to operate in aqueous buffers where the hydrophobic adsorption between analyte and stationary phase is disturbed by an increasing concentration of a miscible organic solvent. The most common application of reversed phase HPLC is in the purification of peptides and proteins where the analyte is often desorbed under gradient elution.
Before proceeding with a discussion on the individual modes of separation it is worth reviewing a brief glossary of terms in association with the stationary phase properties. A simple strategy for selection of the stationary phase will naturally follow, in order to help the reader to choose a supplier wisely. There are two main classes of polymeric support utilized in the preparation of stationary phases for all modes of chromatography, namely silica and polystyrene-divinylbenzene copolymer. For simplicity, from this point on, the former will be referred to as silica and the latter class as polymeric.