Practitioners of reversed-phase liquid chromatography (RPLC) have traditionally used thermodynamic partition coefficients to describe the tendencies of molecules to transfer between the mobile and stationary phases.   Thermodynamics can provide insight into the average equilibrium behavior; single-molecule observations give rich mechanistic insight into the processes that determine the retention time of an analyte.    We have used total internal reflection fluorescence microscopy to perform high-throughput single-molecule tracking of a fluorescent fatty acid analog under conditions typically employed in RPLC.   About 105 molecules were observed on millisecond to minute timescales, and independent measurements were made of the adsorption and desorption rates.  We caused significant changes in the adsorption and desorption rates by changing the amount of organic modifier in aqueous solution.  Interestingly, the adsorption rate scaled logarithmically with the viscosity of the solution.  This work demonstrates the unique potential of single-molecule tracking to uncover the dynamics inside a typical RPLC column.  

Single-molecule dynamics of analytes in reversed-phase liquid chromatography

247th National Meeting (2014)

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