Optimization of the porous structure and polarity of polymethacrylate-based monolithc capillary columns for the LC-MS separation of enzymatic digests

Sebastian Eeltink, Laurent Geiser, Frantisek Svec, Jean Frechet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

The porous structure as well as the polarity of methacrylate ester-based monolithic stationary phases has been optimized to achieve the separation of various peptides originating from enzymatic digestion. The porous structure, determined by the size of both pores and microglobules, was varied through changes in the composition of porogenic solvents in the polymerization mixture, while the polarity was controlled through the incorporation of butyl, lauryl, or octadecyl methacrylate in the polymer backbone. Both the morphology and the chemistry of the monoliths had a significant effect on the retention and efficiency of the capillary columns. The best resolution of peptidic fragments obtained by digestion of Cytochrome c with trypsin in solution was obtained in a gradient LC-MS mode using a monolithic capillary column of poly(lauryl methacrylate-co-ethylene dimethacrylate) featuring small pores and small microglobules. Raising the temperature from 25 to 60°C enabled separations to be carried out at 40% higher flow rates. Separations carried out at 60°C with a steeper gradient proceeded without loss of performance in half the time required for a comparable separation at room temperature. Our preparation technique affords monolithic columns with excellent column-to-column and run-to-run repeatability of retention times and pressure drops.

Original languageEnglish (US)
Pages (from-to)2814-2820
Number of pages7
JournalJournal of Separation Science
Volume30
Issue number17
DOIs
StatePublished - Nov 1 2007

Keywords

  • Column technology
  • High-performance liquid chromatography
  • Monolithic stationary phase
  • Peptides
  • Temperature

ASJC Scopus subject areas

  • Analytical Chemistry
  • Filtration and Separation

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