Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence

Limited by the lack of a sensitive, universal detector, many capillary-based liquid-phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid-phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti-biotin-IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser-induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC-biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC-biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).