By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)
This ebook is meant to familiarize biochemists with HPLC. Theoretical facets of every mode of chromatography are mentioned in chapters 1-9, delivering an realizing of a number of the modes of chromatography that are now attainable utilizing commercially to be had columns, from reversed section to affinity. useful points and instrumentation are coated in bankruptcy 10. the majority of the e-book, which follows, offers examples and purposes of every mode of chromatography in present biochemical perform.
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Extra resources for Applications of HPLC in Biochemistry
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.
Pellicular packings were developed which consisted of glass beads with ionic molecules attached in a layer on the surface. Pellicular packings were well suited to the high pressures but the beads were relatively large and gave a low chromatographic efficiency. They also possessed a relatively low capacity when compared with the original polystyrene-divinylbenzene resin supports. However, through the 1970s, silica-based ion-exchange 44 APPLICATIONS OF HPLC IN BIOCHEMISTRY resins were developed (Knox and Pryde, 1975) and sample capacities have now improved to levels which provide acceptable chromatography, although modem amino acid autoanalysers still use cross-linked polystyrene stationary phases.
These simple guidelines should prevent any major problems associated with the use of halide ions. 1. Proteins: a special case Many globular proteins contain prosthetic groups with metal ions and sensitive thiol groups which may be ‘poisoned’ by slow leakage of metal ions from the welding in the joints of stainless steel equipment. To overcome this potential problem, two manufacturers have designed equipment to minimise contact with any stainless steel. LKB manufacture a reciprocating twin-pump system with pump heads made of KEL-F.