Gel验报告.docxVIP

顾光超生07 2010012303GEL FILTRATION CHROMATOGRAPHYI. Introduction 1.Obejective of the experiment a. Understand and master the major principle and the general procedure of gel filtration chromatography. 2.Principle of the experiment a. Background information on gel filtration The method of gel filtration chromatography exploits the physical property of molecular size to achieve separation. The molecules of nature range in molecular weight from less than 100 to as large as several million. It should be obvious that a technique capable of separating molecules of molecular weight 10,000 from those of 100,000 would be very popular among research biochemists. Gel filtration chromatography is of major importance in the purification of thousands of proteins, nucleic acids, enzymes, polysaccharides, and other biomolecules. In addition, the technique may be applied to molecular weight determination and quantitative analysis of molecular interactions. b. Major principle of gel filtration The operation of a gel filtration column is illustrated in Figure 1. The stationary phase consists of inert particles that contain small pores of a controlled size. Microscopic examination of a particle reveals an interior resembling a sponge. A solution containing solutes of various molecular sizes is allowed to pass through the column under the influence of continuous solvent flow. Solute molecules larger than the pores cannot enter the interior of the gel beads, so they are limited to the space between the beads. The volume of the column accessible to very large molecules is, therefore, greatly reduced. As a result, they are not slowed in their progress through the column and elute rapidly in a single zone. Small molecules capable of diffusing in and out of the beads have a much larger volume available to them. Therefore, they are delayed in their journey through the column bed. Molecules of intermediate size migrate through the column at a rate somewhere between those for large and small mol