ABSTRACT
Iodinated insulins are frequently used as biological tracers as well as in radioimmunoassays and insulin receptor studies. This chapter deals with reversed-phase chromatography (RPC) fractionation of the heterogeneous iodination mixture leading to separation and isolation of very pure monoiodoinsulins. Following appropriate dilution, monoiodoinsulins with high specific radioactivity can be used directly from the RPC fractions in radioimmunoassays, receptor binding analysis, and for chemical analysis. The monoiodoinsulins are isomers with the same overall composition and their different retention times on the RPC columns must therefore reflect differences in hydrophobicity and/or charge distribution. The chapter discusses the effect of mobile phase (buffer substances, pH, organic modifier), stationary phase and separation temperature upon the separation. The influence of individual parameters (column packing, mobile phase, temperature, and column load) upon the separation pattern for the mono- and diiodoinsulins as well as recovery and purity of the isolated monoiodoinsulins can be evaluated.
