Development of Fluorous Solid-Phase Extraction (FSPE) on a Microchip and its Application to Proteomics

Abstract

The origin of fluorous interaction was explored and experimentally examined based on both HPLC and CEC data in this project. It was found that the selective fluorous interaction is a kind of reduced instantaneous or induced dipole interaction compared to the hydrophobic interaction. A series of FPPM preparation parameters were optimized. The optimized FPPM column can resolve the components in a manner that was otherwise not possible with its non-fluorous (hydrocarbon) counterpart. Following, the CEC separation of fluorous analytes on FPPM stationary phase based upon fluorous-fluorous interaction was realized for the first time. It was also found that, quantitatively, hydrophobic stationary phases have better methylene selectivity (〖 α〗_(-CH_2-)), while fluorous stationary phases have better perfluoromethylene selectivity (〖 α〗_(-CF_2-)). Thermodynamically, ∆G_(-CF_2- → -CF_2-)^° : ∆G_(-CH_2- → -CH_2-)^° (Gibbs free energy change of transferring a –CF2– unit to pure fluorous stationary phase versus Gibbs free energy change of transferring a –CH2– unit to pure hydrophobic stationary phase) is approximately equal to 8:1. A new concept, hypothetical water percentage (HWP) based on the comparison of 〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-) was proposed for the first time to quantitatively evaluate the hydrophobicity/fluorophilicity of a stationary phase. A stationary phase can be classified as fluorous stationary phase when the HWP is less than 0 (more negative indicates more fluorous), or as a hydrophobic stationary phase when the HWP is larger than 100. For the range between 0 and 100, the stationary phase can be treated as either fluorous or hydrophobic due to the similar values of〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-). Fluorous tagged peptides and proteins (up to 5800 Da) were effectively separated from their non-fluorous counterparts on the FPPM stationary phase in capillary-based columns and detected both on-line with ESI-MS and off-line with MALDI-MS. Finally, the FPPM solid-phase extraction (SPE) stationary phase was transplanted from the capillary to a microchip format. This microchip exhibits the merits of both selective fluorous interaction and micro total analysis system (µTAS).