Quantitative temperature gradient focusing performed using background electrolytes at various pH values.
Temperature gradient focusing (TGF) has previously been shown to be a practical technique for simultaneous concentration and separation of a variety of samples. In this paper, we demonstrate that TGF can be conducted at a wide range of pH values. Techniques for first-order prediction of the suitability of a given BGE for focusing are discussed. Buffer suitability for TGF is assessed experimentally by simultaneously concentrating and separating a pair of fluorescent analytes. One analyte is held at constant concentration for use as an internal standard while the concentration of the other dye is varied. Peak area is shown to vary linearly with the input dye concentration. A high degree of resolution (R(s) >3) of fluorescein and carboxyfluorescein, as well as for two LysoSensor-based dyes, is also observed. Foucusing and separation by TGF was successfully conducted quantitatively in BGE solutions of pH from 3.0 to 10.5.
Shackman
Jonathan G
JG
Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8394, USA.
Munson
Matthew S
MS
Kan
Cheuk-Wai
CW
Ross
David
D
D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D013696:Temperature
D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein; D019793:Fluorescein