Levure Saccharomyces Cerevisiae

High-performance liquid chromatography (HPLC) is now firmly established

as the premier technique for the analysis and purification of a wide range of

molecules. In particular, HPLC in its various modes has become the central technique

in the characterization of peptides and proteins and has, therefore, played

a critical role in the rapid advances in the biological and biomedical sciences

over the last 10 years.

The enormous success of HPLC can be attributed to a number of inherent

features associated with reproducibility, ease of selectivity manipulation, and

generally high recoveries. The most significant feature is the excellent resolution

that can be achieved under a wide range of conditions for very closely

related molecules, as well as structurally quite distinct molecules. This arises

from the fact that all interactive modes of chromatography are based on recognition

forces that can be subtly manipulated through changes in the elution conditions

that are specific for the particular mode of chromatography. Peptides

and proteins interact with the chromatographic surface in an orientationspecific

manner, in which their retention time is determined by the molecular

composition of specific contact regions. For larger polypeptides and proteins

that adopt a significant degree of secondary and tertiary structure, the chromatographic

contact region comprises a small proportion of the total molecular

surface. Hence, the unique orientation of a peptide or protein at a particular

stationary phase surface forms the basis of the exquisite selectivity that can be

achieved with HPLC techniques. All biological processes depend on specific

From: Methods in Molecular Biology, vol. 251, HPLC of Peptides and Proteins: Methods and Protocols

Edited by: M.-I. Aguilar © Humana Press Inc., Totowa, NJ

interactions between molecules and affinity chromatography exploits these specific

interactions to allow the purification of a biomolecule on the basis of its

biological function or individual chemical structure. In contrast reversed phase

HPLC, ion-exchange and hydrophobic interaction chromatography separate

peptides and proteins on the basis of differences in surface hydrophobicity or

surface charge. These techniques therefore allow the separation of complex

mixtures whereas affinity chromatography normally results in the purification

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