Bioanalytical applications of fluorescence line-narrowing and non-line-narrowing spectroscopy interfaced with capillary electrophoresis and high-performance liquid chromatography
Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are now routinely used for analysis of complex biomolecular samples. Difficulties with detection and identification of the targeted analytes, e.g., biomarkers for disease, often arise because of the limited sensitivity and/or selectivity of the detection modality. Identification of an analyte based on its migration time is not definitive because of the possibility of interferences from structurally similar molecules. To circumvent these problems we have developed new methodologies that interface CE and HPLC with fluorescence line-narrowing (FLN) and non-line-narrowing (NLN) spectroscopy for on-line detection and characterization. With these detection methods, one achieves the high sensitivity afforded by laser-induced fluorescence (LIF) (≈attomole) and high selectivity provided by FLN spectra, whose rich and sharp (≈5 cm-1) vibrational structure serves as fingerprints. Fluorescence line-narrowing is operative at temperatures close to 4 K. Thus, a major challenge was the design of compact cryostats that are compatible with the above separation techniques and allow for rapid cooldown (≈1 minute). After cooldown the stationary analyte plugs can be interrogated indefinitely, which leads to significantly lower detection limits. Photodegradation of the analytes is also markedly reduced relative to laser excitation at room temperature. Automated translation of the cryostat/capillary relative to the spatially fixed laser excitation beam allows for sequential analysis of the separated analytes. Lower resolution but still informative analyses can be performed at 77 K.;Results are presented for depurinating polycyclic aromatic hydrocarbons adducted to DNA isolated from the urine of humans exposed to coal smoke, and from the skin of mice treated with the most potent chemical carcinogen, dibenzo[a,l]pyrene. We anticipate that combining FLN and NLN detection with CE and HPLC will be useful in many more bioanalytical applications.