Analytical study for separation of middle molecules

A 3-step chromatography procedure was employed to perform a profiling analysis of middle molecular substances accumulating in uremia serum. In the first step, gel chromatography on Sephadex G-15 columns (8 x 100 cm) was performed, and the two peak fractions (3,4) appearing near the vitamin B12 elution position were taken, lyophilized, and rechromatography on Bio-Gel P-4 (super fine) column was undertaken on each. The peak 3 from Sephadex G-15 was further fractionated on Bio-Gel P-4 into 4 peaks (a, b, c, d). Moreover, the peak 4 was fractionated into a further two peaks, d and e. Using reversed-phase chromatography on Nucleosil 5C18 (0.4 x 20 cm) with Bio-Gel P-4, the peaks for approximately 100 middle molecules have been separated. It is considered that, through structural determination of each of these middle molecule peaks, their possible toxicity can be ascertained and that the substances to be removed clinically can be clarified.