By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)
This booklet is meant to familiarize biochemists with HPLC. Theoretical points of every mode of chromatography are mentioned in chapters 1-9, delivering an realizing of a few of the modes of chromatography that are now attainable utilizing commercially to be had columns, from reversed part to affinity. sensible elements and instrumentation are coated in bankruptcy 10. the majority of the publication, which follows, offers examples and functions of every mode of chromatography in present biochemical perform.
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Additional info for Applications of HPLC in Biochemistry
1). Alternatively, in cation-exchange chromatography, solute ions Yf compete with mobile phase ions X+ for negative sites (- ) on the stationary phase (these are often sulphonic acid moieties such as sulpho-propyl or carboxymethyl). Separation is achieved because sample ions that interact weakly with the ion-exchange stationary phase will be weakly retained on the column and elute early whereas solutes that interact strongly with the ion-exchange stationary phase will be strongly retained and will elute later.
Consequently, a drop in pH will cause acidic solutes to elute rapidly from an anion-exchange column. 2) to the left and only low concentrations of BH+ will compete for cation-exchange sites. Thus raising the pH will cause basic solutes to elute rapidly from a cation-exchange column. This is shown in Fig. 4 where the effect of pH on retention time for several basic amino acids has been demonstrated. Under extreme conditions of pH, when all the molecules are either totally charged or uncharged, pH will have no further effect.
Mixing of mobile phases for gradient HPLC takes place on the ‘pressurised‘ side of the pumps. The system is compatible with a specialised range of columns introduced for protein separations with recommended operating pressures of less than 500 psi at 1 ml/min. This system can also be used for chromatography using conventional low pressure columns (Pharmacia FPLC system). A recent addition to the range is a reciprocating pump capable of operation at higher pressures and therefore suitable for use with other HPLC columns.