Sensitive Detection and Separation of Fluorescent Derivatives using Capillary Electrophoresis with Laser-Induced Fluorescence Detection with 532 nm Nd:YAG Laser
| Authors | |
|---|---|
| Year of publication | 2006 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Luminescence |
| MU Faculty or unit | |
| Citation | |
| Web | http://www.elsevier.com/wps/find/journaldescription.cws_home/505700/description?navopenmenu=-2 |
| Field | Analytic chemistry |
| Keywords | Electrophoresis; Fluorescence; Derivatization; RBITC; Proteins; Rhodamine; 532 nm Laser. |
| Description | Capillary Electrophoresis with Laser Induced Fluorescence detection (CELIF) is a powerful tool for separation and sensitive determination of fluorescent compounds. Biologically active compounds, such as amino acids, peptides and proteins may exhibit native fluorescence, which is however often low and/or an expensive laser is required for excitation in UV. Therefore, labeling of the analytes with a fluorescent dye is usually necessary. In this work, home-built CELIF instrument was developed and tuned up using rhodamine-based dyes with diode pumped frequency-doubled continuous wave Nd:YAG excitation laser with feedback power regulation (532 nm). First, the suitability and performance of this type of laser for LIF detection in a separation method was evaluated. Detection limit of 2E-13 M was achieved with rhodamine B, which is comparable to previously described CELIF with Ar+ laser. An exemplary separation of rhodamine dyes is given. Rhodamine based derivatizing agent was chosen for labeling of aminoacids and proteins with derivatization techniques suggested by several groups for fluorescein-based dyes. Derivatization conditions such as solvent, pH, time and temperature of derivatization as well as separation conditions were optimized and examples of electropherograms are presented. |
| Related projects: |