TABLE OF CONTENTS Clearly it is not possible to describe every technique, or variation in a particular mode of chromatography, so this chapter will concentrate on the most dominant approaches to optimization of preparative HPLC. The objective here is to maximize the column load and to minimize mobile phase wastage allowing the purification of the largest amount of target in the minimum space.
Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations [5.1]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography. The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography.
In the second example the separation of two optical isomers by batch chromatography using box car injections demonstrates one approach to purification by chiral chromatography.
The purification of synthetic peptide objectives is open to many modes of separation due to the hydrophilic, hydrophobic and charged nature of amino acid side-chains. Employment of ionexchange, hydrophilic interaction, normal phase and reversed phase modes of chromatography all seem perfectly viable. Though ion-exchange has its place in peptide purification, the complex and subtle nature of the impurities observed in peptides prepared by the solid phase approach dictates the use of high performance techniques offered by Normal and Reversed Phase modes of operation.
Synthetic...
