Agilent GF-450 Manuals

Zorbax PrepHT GF-250 and GF-450 are surface-stabilized, hydrophilic gel filtration columns useful for the size separation of biological macromolecules. These columns have been specifically designed to provide superior efficiency, pH stability, and operational lifetime when using typical aqueous buffer solutions (pH 3.0-8.5) as the mobile phase. The GF-250 and GF-450 columns separate compounds by gel filtration (exclusion). Separations depend on the size of the sample molecules and the effective pore diameter of the packing material. Typically, the elution order or retention time obtained for a given molecule is inversely proportional to the logarithm of its molecular size. Molecular size is related to the molecular weight. Very large molecules are excluded from the pores, have the shortest path through the column, and elute first. Medium size molecules partially diffuse into the pores, and elute later. The smallest molecules, which can totally permeate the pore volume of the packing, elute last. The GF-450 and GF-250 columns are complimentary. The GF-450 column provides high performance gel filtration separation of high molecular weight biomolecules which are excluded from the linear separation range of the smaller pore sized GF-250 column. The optimal separation ranges for the columns are 900,000 to 25,000 Daltons (D) for the GF-450 column and 250,000 to 10,000 D for the GF-250, assuming spherical molecules.

Size-exclusion chromatography is a powerful technique for the size-dependent separation of biomolecules. While standard, non-denaturing mobile phases are often desirable, denaturing mobile phases containing guanidine hydrochloride (or SDS), may be used to insure un-aggregated, monomers for proper size determination. As shown below, denaturation shifts molecules toward larger apparent size and smaller retention volume.