Separation of Metabolites

The study of metabolites in biological samples is of high interest for a wide range of biological and pharmaceutical applications. Reversed phase liquid chromatography is a common technique used for the separation of metabolites, but it provides little retention for polar metabolites. An alternative to C18 bonded phases, porous graphitic carbon has the ability to provide significant retention for both non-polar and polar analytes.

The goal of research from the Jorgenson Group, published in the Journal of Chromatography A, is to study the retention and effective diffusion properties of porous graphitic carbon, to see if it is suitable for the wide injection bands, and long run times associated with long, packed capillary-scale separations.

The retention of a set of standard metabolites was studied for both stationary phases over a wide range of mobile phase conditions. This data showed that porous graphitic carbon benefits from significantly increased retention, often more than 100-fold, under initial gradient conditions for these metabolites, suggesting much improved ability to focus a wide injection band at the column inlet.

The effective diffusion properties of these columns were studied using peak-parking experiments with the standard metabolites under a wide range of retention conditions. Under the high retention conditions, which can be associated with retention after injection loading for gradient separations, Deff/Dm ∼ 0.1 for both the C18-bonded and porous graphitic carbon columns.

As C18 bonded particles are widely, and successfully utilized for long gradient separations without issue of increasing peak width from longitudinal diffusion, this suggests that porous graphitic carbon should be amenable for long runtime gradient separations as well.