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Temperature Gradient Focusing with Field-Amplified Continuous Sample Injection for Dual-Stage Analyte Enrichment and Separation

Matthew S. Munson,* Grégoire Danger, Jonathan G. Shackman, and David Ross

Biochemical Sciences Division, National Institute of Standards and Technology, 100 Bureau Drive, MS 8313, Gaithersburg, Maryland 20899

Anal. Chem., 2007, 79 (16), pp 6201–6207

DOI: 10.1021/ac070689r

Publication Date (Web): July 7, 2007

Author to whom correspondence should be addressed. Phone: 301-975-4125. Fax: 307-975-8246. E-mail: matt.munson@nist.gov.

Abstract

We describe the serial combination of temperature gradient focusing (TGF) and field-amplified continuous sample injection (FACSI) for improved analyte enrichment and electrophoretic separation. TGF is a counterflow equilibrium gradient method for the simultaneous concentration and separation of analytes. When TGF is implemented with a low conductivity sample buffer and a (relatively) high conductivity separation buffer, a form of sample enrichment similar to field-amplified sample stacking (FASS) or field-amplified sample injection (FASI) is achieved in addition to the normal TGF sample enrichment. FACSI-TGF differs from FASI in two important respects: continuous sample injection, versus a discrete injection, is utilized; because of the counterflow employed for TGF, the stacking interface exists in a pseudo-stationary region outside of the separation column. Notably, analyte concentration enrichment factors greater than the ratio of separation and sample conductivities (γ) were achieved in this method. For γ = 6.1, the concentration factor for one model analyte (Oregon Green 488) was found to be 36-fold higher with FACSI-TGF as compared to TGF without FACSI. A separation of five fluorescently labeled amino acids is also demonstrated with the technique, yielding an average enrichment of greater than 1000-fold.

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