COLORIMETRIC DETERMINATION OF REACTIVE PRIMARY AMINO-GROUPS OF MACROSOLID AND MICROSOLID SUPPORTS

The several factors that could affect the sensitivity and the accuracy of the determination of solid-supported amino groups using 2-iminothi olane (Traut's reagent) and 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellm an's reagent) are described. The authors found that by using 0.1M phos phate buffer, pH 8.0, instead of ethanol as solvent for the reaction o f the solid supports with the 2-iminothiolane, using 0.1M phosphate bu ffer 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 cap able of quantitatively determining the surface amino groups of very di fferent types of samples. The assay is well adapted for quantitative d etermination of amino-carrying plastic beads, permitting the determina tion of nanomolar quantities. In addition, the assay is well suited fo r microparticulated solid supports (e.g., AH-Sepharose).