Study of the potentialities of a Laser Induced Native Fluorescence Detector for Capillary Electrophoresis
Résumé
Capillary Electrophoresis (CE) is a miniaturised separation technique that has high potentialities for the analysis of biological matrices. However, in some cases, the detector sensitivity is not sufficient and a sample concentration must be performed before the analysis. Another way to overcome detection problems is to use laser induced fluorimetric detection that allows us to improve the detection limits obtained with classical UV detection. Nevertheless, a derivatization step is generally necessary before separation because, at native state, only a few solutes possess a fluorophoric moiety that can be excited by commercially available visible lasers. Unfortunately, this derivatization may be difficult because of the low sample volume. Consequently, it may be interesting to perform laser induced detection by using the newly available UV lasers. The latter lead to new potentialities for sensitive detection because a lot of solutes have a fluorophoric moiety that can be excited in UV range. In this work, we studied the opportunities offered by such lasers, and, in particular, by a 224 nm Ag-He laser integrated in a commercial detector. After studying the response linearity of the detector and optimizing the preparation of the detection window, we used this detector in CE. In this approach, we have modified the cartridge containing the separation capillary for performing, successively on the same analysis, the laser induced native fluorescence detection (LINFD) and, secondly, a classical UV detection. In this original configuration, we demonstrated that LINFD enabled us to improve the detection sensitivity by a factor 50 to 500 compared to absorption UV detection for reduced immunoglobulin G and ibuprofen respectively.