Fast separation of native proteins using sub-2 μm nonporous silica particles in a chromatographic cake

A novel method for the fast separation of native proteins was investigated using sub-2 μm nonporous silica packing inside a chromatographic cake having a diameter much larger than its thickness. Various silica-based particles ranging from 630 nm to 1.2 μm were synthesized and chemically modified with polyethylene glycol 600. The packing material was laterally packed into a series of chromatographic cakes containing the same diameter (10mm) and different thicknesses, ranging from 2 to 10 mm, and tested by hydrophobic interaction chromatography. The results showed that the sub-2 μm NPS particles in a small chromatographic cake were found to have a high efficiency at a flow rate of 10 mL/min and a backpressure of <20 MPa. The effect of the thickness of the chromatographic cake on the resolution of the proteins was also investigated and it was found that too short a column length could dramatically decrease the protein resolution; the minimum column length was also qualitatively evaluated. The presented method is expected to be useful for routine analysis of native and/or intact proteins in hospitals and as a tool for the fast screening protein drugs and optimization of experimental laboratory conditions.