Effect of experimental parameters on the HPLC separation of peptides and proteins

Haleem J. Issaq*, Stephen D. Fox, Megha Mahadevan, Thomas P. Conrads, Timothy D. Veenstra

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


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.

Original languageEnglish
Pages (from-to)2255-2283
Number of pages29
JournalJournal of Liquid Chromatography and Related Technologies
Issue number14
StatePublished - 2003
Externally publishedYes


  • ESI-MS
  • Peptides
  • Proteins
  • Proteomics
  • μHPLC


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