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

• Published in: Electrophoresis 1999-01, Vol.20 (1), p.57-66
• Main Authors: Thomas, David H., Rakestraw, David J., Schoeniger, Joseph S., Lopez-Avila, Viorica, Van Emon, Jeanette
• Format: Article
• Language: English
• Subjects: Buffers; Capillary; Chromatography, Affinity - methods; Chromatography, Liquid - methods; Electrochromatography; Electrophoresis, Capillary - methods; Evaluation Studies as Topic; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescent Dyes; Immunoaffinity; Lasers; Miniaturization
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/(SICI)1522-2683(19990101)20:1<57::AID-ELPS57>3.0.CO;2-J

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).