High performance gel permeation chromatography of bovine thyrotropin (TSH): effect of column stability and mobile...

Because of previously observed variability in the elution patterns of radiolabeled bovine TSH (bTSH) and bTSH bioactivity (in a particular bTSH preparation) on gel permeation high performance liquid chromatography (GPC), studies were conducted to examine the effect of different conditions on the elution of this material. Continued use of a Waters' I-125 column at pH 7.2 demonstrated progressive retardation in the elution of bioactivity and radioactivity in this highly-purified bTSH preparation, with progressive but incomplete separation of these two functions. With decreasing mobile phase pH the elution positions of TSH bioactivity and radioactivity advanced to a coincident peak near to the void volume (pH 4). Addition to the mobile phase of the ion pair reagents tetrabutyl-ammonium phosphate (PIC-A) and pentane sulfonic acid (PIC B5) produced marked alterations in the elution positions of TSH radioactivity and bioactivity, with separation of these functions in the presence of the former reagent. These data indicate the present limitations of GPC for the purification of TSH.