Single and multicomponent protein adsorption and diffusion in cationic polyacrylamide hydrogels: Visualization and analysis

Gel-filled particles are an important part of many biological separation processes and offer desirable adsorption behavior in protein chromatography. The determination of transport mechanisms in these materials is critical. However, it is difficult to acquire a detailed understanding of the transport mechanisms from macroscopic particle studies. In contrast, protein transport can be directly observed on a microscopic level by synthesizing these gels within transparent capillaries. In this case, protein transport can be visualized either by using a colored protein or by using proteins labeled with a fluorescent probe that minimally changes the size and effective charge. This work extends previous single component protein adsorption studies in anionic polyacrylamide gels to the case of cationic gels. More importantly, it extends the prior work to the case of multicomponent protein systems. Initial studies focused on single component transport using myoglobin, alpha-lactalbumin, ovalbumin, and BSA. The quantitative results obtained in this work shed light on the mechanism of protein transport in these gels, paving the way for the development of transport models and the design of molecularly engineered stationary phases for protein chromatography. For example, the diffusivity and binding capacity of the proteins determined from the microscopic data compared favorably to those determined using Q-Hyper D, a gel-filled particle chemically similar to the gels synthesized in these experiments. Based on this, the stationary phase could be altered to offer optimal transport for proteins in this media.; Concentration profiles for transient adsorption involving multiple proteins were also obtained by labeling the proteins with non-interfering fluorescent probes. Proteins were introduced either sequentially or simultaneously in the experiments. A Fickian model combined with the Steric Mass Action (SMA) model were used to predict the multicomponent behavior based on the single component data. Based on this model, predictions accurately described the co-adsorption behavior. The concentration profiles were also qualitatively predicted for sequential cases. This suggests that a simple transport model can describe the binary protein transport mechanism and provides a useful benchmark for future multicomponent modeling work.