Colorimetric determination of reactive primary amino groups of macro- and microsolid supports |
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Authors: | Philemon E. Tyllianakis Sotiris E. Kakabakos Gregory P. Evangelatos Dionyssis S. Ithakissios |
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Affiliation: | 1. Institute of Radioisotopes and Radiodiagnostic Products, Radioimmunochemistry Laboratory, N.C.S.R. “Demokritos”, 153 10, Athens, Greece 2. Department of Pharmacy, Pharmaceutical Technology Laboratory, University of Patras, 261 10, Patras, Greece
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Abstract: | The several factors that could affect the sensitivity and the accuracy of the determination of solid-supported amino groups using 2-iminothiolane (Traut's reagent) and 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) are described. The authors found that by using 0.1M phosphate buffer, pH 8.0, instead of ethanol as solvent for the reaction of the solid supports with the 2-iminothiolane, using 0.1M phosphate buffer adjusted at pH 7.27 instead of 8.0 as diluent of 5,5′-dithiobis-(2-nitrobenzoic acid), and selecting carefully the concentration of the latter reagent, it was possible to produce a very sensitive assay capable of quantitatively determining the surface amino groups of very different types of samples. The assay is well adapted for quantitative determination of amino-carrying plastic beads, permitting the determination of nanomolar quantities. In addition, the assay is well suited for microparticulated solid supports (e.g., AH-Sepharose). |
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