A Novel Open Tubular Capillary Electrochromatographic Column With Rough Inner Surface For Proteins Separation

Open tubular capillary electrochromatographic (OTCEC) column using porogen to form rough inner surface was developed. The rough layer was coated on the capillary inner wall by in situ polymerization of methacrylate and ethylene glycol dimethacrylate in the presence of porogen. The roughness of polymer was optimized by different types and proportions of porogenic solvents in polymerization mixture in this study. The results show that the column prepared with porogenic solvent is rougher and more appropriate for separation than the column without porogenic solvent. Furthermore, the columns with sole porogen have higher separation efficiency than the column with co-porogen due to more uniform surface. The best separation of BSA and Cty-c with higher resolution is obtained in the poly (butyl methacrylate) OTCEC column using 1-propanol as sole porogen and in the poly (lauryl, methacrylate) OTCEC column using 1-dodecanol as sole porogen. Hildebrand solubility parameter (8) of 1-propanol and 1-dodecanol close to their polymer would bring about a small pore size with a large specific surface area. In addition, the higher the temperature, the more stable coating and the smaller pore size. The results showed that the best separation performance was achieved when the polymerization temperature was at 65℃. The two types of prepared OTCEC columns show good column-to-column (less than 3.0%,11.0%) and run-to-run (less than 1.0%,9.0%) repeatability of retention times and peak areas. Additionally, the result of protein separation by methacrylate-based porous OT column and dynamically coated column with polyvinylpyrrolidone (PVP) is compared. The separation efficiency is similar for the two columns. While column-to-column repeatability of the OT column is slightly better than the dynamically coated column, the sensitivity of the OT column is obviously improved than the dynamically coated column. When compared to the methacrylate-based monolithic column, the OT column significantly shortened the separation time. The results show that the proposed OTCEC system is useful for the fast separation of simple protein samples.