Novel and sensitive CE methods coupled with LIF and LINF detection using a series of separation modes have been developed for profiling organic metabolites containing amines and carbohydrates in mammalian and plant biofluids. In this study, metabolites containing an amine group were derivatized with 4-Fluoro-7-nitro- benzofurazan (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection (LIF). Under the optimized conditions most of the amine-containing metabolites in human biofluids such as plasma, urine and saliva could be identified by reference to standard compounds and the concentrations measured were found to be in agreement with literature values. Furthermore, 17 carbohydrates including mono-, di- and oligosaccharides are also simultaneously derivatized via a two-step reaction involving reductive amination with ammonia followed by condensation with NBD-F. Under the optimized derivatization conditions all carbohydrates were successfully derivatized within 2.5 h and separated within 15 min using borate buffer (90 mmol Lā1, pH 9.2). The method was applied to measure sugars in nanoliter volume samples of phloem sap obtained by stylectomy from wheat and to honeydew samples obtained from aphids feeding from wheat and willow. Finally, an on-line sample concentration technique, sweeping-micellar electrokinetic chromatography (sweeping-MEKC), in conjunction with UV laser-induced native fluorescence detection (LINF) was developed and applied to the detection of native fluorescent analytes in biofluid samples such as plant phloem sap, human plasma and urine samples. The concentration limits of detection of analytes were in the range 7ā100 nmol Lā1, which were 250ā3600-fold improvement for dopamine, DOPA and epinephrine compared with conventional capillary zone electrophoresis (CZE). The results indicated that a long-term limitation of relatively low detection sensitivity in CE-UV analysis arisen from the small injection volume and short optical path-length could be much improved, while no apparent loss in separation efficiency occurred.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:508149 |
| Date | January 2009 |
| Creators | Tseng, Hua-Ming |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/10873/ |
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