Studies on indirect absorbance detection of anions by capillary electrophoresis
Creators: name: Doble, Philip
Publisher: name: University Of Tasmania
Year: 2023

description: This work presents studies on separations and indirect absorbance detection of anions by capillary electrophoresis. Transfer ratios (i.e. the number of moles of the UV-absorbing probe anion displaced by one mole of analyte anion) were determined for the separation of inorganic and organic anions by capillary zone electrophoresis using indirect UV absorbance detection. When the electrolyte was buffered and contained only the probe anion and a single counter-cation, transfer ratios calculated from Kohlrausch theory were found to agree well with values obtained experimentally from accurately determined mobility data. Background electrolytes (BGEs) containing multiple co-anions were investigated as possible means to control peak symmetries and improve the sensitivity of indirect detection in the separation of a mixture of inorganic and organic anions having a range of electrophoretic mobilities. In general, it was found that an analyte mainly displaced the BGE component to which its mobility was closest, and exclusively displaced any BGE component having the same mobility. This behaviour was utilised to design BGEs containing multiple probes so as to improve peak shapes by matching the mobilities of the BGE components with those of the analytes. System peaks were observed for each multiple component BGE and for n BGE co-anions, n-1 system peaks were induced. A simple linear function relating the mobility of the system peak for a two co-anion BGE to the mobilities and relative concentrations of each of the co-anions was derived empirically. High sensitivity for indirect detection was achieved by utilising highly absorbing species as the displaced co-ion. Two highly absorbing dyes, bromocresol green and indigo tetrasulfonate, were investigated as potential probes in the determination of small organic and inorganic anions. Minimal detectable amounts were in the low attomole region (1 x 10 -18 mole), corresponding to sub 1.1.M vacuum injected solution concentrations. These detection limits were an order of magnitude lower than the general detection limit reported for indirect photometric detection, and were comparable with detection limits achieved with indirect fluorescence detection. Two approaches for buffering indirect electrolytes were investigated using chromate as a model system. The first system utilised a suitable buffering base as a counter-ion of the chromate. Two buffered electrolytes were investigated containing tris(hydroxymethyl)aminoethane (Iris, pKa=8.5) or diethanolamine (pKa=9.2). The analytical performance characteristics of the Tris buffered electrolyte were compared with the unbuffered chromate electrolyte. Both systems showed similar separation selectivity, efficiency and detection sensitivity, but the buffered electrolytes showed superior repeatability for migration times and peak areas, as well as a significantly greater tolerance to alkaline sample matrices. The second buffering system investigated the suitability of the relatively slow (low absolute value of mobility) co-anionic buffer 2-(cyclohexylamino)- ethanesulfonic acid (CHES). Within its useful pH buffering range CHES acted as a competing probe co-anion. System peaks were induced which had deleterious effects on detection sensitivity of low to medium mobility anions. The mobility of the system peak was determined by the effective mobility of CHES, and increased with increasing pH. The peaks of analytes that migrated near or on the system peak were distorted and lost all quantitative properties. Analytes that migrated after the system peak either were not detected or exhibited a detector response which was reversed in direction. Analytes that migrated well before the system peak were unaffected.