An Investigation of a Novel Monolithic Chromatography Column, Silica Colloidal Crystal Packed Columns

Abstract

Many researchers have investigated ways to improve the separation power of conventional chromatography, most notable is the development of ultra-high performance liquid chromatography (UHPLC). However, only slight improvements in separation efficiency have been achieved up to this point, and unfortunately, modern reversed phase liquid chromatography (RPLC) methods do not have high enough resolving power to analyze complex proteomic mixtures.Uniformly sized silica particles from 10 nm to 1 micron are known to self-assemble into a highly ordered face centered cubic crystal. Silica colloidal crystals have shown recent promise in biological applications such as permselective nanoporous membranes, DNA sieving, reversed phase separation of small molecules on planar substrates, protein sieving, microarrays, total internal reflection fluorescence microscopy of live cells, and 3-D scaffolds for supported lipid films. In this work, silica colloidal crystals packed in capillaries are explored for their potential improvement in the efficiency of reversed phase chromatography.The silica colloidal crystal columns were chemically stabilized by with trichlorosilanes. The trichlorosilanes form chemical bonds between the particles and the particles and the substrate creating an increase in mechanical stability, and at the same time, providing an excellent chromatographic monolayer. After stabilization the fritless columns were able to withstand the pressure limit of the commercial UHPLC. Next, the columns were characterized using a small dye molecule, 1,1' - Didodecyl - 3,3,3',3' - tetramethylindocarbocyanine (DiIC12). The dye was run under capillary electrochromatography (CEC), and sub-micron plate heights were achieved. Further, a van Deemter plot of the dye molecule indicates that the plate height is largely due to the molecule's diffusion. This result suggests that the plate heights for proteins would be even smaller, since proteins have diffusion coefficients an order of magnitude smaller. The analysis of proteins by CEC yielded nanometer plate heights. Finally, pressure driven flow separations coupled with nano-electrospray ionization (n-ESI) MS have also been explored. The Poiseuille flow profile has been shown not to perturb the low plate heights. Gradient elution of peptides was also achieved, and the results demonstrate the highest chromatographic peak capacities for short analysis times to date.