Electrophoretic molecular sieving

Though electrophoretic separations were historically first studied in free solutions, more recent developments have extended its application to solid supports, including polyacrylamide, agarose, and starch gels. The purpose of a solid support is to suppress convection current and diffusion so that sharp separations may be retained. In addition, support gels of controlled pore sizes can serve as size-selective molecular sieves to enhance separation - smaller molecules experience less frictional resistance and move faster, while larger molecules move slower. Therefore, separation can be achieved based on molecular size. 

Which of the following electrophoretic media show molecular sieving effects ... 

Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacrylamide (Fig. 3-19). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility of the molecule, g, is the ratio of the velocity of the par-... 

Earlier chapters have been devoted to both these topics, obviating the need for further discussion. Although preparative electrophoretic methods do exist they are not often found in purification schemes. Molecular sieve chromatography, on the other hand, is used extensively. However, owing to restrictions of sample size this method is usually one of the last techniques to be employed. 

Electrophoretic separations are nearly always carried out in gels (or on solid supports such as paper) because the gel serves as a molecular sieve that enhances separation (Figure 4.7). Molecules that are small compared with the pores in the gel readily move through the gel, whereas molecules much larger than the pores are almost immobile. Intermediate-size molecules move through the gel with various degrees of facility. Electrophoresis is performed in a thin, vertical slab of polyacrylamide. The direction of flow is from top to bottom. Polyacrylamide gels, formed by the polymerization of... 

Electrophoresis using support media like starch gel or polycrylamide gel has now largely superseded thin sheet electrophoresis systems for the separation of biomolecules such as proteins. The gel methods have the great advantage that besides their useful chracteristics purely as support media, gels act as molecular sieves so that the movement of components is controlled by the size and shape of molecules in addition to charge and this results in more efficient electrophoretic separations. 

This is a CE analog of conventional zone gel electrophoresis for the separation of macromolecules based on size. The capillary is filled with a porous polymer gel, and molecular sieving occurs as the molecules move through the gel, that is, separation is based on both electrophoretic mobility and molecular size. Very high resolution is achieved. The trend is to fill the capillary with a liquid gel matrix (pumpable gel solutions, such as deriyatized celluloses dissolved in the run buffer). This allows replacement of the gel in the capillary to eliminate contamination problems from the sample matrix that occurs with fixed gels.. This technique is widely used for separation of nucleotides in deoxyribonucleic acid (DNA) sequencing (Chapter 25). 

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