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Studies on indirect absorbance detection of anions by capillary electrophoresis


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Doble, Philip (1998) Studies on indirect absorbance detection of anions by capillary electrophoresis. PhD thesis, University of Tasmania.

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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

Item Type: Thesis (PhD)
Keywords: Anions, Capillary electrophoresis
Copyright Holders: The Author
Copyright Information:

Copyright 1998 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 1998. Includes bibliographical references

Date Deposited: 09 Dec 2014 00:04
Last Modified: 20 Jul 2016 00:33
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