(212d) Electric Field Gradient Focusing In Monolithic Columns Using Nonfouling Materials

The need to improve methods for the fractionation of biomolecules in complex mixtures continues to grow. Electric field gradient focusing (EFGF) is a developing technique that allows both separation and enrichment of species such as proteins. EFGF utilizes an electric field gradient combined with laminar flow in the opposite direction to focus molecules into discrete fractions based on their electrophoretic mobilities. We have been evaluating EFGF systems having open capillaries embedded in an ionically conductive polymer, which has a cross-sectional area that varies along the length of the focusing channel [1]. These focusing devices can separate proteins and enrich them by over 10,000-fold. We have also been applying microfabrication methods to construct EFGF systems with smaller channel dimensions, leading to reduced band broadening [2]. Moreover, we have optimized procedures for the fabrication of EFGF systems in poly(ethylene glycol)-based materials, which provide greatly reduced nonspecific adsorption of protein analytes [3]. Finally, we are polymerizing monolithic beds within the focusing column to reduce the band dispersion due to the laminar flow profile. Monolith-filled focusing channels offer enhanced protein separation performance relative to open capillaries of the same dimensions. These improved systems will enable greater capabilities and broader application of EFGF in the analysis of proteins.

References

[1] Humble, P.H; Kelly, R.T.; Woolley, A.T.; Tolley, H.D.; Lee, M.L. Anal. Chem. 76, 5641-5648 (2004).

[2] Kelly, R.T.; Li, Y.; Woolley, A.T. Anal. Chem. 78, 2565-2570 (2006).

[3] Liu, J.; Sun, X.; Lee, M.L. Anal. Chem. 79, 1926-1931 (2007).