A systematic investigation of recovery in preparative reverse phase high performance liquid chromatography/mass spectrometry

In this paper we report a systematic recovery study based on reversed phase high performance liquid chromatography (RP-HPLC) separation and mass spectrometric (MS) based fractionation. Factors including a compoundʹs physicochemical properties, column mass loading and presence of impurities were investigated through commercially available compounds. Results suggest that the delay time between MS peak detection and fraction collection, fraction detectorʹs signal-to-noise ratio and compoundʹs base peak width in the chromatogram have the biggest impacts on purification recovery. In an effort to assess sample recovery within our high throughput purification process, re-purification was performed on four compound libraries that were synthesized in-house. Reproducible recoveries (>80%) were achieved in all tests.