Chromatography Essay

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Chromatography Chromatography encompasses a variety of techniques that involve a mobile phase flowing through a bed of stationary phase. A mixture of solutes is resolved or separated as a result of such differences in "affinity" of the solutions for the mobile phase relative to the stationary phase. A solute that has more "affinity" for the mobile phase moves forward with the mobile phase whereas a solute with more "affinity" for the stationary phase is retarded. Thus, solutes are separated into bands based on their distribution between phases. Molecules of biological interest are separated or resolved by a variety of chromatography techniques including adsorption chromatography, ion-exchange chromatography, partition chromatography (paper or thin layer chromatography), gas-liquid chromatography, and molecular exclusion chromatography (molecular sieve or gel filtration). Gel Filtration Chromatography Gel filtration separates molecules based solely on their size. Larger molecules elute first from the column. Smaller molecules elute later from the column. Gel filtration chromatography is used primarily to purify proteins or other molecules of interest and can be used to estimate the size of unknown proteins. Principles of Gel Filtration Chromatography In gel filtration or molecular exclusion chromatography, molecules in solution are separated by size as they pass through a column of cross-linked beads that form a three-dimensional network. These polymer beads (frequently made of dextran, agarose, or acrylamide) comprise the stationary phase. The liquid phase is the solvent that is found both around the beads and in the pores of the stationary phase matrix. As a sample passes through the column, there are two routes that molecules can take through the column, depending on their size and the size of the pores in the beads. Molecules

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