(443g) Protein Transport in Charged Agarose Gels Studied by Optical Microscopy in Microfluidics Devices

Understanding protein transport in ion exchange matrices is essential for the rational design of improved stationary phases and for the development of models to describe rate processes in chromatography columns. Macroscopic measurements of mass transfer rates can provide quantitative information about transport behavior. However, such measurements are subject to multiple mechanistic interpretations since different models often result in qualitatively similar macroscopic behavior. On the other hand, the measurement of concentration profiles within stationary phases at the microscopic level is thought to provide greater insight in the nature of the transport mechanism. In the past, such measurements have been conducted for charged polyacrylamide gels supported within thin capillaries and for spherical ion exchange particles by confocal microscopy using fluorescently labeled proteins. In this work, we extend the visualization of concentration profiles to charged agarose gels supported in microfluidics chips using optical microscopy with chromatophoric proteins. In order to directly view concentration profiles during transient adsorption and desorption, thin (100 um) slabs of anionic agarose gels are synthesized on etched glass microfluidics chips. A protein solution flows past the exposed edge of the gel slab and a microscope with CCD camera is used to monitor the evolution of concentration profiles in the slab. The latter is converted to protein concentration using a calibration curve. We have studied the behavior of three proteins, cytochrome c, myoglobin, and hemoglobin. Because of the rectilinear, semi-infinite geometry, boundary effects are de-emphasized compared to the case of spherical particles and a simple mathematical treatment can be used to determine the protein diffusivity in the gel as a function of protein concentration. The effects of solution protein concentration, ionic strength, and flow rate have been measured for different proteins. For comparison purposes, agarose spheres are also prepared allowing conventional macroscopic measurement of adsorption isotherms and uptake rate. Microscopic and macroscopic measurements are then compared with different mass transfer models. Agreement between these measurements suggests that fundamental data obtained at the microscopic level can be used to predict the macroscopic behavior of practical stationary phases.