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


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Authors
Bapiro, Tashinga E 
Richards, Frances M 
Jodrell, Duncan I 
Abstract

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/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.

Description
Keywords
0301 Analytical Chemistry
Journal Title
Anal Chem
Conference Name
Journal ISSN
0003-2700
1520-6882
Volume Title
88
Publisher
American Chemical Society (ACS)
Sponsorship
Cancer Research UK (CB4270)
This work was funded by the Cancer Research UK Cambridge Institute (Grant NO. C14303/A17197).