An application of GPC spin column/ESI-MS methodology is to re-equilibrate the eluate of a spin column and pass it again through a second spin column to detect by ESI-MS the enriched tighter binding ligands at the expense of the weaker binding ligands . This approach is applicable to mixtures of unknown structures and unknown concentrations for differentiating strong from weak binders. For these systems, the relative ESI-MS responses for different unknown compounds are not indicative of their relative binding affinities since their concentrations in the original mixture are unknown. Cycling the initial eluate through fresh GPC spin columns can deconvolute this. The strong binders will be selectively found in the eluates at the expense of the weak binders. After each pass of the eluate through the GPC spin column, the relative change in mass spectral response for the strong binder will be considerably less than that for the weak binder.
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