Quantitation of radiolabeled biological molecules separated by high-performance liquid chromatography

Several techniques are compared for the quantitation of radiolabeled molecules separated by high-performance liquid chromatography (HPLC). The first technique is the direct fraction collection of the HPLC eluent at pre-set times in individual tubes. Aliquots are removed from each tube, placed in scintillation vials, scintillation fluid is added and the radioactivity is counted in a liquid scintillation counter. The second technique is the direct interface (immediate analysis and plotting of data) of the eluent from the HPLC to a flow-through radioactivity detector. The third technique is to split the eluent from the HPLC with a microbore electronic stream splitter (flow/no flow) with a certain percentage directed to the flow-through radioactivity detector and the remainder collected in a fraction collector for further chemical characterization (gas chromatography-mass spectrometry, recrystallization, further purification). A direct comparison of the chromatograms, radioactivity counting efficiencies and sensitivities for these three techniques is presented.