Premium
On‐line coupling of size‐exclusion chromatography and capillary zone electrophoresis via a reversed‐phase C18 trapping column for the determination of peptides in biological samples
Author(s) -
Stroink Thom,
Schravendijk Pim,
Wiese Gerard,
Teeuwsen Jan,
Lingeman Henk,
Waterval Joop C. M.,
Bult Auke,
de Jong Gerhardus J.,
Underberg Willy J. M.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390132
Subject(s) - chromatography , chemistry , capillary electrophoresis , elution , size exclusion chromatography , capillary action , detection limit , reproducibility , acetonitrile , analytical chemistry (journal) , phase (matter) , electrophoresis , materials science , composite material , enzyme , biochemistry , organic chemistry
Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size‐exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm×3 mm ID reversed‐phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart‐cut of 200 μL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 μL plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 μg/ mL with an injection volume of 50 μL).