凝胶柱层析实验报告(生物化学).pdfVIP

Gel Filtration Lin ChengyuBio 04 2010030007 Cooperator: Liu Yidi Experiment Date: 2012-03-05 2012-03-19 Submitting Date: 2012-04-14 1 Introduction 1.1 Background information The method of gel filtration chromatography exploits the physical property of molecular size to achieve separation. It has been 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. 1.2 Major principles 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 molecules. Therefore, the order of elution of the various solute molecules is directl