Instrumental development of novel detection and separation methods for capillary electrophoresis
Is Version Of
In Section I the use of indirect fluorescence detection (IFD) is extended to the visible region. Indirect fluorescence detection is shown to be a universal and sensitive detection technique for CZE. Detection is based on charge displacement and is not based upon any absorption or emission properties of the analyte, therefore the need for chemical derivitization is eliminated. CZE-IFD can separate and detect almost any compound that contains a charge. This was demonstrated by the separation and detection of a mixture of sugars which are only weakly acidic. The mass detection limit of fructose was 2 fmol using a 5 [mu]m diameter capillary. This peak had an efficiency of more than 600,000 theoretical plates;In Section II a novel detection system for CZE is explored. Normally, only highly fluorescent materials can be detected at low concentrations in the small detection volumes typical of capillary electrophoresis in a laser-based fluorometer. We report here the detection of absorbing but non-fluorescing analytes by laser excited fluorescence. This is possible if the excited analytes transfer their energy to a fluorescent additive in the running buffer to increase the background fluorescence level. Two different fluorophores and four different absorbing analytes were tested in this detection scheme. Concentrations as low as 1.5 x 10[superscript]-8 M and amounts as small as 9 amol at injection are detectable. The data support a long range energy transfer scheme, but the transfer efficiency is much larger than those reported for other donor-acceptor pairs;Section III describes a method for the CZE separation of compounds with similar mobilities. Increased selectivity is shown for compounds of this type when other modes of separation are added to the system. A capillary coated with a hydrophobic stationary phase is shown to be a dynamic ion exchanger when a quaternary ammonium compound is added to the running buffer. Compounds are shown to have a decrease in retention when the concentration of the running buffer is increased. The effect of adding an organic modifier and the effect on the concentration of the surface active reagent is also studied.