HOLOGRAM-BASED THERMOOPTIC ABSORBENCY DETECTION IN CAPILLARY ELECTROPHORESIS - SEPARATION OF NUCLEOSIDES AND NUCLEOTIDES

On-column thermooptical absorbance (TOA) detection in capillary electr ophoretic 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-double d 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, Abso rption 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 t hree to four decades. As higher pumping power is required to enhance t he thermooptical sensitivity, photobleaching appears as a major proble m in the quest for lower detection limits for some of the substances s tudied such as deoxyuridine and uridine. Concentration detection limit s as low as 50 nM for adenosine monophosphate, corresponding to a mass detection limit of 0.4 fmol, and separation efficiencies up to 320 00 0 theoretical plates are measured. A theoretical model which translate s the obtained TOA signals into absorbances, is proposed and describes the TOA effect for smaller capillaries rather well.