Optimisation of probe concentration in indirect photometric detection in capillary electrophoresis using highly absorbing dyes
Johns, CA and Macka, M and Haddad, PR, Optimisation of probe concentration in indirect photometric detection in capillary electrophoresis using highly absorbing dyes, Electrophoresis, 23, (1) pp. 43-48. ISSN 0173-0835 (2002) [Refereed Article]
In indirect photometric detection in capillary electrophoresis, the concentration of the absorbing probe ion in the background electrolyte should be as high as possible in order to increase the dynamic range of the detection method. For relatively low absorptivity probes (ε < 2000 L mol-1cm-1) used under typical conditions (75 μm ID capillary) the maximum probe concentration is normally limited by the separation current. However, for medium (ε 2000-15 000 L mol-1cm-1) and especially for high (ε > 15 000 L mol-1cm-1) absorptivity probes such as dyes, the maximum concentration may be limited by the background absorbance of the electrolyte which must fall within the linearity range of the detector. In this work, it is shown that another practical factor limiting the probe concentration is the adsorption of probe onto the capillary wall at higher concentrations, resulting in unstable baseline and increased noise. Use of a zwitterionic surfactant to suppress adsorption enabled the concentration of a model probe anion (tartrazine) to be increased by a factor of six times (to 3 mM). This resulted in significant improvements in peaks shapes, resolution between peaks, detection sensitivity and linear calibration range for the analyte anions. Baseline separation of a test mixture was maintained up to 7.5 mM total concentration of sample coions injected (13.7 nL) for the 3 mM electrolyte, with detection limits ranging from 0.63 to 0.94 μM. Peak height reproducibility (over 20 consecutive injections) was improved (values ranging from 1.1 to 1.9%) compared with electrolytes containing lower concentrations of the probe. Overall, the optimised, higher concentration probe electrolyte provided the sensitivity benefits of highly absorbing probes with the additional benefits of ruggedness and improved stacking, peak shapes and resolution.