Advances such as grafted functional polymers in chromatographic media have greatly enhanced capacity and uptake.  Slow displacement rates and large elution pools remain major drawbacks, however, and likely result from transport or kinetic limitations that are incompletely understood.  While measuring uptake rates in chromatographic media is straightforward, measuring rates of mobility in fully-loaded particles remains challenging.  As models of multicomponent separations rely on rates of protein displacement and elution, a straightforward method is desired to measure the mobility of bound protein.  We have adapted fluorescence recovery after photobleaching (FRAP) methods to study transport and exchange of protein at equilibrium in a single particle.  Further, we have developed a mathematical model to capture diffusion and desorption rates governing fluorescence recovery.  We will describe an investigation of how these rates vary as a function of protein size, binding strength and media, with a focus on explaining differences between polymer-modified and traditional media.

Protein mobility in chromatographic media from fluorescence recovery after photobleaching (FRAP) methods

245th National Meeting (2013)

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