Effect of Experimental Parameters on the HPLC Separation of Peptides and Proteins

Abstract

This manuscript examines the effects of different experimental parameters on resolution, peak symmetry, peak width, and selectivity (peak elution order) of peptides by micro high performance liquid chromatography. The experimental parameters are: mobile phase composition, flow rate, organic modifiers, ion‐pairing agents, column temperature, the effect of packing material properties: particle size, particle porosity and reversed‐phase (RP) alkyl chain length, and column dimensions. When a mass spectrometer (MS) is used as the detector in micro‐HPLC, certain experimental parameters such as mobile phase flow rate and buffer composition have to be adjusted in order to meet the requirements of the MS procedure employed. When electrospray ionization (ESI) is the selected MS mode of operation the mobile phase flow rate should be in nL min⁻¹ and a volatile buffer should be used to achieve maximum sensitivity. Also, it was found that the elution order (selectivity) of peptides in RP‐HPLC is affected by type and concentration of ion‐pairing agent, organic modifier, column temperature, pH of the buffer, and the alkyl chain length of the derivatizing agent in RP. This manuscript includes work that has been done in our laboratory and is supplemented by data published by other researchers.

Keywords:

• Proteomics
• Proteins
• Peptides
• μHPLC
• ESI–MS

Acknowledgments

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1‐CO‐12400.