Capillary zone electrophoresis with time-resolved laser-induced fluorescence detection

Karen J Miller, Purdue University

Abstract

Due to high separation efficiency and low sample volume requirements, capillary zone electrophoresis (CZE) has become an established analytical tool in the decade since its introduction. Among the many detection methods used in CZE, laser-induced fluorescence (LIF) has achieved some of the best detection levels. When applying LIF detection to CZE, background signal caused by laser scatter from the capillary walls can be a significant problem. By using a pulsed laser source and time-gated detection, the collection of this scatter can be reduced. Time-resolved detection can also resolve short-lived interferences in biological sample matrixes from long-lived fluorophores. A CZE system with time-resolved LIF detection was developed using a diode-pumped solid-state laser. The detection limit for the new instrument was $10\sp{-9}$ M for 4-methoxy-$\beta$-naphthylamine, a long-lived fluorophore. The instrument was applied to the detection of leucine aminopeptidase, a clinically significant enzyme, in biological matrixes. Results for serum and urine were within the ranges of expected values found in the literature. A low concentration of $6\times 10\sp{-13}$ M enzyme in buffer was detected. The CZE-based analysis was faster and consumed far less sample than the conventional photometric method. The instrument was also used to profile the aminopeptidase enzymes of immobilized yeast cells. The cells were trapped on the capillary using a mid-capillary frit of novel construction. Profiles were obtained using just 500 yeast cells. The new CZE-based profiling method was found to have several significant advantages over the older cuvette/fluorometer method.

Degree

Ph.D.

Advisors

Lytle, Purdue University.

Subject Area

Analytical chemistry

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