Temperature gradient focusing for microchannel separations

Microfluidic devices are currently evolving beyond a concept technology into a mature field, with examples already evident in commercial applications. Microchips employing free solution electrophoresis have benefited from over 20 years of development in capillary electrophoresis (CE) aimed at enhancing selectivity and sensitivity. Like conventional CE, these devices suffer from relatively poor detection limits because of the extremely small quantities of analyte that are injected onto the separation column and the very short (10–100 μm) optical detection path lengths. These issues become exacerbated in microfluidics, which can have integrated injection schemes allowing for even smaller injection plugs than traditionally afforded, as well as exceedingly small cross-sectional channel areas (for a review, see [1]). Additionally, many of the microchip substrate materials, whether glass-based or polymeric, can have high backgrounds for fluorescence or other optical detection methods and so degrade detection limits relative to fused silica employed in conventional CE. Sensitivity enhancements have been realized through instrumental detection improvements, novel detection geometries, and detection methods other than fluorescence or absorbance. Several improvements in sample preconcentration prior to the separation step have also evolved to improve detection limits, including sample stacking methods, sweeping methods, and isotachophoresis, as well as chromatographic methods. Another group of analyte concentration methods have been termed equilibrium gradient focusing methods, with the most prevalent example in microchannels being isoelectric focusing, where the separation arises from variations in analyte isoelectric points along a pH gradient. Gradient methods combine concentration and separation steps by forcing analytes to a unique equilibrium point along the separation axis; analytes focus at their unique point of null velocity, with the separation based upon analytes having differing equilibrium points. Due to the nature of the focusing, peaks become both narrower and more concentrated throughout the separation, allowing for high resolution and sensitivity. A novel new equilibrium method was introduced for proteins by Koegler and Ivory using a variable electric field [2, 3]. Electric field gradient focusing (EFGF) has been further developed in recent years by the groups of Lee and Woolley [4, 5], as well as by Myers and Bartle [6]; at present the method has been successfully integrated into microfluidic devices [7–9]. As an alternative to pH or electric field gradients, a temperature gradient can be employed, as has been shown with temperature gradient focusing (TGF), which is based upon balancing analytes’ electrophoretic mobilities against a bulk flow containing both hydrodynamic and electroosmotic flow components (Fig. 1). TGF has been applied to a wide variety of analytes, including proteins, DNA, small dye molecules, and amino acids, in both capillary and microfluidic formats (Fig. 2; [10–16]; Kamande MW, Ross D, Locascio LE, Warner IM, 2006, submitted to Anal Chem; Huber DE, Santiago JG, 2006, submitted to Electrophoresis). Anal Bioanal Chem (2007) 387:155–158 DOI 10.1007/s00216-006-0913-4