Porous Graphitic Carbon for Separations of Metabolites

Abstract

The analysis of the low molecular weight metabolites produced from cellular activity has a broad range of applications in systems biology. One method used to characterize these complex samples is liquid chromatography-mass spectrometry. Traditionally, reversed phase separations using n-octadecyl (C18) bonded silica stationary phases have been used for metabolite samples. Human metabolite samples contain a significant population of polar metabolites, which are not well retained on reversed phase columns. As an alternative to bonded silica, porous graphitic carbon (PGC) stationary phases have been shown to be useful for the analysis of polar and non-polar solutes. PGC offers alternative retention mechanisms combining dispersion interactions with electrostatic interactions. Experiments here explore the applicability of PGC for separations of metabolites using long packed capillary columns. When moving to capillary scale separations, it is common to use large volume injections relative to the column volume. Due to the lack of retention on C18 bonded silica, these injections will cause polar metabolites to elute as broad peaks early in the gradient. PGC offers significantly increased retention for model metabolites when compared to C18 bonded silica, and PGC also would be suitable for focusing large volume injections. When performing gradient separations with long capillary columns run times can be very long. If a solute has rapid diffusion in the stationary phase during these runs, the bands could elute as broad peaks. It was found that PGC possesses a low level of stationary phase diffusion across a range of temperature and retention conditions, allowing for sharp peaks to elute even after long gradients. Using gradient prediction models, it was found that experimental PGC gradient separations behaved as predicted based on retention data. Long capillary columns packed with PGC particles produce less efficient columns than C18 bonded silica. As the main interest in PGC was its ability to retain polar metabolites, this was not of particular concern. Application of these columns to liquid chromatography-mass spectrometry of urinary metabolites showed that PGC offered increased peak capacity, largely due to the ability to separate the metabolites over a significantly wider range of mobile phase strengthsDoctor of Philosoph