HOLOGRAM-BASED THERMOOPTICAL ABSORBANCE DETECTION IN CAPILLARY ELECTROPHORESIS - SEPARATION OF NUCLEOSIDES AND NUCLEOTIDES

摘要

On-column thermooptical absorbance (TOA) detection in capillary electrophoretic separations of various nucleoside and mono- and diphosphate nucleotide mixtures absorbing at 257 mn is demonstrated in 20 mu m i.d. capillaries. The analytes are optically pumped by a frequency-doubled argon ion laser and probed by a laser diode or by a He/Ne laser beam guided to the detection volume by a holographic optical element, Absorption detection limits of 2.2 mu AU using time constants of 0.3 s and 20 mW of UV power are obtained over a linear dynamic range covering three to four decades. As higher pumping power is required to enhance the thermooptical sensitivity, photobleaching appears as a major problem in the quest for lower detection limits for some of the substances studied such as deoxyuridine and uridine. Concentration detection limits as low as 50 nM for adenosine monophosphate, corresponding to a mass detection limit of 0.4 fmol, and separation efficiencies up to 320 000 theoretical plates are measured. A theoretical model which translates the obtained TOA signals into absorbances, is proposed and describes the TOA effect for smaller capillaries rather well.