Two-dimensional reverse phase-reverse phase chromatography: A simple and robust platform for sensitive quantitative analysis of peptides by LC/MS. Hardware design

Eduard Rogatsky, Daniel T. Stein

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We have revised current two-dimensional RP-RP approaches and developed a new robust 2-D RP-RP platform. This platform was implemented on an Agilent 1100 2-D liquid chromatography system and is based on high pressure switching between two high-resolution RP columns. An independent binary gradient was implemented for each dimension. The powerful combination of dual analytical columns with independent gradient elution achieves high analyte purity, effectively eliminates matrix effects, and maximizes MS sensitivity in Q1 SIM comparable to the sensitivity enhancements of MS/MS-based methods. Implementation of dual simultaneous gradient profiles (overlapped gradients) reduces 2-D method run-time to the scale of 1-D method run-times. This robust and sensitive approach is particularly suitable for hydrophobic peptides and small proteins and can be used as a routine standard technique for enhanced on-line peptide purification coupled with mass spectrometric detection.

Original languageEnglish (US)
Pages (from-to)539-546
Number of pages8
JournalJournal of Separation Science
Volume29
Issue number4
DOIs
StatePublished - Mar 1 2006

Keywords

  • Carryover
  • Column switching
  • Mass spectrometry
  • Matrix effects
  • Peak focusing

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

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