By A. Fallon, R.F.G. Booth and L.D. Bell (Eds.)

This booklet is meant to familiarize biochemists with HPLC. Theoretical features of every mode of chromatography are mentioned in chapters 1-9, supplying an knowing of a number of the modes of chromatography that are now attainable utilizing commercially to be had columns, from reversed part to affinity. functional features and instrumentation are coated in bankruptcy 10. the majority of the publication, which follows, provides examples and functions of every mode of chromatography in present biochemical perform.

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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. Gradients are formed by a low volume mixer whch is located on the low pressure side of the pump, thereby 30 APPLICATIONS OF HPLC IN BIOCHEMISTRY eliminating some of the solvent degassing problems.

However, since resolution depends on fi,it can be impractical to significantly increase the resolution by means of this alone. 6. Separation time It is of value to the chromatographer to observe the influence of changing various parameters on the separation time, so that an optimum separation can be achieved in minimum time (in addition, this approach will minimise solvent consumption). It can be shown that the time taken to elute the last peak is given by: t, = 16 X + RTx [a/(. 16) 20 APPLICATIONS OF HPLC IN BIOCHEMISTRY In this form the equation shows that whilst other terms are constant, doubling the resolution causes a four-fold increase in separation time.

For a complex mixture of solutes it is generally not possible to achieve good resolution between all components with a maximum k’ value of 5. Typically k‘ values of up to 10 offer a practical solution. Since the term containing the capacity factors plays a significant part in this equation, the spread of k’ values can be minimised by using either gradient elution or by coupled columns. 7. Preparative separations Scaling-up an analytical separation allows the isolation and purification of a particular component from a mixture of solutes.

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