Microsecond analysis of transient molecules using bi-directional capillary electrophoresis.
We demonstrate the feasibility for minimizing electrophoretic analysis times of transient chemical species by inducing nascent, oppositely charged photochemical products to migrate in opposite directions from their point of creation. In this approach, separate probe sites are positioned within an electrophoretic channel both upfield and downfield from a photoreaction site formed by high-numerical-aperture optics, with positively charged (and in some cases neutral) components migrating toward one probe site and negatively charged species migrating in the opposite direction, toward the second probe site. As a proof-of-concept, fluorescent photoproducts of the hydroxyindoles, 5-hydroxytryptamine (serotonin), 5-hydroxytrptophan, and 5-hydroxyindole-2-carboxylic acid, are formed within a geometrically modified capillary and are transported electrophoretically and electroosmotically to probe sites several micrometers away. Although it is possible to detect all components in a single channel, or to use a two-channel imaging approach to independently detect positive and negative components, we have found the most rapid analysis approach involves a protocol in which laser light is alternately directed to opposing probe sites at high frequency (1 kHz), a strategy that allows positive and negative species to be detected with no cross-talk, even when components have overlapping detection times. Fluorescence-signal-averaging is performed on each temporal channel via summation of the two sequences of interdigitized electrophoretic traces. This approach allows photoproducts to be detected free from interferences from oppositely charged species, enabling positive and negative species in a mixture to be analyzed electrophoretically in ca. 6 micros, a period several-fold faster than was previously feasible using unidirectional electrophoresis.
Ritschdorff
Eric T
ET
Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, USA.
Plenert
Matthew L
ML
Shear
Jason B
JB
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007211:Indoles / Q000737:chemistry*; D012701:Serotonin / Q000737:chemistry*; D013997:Time Factors
D005456:Fluorescent Dyes; D007211:Indoles; C026785:5-hydroxyindole; D012701:Serotonin; D006916:5-Hydroxytryptophan