Abstract
Many modem analytical methods deal with the trace-level determination of compounds of interest in highly complex biomedical and environmental samples by means of chromatographic techniques. The introduction of a "clean" sample into an analytical instrument can make analyses easier and prolongs equipment life. The use of solid-phase extraction (SPE) has grown and is a fertile technique of sample preparation as it provides better results than those produced by liquid-liquid extraction (LLE). The application of SPE can give selectivity of extraction providing a purified and concentrated extract. In the field of SPE techniques, trace enrichment and sample clean-up via the use of bonded silica cartridges and immobilised antibodies are discussed. SPE using bonded silica has been optimised with respect of sample pH, sample concentration, elution solvent strength, sample volume, and elution volume. In this investigation a variety of non-polar sorbent cartridges were also screened. The octadecyl bonded silica cartridge (C18) has proven successful in simplifying sample preparation. Although bonded phases are commercially available, and can be useful for broad-range screening, they do not have a good selectivity. The use of immuno-extraction procedure as a novel solid phase extraction system utilizing immobilised antibodies has been evaluated. In the present work, it was possible to use antisera covalently bonded as a selective SPE and enrichment method. Several feature of sample preparation especially retention and elution processes are evaluated and the general applicability of the system is also demonstrated. A new approach in the present method proved that chlortoluron and isoproturon could be retained on immuno-columns based on specific analyte-antibody interaction. Further study employed a buffer solution of PBS and ethanol to extract the analytes selectively from a variety of spiked matrices and gave a clean sample for HPLC-UV. A comparison study showed that, the retention was not occurring if activated silica and non-immune antibody were used. The method enabled the pre-concentration of a large sample volume of water (1000 ml) followed by elution of the analytes in 1 ml of eluent. The effect of different eluents, elution solvent pH, sample pH, sample concentration, and sample flow-rate were also optimised. A mass range of 5 to 2000 ng for chlortoluron and 5 to 500 ng for isoproturon could be retained and eluted, resulting in a very low detection limit using a large sample volume. The specificity of the immuno-columns towards closely related compounds have also been evaluated. The columns proved to be selective if different groups of compounds are applied. They were reusable for more than sixty times without losing their bioactivity. The method were optimised for water and showed good accuracy and precision day-to-day and within-day. They were validated with 3 different pools of spiked samples and good reproducibility over 6 consecutive days. The feasibility of using antibodies as a tool in sample preparation is now encouraging further study in environmental and biological analysis.