Temperature gradient focusing with field-amplified continuous sample injection for dual-stage analyte enrichment and separation.
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 (gamma) were achieved in this method. For gamma=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.
Munson
Matthew S
MS
Biochemical Sciences Division, National Institute of Standards and Technology, 100 Bureau Drive, MS 8313, Gaithersburg, Maryland 20899, USA. matt.munson@nist.gov
Danger
Grégoire
G
Shackman
Jonathan G
JG
Ross
David
D
D002021:Buffers; D004553:Electric Conductivity; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D013696:Temperature
D002021:Buffers