Department of Chemical & Biomolecular Engineering THE NATIONAL UNIVERSITY of SINGAPORE
Chemical Engineering Process Laboratory II
Experiment B2 Chromatography for Protein Purification
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A. Learning objectives 1. 2. 3. 4. Establish chromatographic assay to determine protein concentrations in a mixture. Appreciate the importance of resolution in protein chromatography. Understand the tension between purity and yield in protein chromatography. Understand the importance of mass balance closure in protein purification.
B. Introduction I. Fast Protein Liquid Chromatography (FPLC) High Performance Liquid Chromatography (HPLC) is the workhorse for any biopharmaceutical protein downstream processing train, featuring at least twice within the train. You must recall experiencing the HPLC in one of the experiments in your CN2108 module. Read up on the essential parts of the HPLC system. In this experiment, you will use a modification of the HPLC, the FPLC (Fast Protein Liquid Chromatography System) to separate and purify a mixture of two proteins. The FPLC has been developed to specifically take advantage of the resolution capability of the HPLC for protein purification and collection. II. Concepts in LC When a mixture of proteins is injected into an LC column, the proteins interact with the stationary phase based on their respective chemistries and move through the column at different speed. Based on this differential migration, the proteins elute from the end of the column at different times and therefore become separated. This process is usually facilitated by following the proteins with a mobile phase. Although the protein mixture will have entered as a narrow, concentrated peak, it will exit dispersed and diluted by the mobile phase. This is called bandspreading. Bandspreading (which is an inverse indication of the column efficiency) must be minimized especially for large-scale protein...
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