Journal
JOURNAL OF SEPARATION SCIENCE
Volume 30, Issue 17, Pages 2814-2820Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.200700185
Keywords
column technology; high-performance liquid chromatography; monolithic stationary phase; peptides; temperature
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Funding
- NIGMS NIH HHS [R01 GM044885, R01 GM044885-15, GM44885] Funding Source: Medline
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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 methaciylate-co-etllylene dimethacrylate) featuring small pores and small microglobules. Raising the temperature from 25 to 60 degrees C enabled separations to be carried out at 40% higher flow rates. Separations carried out at 60 degrees 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.
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