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HPLC- High-Performance Liquid Chromatography

The High-Performance Liquid Chromatography Technique

The analysis of peptides and proteins has been revolutionized by the introduction of HPLC. HPLC allows for the rapid and sensitive analysis of peptide and protein structure. It has simplified our analysis in understanding of biological processes and in the development of peptide and protein based pharmaceuticals.

But HPLC doesn't stop there, it's applications in peptide and protein purification continues to expand at an extremely rapid rate. For example solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides and proteins that need to be highly purified. Also another very important application for HPLC in the post-genomic age is that its techniques are used extensively in the isolation and characterization of novel proteins.

HPLC's most significant feature would be its excellent resolution that is easily achieved under a wide range of conditions for very closely related molecules, as well as structurally quite distinct molecules. This feature is 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 orientation specific 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. All biological processes depend on specific 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.

Retention and Bandwidth Relationships of HPLC

Retention time or tr in HPLC is the time taken for a solute to pass through a chromatographic column . This retention time is measured as the time taken by the solute, following injection, to emerge from the column and to be detected. In order to allow retention times to be compared to different columns or under different conditions, the retention time of a solute is normally compared with the retention time of a molecule which is not retained on the specific column of interest. This allows the unitless capacity factor k' of a solute to be expressed in terms of the retention time tr, through the relationship