Sizeexclusion chromatography gel filtration

Size-exclusion chromatography, also termed gel-permeation or gel-filtration chromatography, separates proteins on the basis of their size and shape. As most proteins fractionated by this technique are considered to have approximately similar molecular shape, separation is often described as being on the basis of molecular mass, although such a description is somewhat simplistic.

Fractionation of proteins by size-exclusion chromatography is achieved by percolating the protein-containing solution through a column packed with a porous gel matrix in bead form (Figure 6.8). As the sample travels down the column, large proteins cannot enter the gel beads and hence are quickly eluted. The progress of smaller proteins through the column is retarded, as such molecules are capable of entering the gel beads. The internal structure of the matrix beads could be visualized as a maze, through which proteins small enough to enter the gel must pass. Various possible routes through this maze are of varied distances. All proteins capable of entering the gel are thus not retained within the gel matrix for equal time periods. The smaller the protein, the more potential internal routes open to it and, thus, generally, the longer it is retained within the bead structure. Protein molecules, therefore, are usually eluted from a gel-filtration column in order of decreasing molecular size.

In most cases the gel matrices utilized are prepared by chemically cross-linking polymeric molecules such as dextran, agarose, acrylamide and vinyl polymers. The degree of cross-linking controls the average pore size of the gel prepared. Most gels synthesized from any one polymer type are thus available in a variety of pore sizes. The higher the degree of cross-linking introduced, the smaller the average pore size and the more rigid the resultant gel bead. Highly cross-linked gel matrices have pore sizes that exclude all proteins from entering the gel matrix. Such gels may be used to separate proteins from other molecules that are orders of magnitude smaller, and are often used to remove low molecular weight buffer components and salts from protein solutions (Figure 6.9).

Size-exclusion chromatography is rarely employed during the initial stages of protein purification. Small sample volumes must be applied to the column in order to achieve effective resolution. Application volumes are usually in the range of 2-5 per cent of the column volume. Furthermore, columns are easily fouled by a variety of sample impurities. Size-exclusion chromatography is thus often employed towards the end of a purification sequence, when the protein of interest is already relatively pure and is present in a small, concentrated volume. After sample application, the protein components are progressively eluted from the column by flushing with an appropriate buffer. In many cases, the eluate from the column passes through a detector. This facilitates i mmediate detection of protein-containing bands as they elute from the column. The eluate is normally collected as a series of fractions. On a preparative scale, each fraction may be a number of litres in volume. Although size-exclusion chromatography is an effective fractionation technique, it generally results in a significant dilution of the protein solution relative to the starting volume applied to the column. Column flow rates are also often considerably lower than flow rates employed with other chromatographic media. This results in long processing times, which, for industrial applications, has adverse process cost implications.

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