HPLC analysis and purification of peptides

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chromatography

Background:

• High-performance liquid chromatography (HPLC) is a cornerstone technique for peptide isolation and purification.
• Peptides vary significantly in source, quantity, and complexity, necessitating diverse separation strategies.

Purpose of the Study:

• To review major HPLC modes for peptide separation: size-exclusion, ion-exchange, and reversed-phase.
• To introduce a novel mixed-mode hydrophilic interaction/cation-exchange chromatography for peptide analysis.
• To highlight HPLC's utility in peptide and protein structural characterization and preparative purification.

Main Methods:

• Exploration of size-exclusion chromatography (SEC) for assessing peptide/protein oligomerization.
• Application of reversed-phase HPLC (RP-HPLC) for monitoring peptide hydrophilicity/hydrophobicity.
• Demonstration of capillary electrophoresis (CE) for peptide separations.
• Development of preparative RP-HPLC protocols for sample loads up to 200 mg.

Main Results:

• Established the versatility of SEC, ion-exchange, and RP-HPLC for diverse peptide separations.
• Showcased a novel mixed-mode hydrophilic interaction/cation-exchange approach for enhanced peptide resolution.
• Validated HPLC techniques for crucial structural characterizations, including oligomerization and hydrophobicity.
• Presented effective preparative chromatography protocols for significant peptide and protein quantities.

Conclusions:

• HPLC, encompassing various modes and novel approaches, is indispensable for peptide purification and characterization.
• The reviewed techniques provide valuable insights into peptide structure and facilitate the development of new therapeutics, such as antimicrobial peptides.
• Preparative HPLC methods enable efficient purification of substantial amounts of peptides and recombinant proteins.