[Preparation and characterization of sinopak-s-DEAE weak-anion exchange packing for high performance liquid...

High-performance ion exchange chromatography (HPIEC) is extensively used in the separation of peptides and proteins, especially in the biotechnology process. The principle of separation of proteins is based on the changes of pH and salt concentration in the mobile phase for the chromatographic model. A new synthetic method with the help of a catalyst for the bounding of diethylaminoethyl group on a home-made macro-pore silica sphere (the trade mark is Sinopak-s, with sphere size of 5 microm and pore diameter of 100 nm) was developed in our laboratory for the application of the scale-up separation of biotechnological target products in China. The Sinopak-s-DEAE weak anion ion exchange matrix for HPLC was prepared and characterized with various proteins. The pH value and reaction time were discussed for the reaction efficiency of ligand to the silica sphere. The coverage of the DEAE ligand on the silica surface were among 1.6 to 2.1 micromol/m2 for six batches of packings. The influences of the pH value and the salt concentration in mobile phase upon the retention of proteins on the DEAE column were also discussed. A bio-activity recovery up to 98% for trypsin was arrived after purification with the DEAE column under the chosen chromatographic conditions. The capacity of matrix for BSA was 80 mg/g. The column was successfully applied to separate a mixture of several standard proteins in a linear gradient elution condition from 0 to 0.4 mol/L of NaCl in a 50 mmol/L of Tris/HCl buffer (pH 7.0) at 1.0 mL/min flow rate and detected at 280 nm wavelength.