Retention and effective diffusion of model metabolites on porous graphitic carbon.

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 this work 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 >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.

Recent advances in capillary ultrahigh pressure liquid chromatography.

In the twenty years since its initial demonstration, capillary ultrahigh pressure liquid chromatography (UHPLC) has proven to be one of most powerful separation techniques for the analysis of complex mixtures. This review focuses on the most recent advances made since 2010 towards increasing the performance of such separations. Improvements in capillary column preparation techniques that have led to columns with unprecedented performance are described. New stationary phases and phase supports that have been reported over the past decade are detailed, with a focus on their use in capillary formats. A discussion on the instrument developments that have been required to ensure that extra-column effects do not diminish the intrinsic efficiency of these columns during analysis is also included. Finally, the impact of these capillary UHPLC topics on the field of proteomics and ways in which capillary UHPLC may continue to be applied to the separation of complex samples are addressed.

Evaluation of preparative hydrodynamic chromatography of silica stationary phase supports.

Reducing the particle size distribution (PSD) of sub-2 µm chromatographic packing materials can improve the performance of capillary UHPLC columns, but several size refinement methods are only partially effective in this size range. To this end, a preparative scale hydrodynamic chromatography (HDC) method was developed to size-refine C18 functionalized sub-2 µm particles, but suffered from poor reproducibility and particle aggregation issues. Presented here are improvements based on the use of an ammonium hydroxide as the mobile phase. This mobile phase makes the method reproducible, decreases column conditioning requirements, and focuses on the preparation of bare silica material which allows for a wider variety of stationary phase bondings. Additionally, particle recovery for both non-porous silica size standards and bridged-ethyl hybrid (BEH) particles are detailed to highlight the advantages of this method. The data presented demonstrates the capability of this method to reduce the relative standard deviation (RSD) of the PSD of BEH particles by 33% in under 2 h with sufficient yield to pack several capillary columns.