Fluorescence microscopy is a powerful technique for analyzing beads with very low loadings of fluorophores; however, the method is flawed when looking at more highly loaded beads as a result of severe problems with absorption. To probe distributions at higher loading levels, Raman spectroscopy avoids many of these issues. These studies show that there is a uniform distribution of reactive sites throughout the beads but that the spatial distribution of reacted sites depends on the polymer type, with a fine balance between reaction and diffusion rate.
Scanning confocal Raman spectroscopy was used to study the distribution of reactive sites within a resin bead used for solid-phase synthesis. The distribution of NH2 groups in aminomethylated polystyrene resin (APS) was determined by doping with varying amounts of 4-cyanobenzoic acid. The extent of loading was determined by both elemental analysis and ninhydrin assays. The spatial distribution of the coupled 4-cyanobenzamide within the bead was determined to an in-plane resolution of 1 microm and depth resolution of about 4 microm, using the strong Raman CN stretching vibrational transition at 2230 cm(-1). Dry and swollen beads were studied and the distribution was found to be essentially uniform throughout the bead in all cases.
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