Understanding the Complexity of Porous Graphitic Carbon(PGC) Chromatography: Modulation of Mobile-Stationary Phase InteractionsOvercomes Loss of Retention and Reduces Variability

摘要

Porous graphitic carbon (PGC) is an important tool in a chromatographer’s armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandemmass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolitesextracted from tumor tissue, we demonstrate reproducible chromatographyon three PGC columns of different ages. This approach simplifies useof the PGC to the same level as that of a C-18 column, removes theneed for column regeneration, and minimizes run times, thus allowingPGC columns to be used to their full potential.