Near-infrared laser induced fluorescence as an ultra sensitive method of detection for capillary electrophoresis

This dissertation describes the advantages and feasibility of using near-infrared laser induced fluorescence as an ultra sensitive method of detection for capillary electrophoresis (CE). Instrumental advantages of working in the near-infrared region and availability of fluorescent labels suitable for use with popular CE modes are demonstrated.;A simple and reliable interface was designed for an avalanche photodiode based, laser induced fluorescence (LIF) detector and a commercial automated CE instrument. The limit of detection (SNR = 3) for a NIR labeling dye (NN382) was 80 zeptomole (10-21mole) injected on-column, using 100% aqueous conditions. Detector sensitivity was improved about 400 fold in the NIR region around 820 nm compared to a commercial PMT based LIF detector.;NIR fluorescent dye NN382 was evaluated as an ultra sensitive peptide-labeling reagent for use with CE. Six Angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related deca-peptides were labeled with the NIR dye, separated using CE, and detected by NIR LIF. Derivatization of the peptides was achieved under aqueous conditions using 2.5 to 500 pmol of Ang-I in a 50 muL sample (5 x 10-8 to 1 x 10-5 M). The fluorescence response was linear over a 200 fold range (R ≥ 0.9986) and the SNR of a 1.3 amol injection of the Ang-I variants was ≥15, indicating a limit of detection (SNR = 3) of ≤240 zmol. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis. Two pairs of co-eluting peptides were successfully resolved using micellar electrokinetic chromatography. Two neutral NIR dyes were identified as suitable markers for measuring electroosmotic and micellar flow.;Capillary gel electrophoresis (CGE) with LIF detection was utilized to assess the purity of NIR labeled oligonucleotides, The CGE method showed great sensitivity, with limits of detection around 90 zeptomole, and a 1000 fold linear dynamic range. The number of theoretical plates obtained for the primers exceeded 300,000. The excellent sensitivity and high separation efficiency observed for the NIR labeled primers indicate their suitability for many other CGE applications.