Department of Chemical & Biomolecular Engineering THE NATIONAL UNIVERSITY of SINGAPORE Chemical Engineering Process Laboratory II Experiment B2 Chromatography for Protein Purification Name Matric No. Group : : : Date of Expt. : GRADE : 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 purification. When bandspreading is severe, the proteins may not be sufficiently resolved within a reasonable time-frame. The degree of separation of one component from another is referred to as the resolution (RS), determined based on equation 1 (refer to Fig. 1): RS = VB ? V A 0. (W A + WB ) …Eqn. (1) Injection wA VA VB wB Figure 1. Typical protein chromatogram Note that resolution can also be defined based on retention times, instead of volumes. There are various ways to improve resolution, the most straightforward of which is to vary the mode of elution – isocratic versus gradient. Both of these modes are based on the strength of the mobile phase, which directly affects the interactions between the proteins and the stationary phase. In protein chromatography, it is desirable to have high yield as well as high purity of the collected fraction.
Yield is the amount of a protein collected as a fraction of the total amount of the same protein fed, while purity is a measure of how much of that protein is in the fraction collected. C. Experimental I. Protein Quantification You will design an experiment to obtain the calibration curves for the two proteins provided using FPLC. You are provided with the following for this experiment: 1. An FPLC system which has been properly set up and equilibrated. You only need to inject 100 µL of each of your samples, and your data will be recorded and analysed by the computer.
Note the profile of the mobile phase programmed. 2. A protein mixture containing two proteins (S1 and S2) at concentrations of 1. 0 mg/mL each. II. Protein Purification and Collection You are to perform a chromatographic purification of 1mL of the protein mixture provided. You can expect the chromatogram shown in Fig. 2. Myoglobin Lysozyme Figure 2. Chromatogram of two proteins from FPLC Based on Figure 2, you are to conduct the following: 1. Collect one fraction of the highest yield that is 100% pure S1, and the balance in another fraction. 2.
Collect one fraction containing as much of S1 fed as possible. D. Discussion 1. 2. Briefly describe the experiment that you designed in CI. Explain your choice of the collection times for each of your collection in experiment CII. Determine the yield and purity of each of your collected fractions. Perform a material closure for each of CII (1) and CII (2). Based on your results in (2) above, rationalize the importance of resolution in chromatographic separations. Given the tension between yield and purity, which, in your opinion, is more crucial, yield or purity?
How do you propose to improve the resolution of S1 and S2 in this chromatographic purification? 3. 4. Useful Notes 1. Reagents: a. Mixture of two proteins b. Mobile phase – 2M ammonium sulfate in 100mM Phosphate buffer pH 7. 0 c. Elution – 100mM Phosphate buffer pH 7. 0 FPLC to be set up with the appropriate parameters: a. detection wavelength at 280nm b. sample loop – 100 µL c. HIC column for protein separation. d. mobile phase – 2 M ammonium sulfate e. mobile phase flow rate: 1 mL/min f. gradient elution – linear gradient 100% to 0% over 10 column volume. 2.
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