Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) are enabling rapid and highly efficient separations. Various analytical strategies have been proposed, for example the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 microm superficially porous particles (fused core) or with sub-2 microm porous particles for use in ultra-high-pressure LC (UHPLC). The purpose of this review is to describe and compare these approaches in terms of throughput and resolving power, using kinetic data gathered for compounds with molecular weights ranging between 200 and 1300 g mol(-1) in isocratic and gradient modes. This study demonstrates that the best analytical strategy should be selected on the basis of the analytical problem (e.g., isocratic vs. gradient, throughput vs. efficiency) and the properties of the analyte. UHPLC and fused-core technologies are quite promising for small-molecular-weight compounds, but increasing the mobile phase temperature is useful for larger molecules, for example peptides.
In this paper, a new fluorescence polarization (FP) assay strategy is described reporting the first demonstration of a noncompetitive FP technique dedicated to the small molecule sensing. This approach was based on the unique induced-fit binding mechanism of nucleic acid aptamers which was exploited to convert the small target binding event into a detectable fluorescence anisotropy signal. An anti-L-tyrosinamide DNA aptamer, labeled by a single fluorescent dye at its extremity, was employed as a model functional nucleic acid probe. The DNA conformational change generated by the L-tyrosinamide binding was able to induce a significant increase in the fluorescence anisotropy signal. The method allowed enantioselective sensing of tyrosinamide and analysis in practical samples. The methodology was also applied to the L-argininamide detection, suggesting the potential generalizability of the direct FP-based strategy. Such aptamer-based assay appeared to be a sensitive analytical system of remarkable simplicity and ease of use.
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