High-sensitivity radioassay in chromatographic effluents

Abstract

We have devised convenient systems for fractionating the effluent of high-performance liquid chromatography (HPLC) columns and concentrating the fractions for radioassay. A succession of aliquots of the HPLC effluent is deposited in wells formed in non-wetting fluorocarbon film and evaporated to near-dryness where the aliquots form droplets of uniform size. The droplets are then quantitatively transferred to filter paper impregnated with scintillator, where they form uniform circles 2–3 mm in diameter. Papers containing samples and standards are then exposed to photographic film for a time dependent on the radioactivity of the sample, the film is developed and teh autoradiographs are scanned with a thin-layer chromatography scanner used as a densitometer. The density of the spots was proportional to the radioactivity and could be quantified by comparison with the density produced by the standards. Scans of successions of spots from samples collected during elution of amino acids from HPLC columns reproduced the shapes of the peaks recorded by the UV-absorbance detector flow-cell, demonstrating that the resolution of the analysis was preserved. Film blackening sufficient for quantification was obtained with samples containing 14C in the 100–1000 dpm range in as little as 6 h of exposure with further increases in sensitivity in proportion to the increase in time of exposure. Hundreds of fractions from HPLC effluent and samples from several effluents could be assayed simultaneously, thus offering a large workload capability along with high sensitivity. We postulate that the same technique will produce similarly high sensitivity with little loss of resolution when used in conjunction with methods previously described for stripping radioactive compounds from gas—liquid chromatographic effluent into flowing liquid streams.