Agilent GF-250 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.

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This document introduces a protein separation method based on high-performance liquid chromatography. The method uses non-covalent affinity chromatography columns and polyacrylamide gel chromatography columns, which can effectively separate proteins of different molecular weights.

This document introduces ZORBAX GF-250 column in the separation of Bio-Rad molecular weight markers

This document describes how to calibrate a ZORBAX GF-250 column using Bio-Rad gel filtration standards.

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.

This document describes the characteristics and usage of the ZORBAX GF-250 column. The column has a wide separation range and is suitable for separating proteins with different PI and hydrophobicity.

The document introduces the use of Gel-Filtration standards and the performance testing of ZORBAX GF-250 columns. The ZORBAX GF-250 column can effectively separate the components of the Bio-Rad® gel-filtration standard between thyroglobulin (670 KDa) and Vitamin B12 (1.35 KDa). The document also discusses the application and results of using gel-filtration test mixtures to test ZORBAX GF-250 columns.