Highly sensitive indirect photometric detection of anions and cations in capillary electrophoresis
Creators: name: Johns, CA
Publisher: name: University Of Tasmania
Year: 2023

description: The aim of this work was to substantially improve the detection sensitivity and corresponding limits of detection (LODs) of methods for separation of anions and cations by capillary electrophoresis using indirect photometric detection. The approach taken in this project was to employ highly absorbing dyes as indirect detection probes as an effective way to increase the detection sensitivity. The benefits of using dyes as indirect detection probes in electrolytes buffered with isoelectric ampholytic buffers to enhance detection sensitivity have been demonstrated. Electrolytes containing the dyes, tartrazine and naphthol yellow S (˜í¬µ= 21,350 and 23,125 L mol‚ÄövÖ¬™¬¨œÄ cm‚ÄövÖ¬™¬¨œÄ respectively), were properly buffered without the addition of co-ions by the use of the ampholytic buffer histidine at its isoelectric point. The increased absorptivity offered by both dyes in comparison to traditionally used anionic probes was reflected by the very low limits of detection (typically sub ˜í¬¿M) achieved. Excellent peak shapes and separation efficiencies (up to 300,000 theoretical plates) were observed. When electrolytes were buffered with low conductivity buffers, such as an ampholyte at its isoelectric point, the limiting factor regarding the probe concentration was the background absorbance and not the separation current. A detailed study on detection linearity of various CE instruments and detection configurations showed that most modern instruments retained a linear absorbance response far in excess of the absorbance range normally used for indirect photometric detection. The upper detector linearity limit (absorbance at which 95% of sensitivity is retained) of five commercially available instruments fitted with a 75 ˜í¬¿m ID capillary ranged from 0.3 to 1.2 AU. An estimate of effective pathlengths of each system gave values ranging from 49.7 to 64.6 p.m for a 75 ˜í¬¿m ID capillary. Upper detector linearity limits and effective pathlengths of an Agilent Extended Light Path Capillary and High Sensitivity Detection Cell were also determined, with values of 1.49 and 2.19 AU, and 128 and 1068 ˜í¬¿m, respectively being obtained. The effects of increasing the probe concentration whilst still remaining within the linear response range of the detector were investigated. The concentration of tartrazine as probe was increased from 0.5 to 3 mM, which resulted in improved resolution between peaks, enhanced peak shapes, extension of the linear analyte response range and the ability to handle more highly concentrated samples without loss of high detection sensitivity. The combination of high sensitivity and improved stacking effects from the use of dyes at increased concentration were demonstrated by the use of a new dye, Orange G (˜í¬µ= 24,345 L mol‚ÄövÖ¬™¬¨œÄ cm‚ÄövÖ¬™¬¨œÄ) for the analysis of a range of inorganic and small organic anions. After optimisation, a concentration of 4 mM was found to provide the highest sensitivity. Limits of detection with the optimised system varied from 0.22-0.91 ˜í¬¿M. The increased probe concentration resulted in more efficient separations with efficiencies ranging from 128,000-297,000 theoretical plates. A novel approach was used to minimise adsorption problems associated with the use of the dye at high concentrations. A combination of a polyethylenimine coated capillary, addition of a neutral polymer to the BGE and the use of a 50 ˜í¬¿m ID capillary provided flat baselines at high Orange G concentrations. The optimised system was used for the determination of anions present in air filter samples and good agreement was obtained with results from ion chromatography. The use of cationic dyes for the detection of cations is performed even less than for the detection of anions. Based on previous experience and principles gained from work on separation and detection of anions, a new cationic dye, chrysoidine (˜í¬µ= 23,427 L mol‚ÄövÖ¬™¬¨œÄ cm‚ÄövÖ¬™¬¨œÄ) was investigated. In conjunction with the use of 2-hydroxyisobutyric acid (HIBA) and lactic acid as complexing agents, a mixture of alkaline earth metals, transition metals and lanthanides was separated with limits of detection from 0.22-0.61 ˜í¬¿M (for HIBA) and 0.12-1.43 ˜í¬¿M (for lactic acid). The use of complexing agents with cationic dyes had not been previously reported. Separation efficiencies ranged from 40,000 to 153,000 theoretical plates.