Discovery BIO GFC 3 μm Packings

By: Hillel Brandes, Hugh Cramer, Reporter US Volume 27.4

Hillel Brandes and Hugh Cramer

hillel.brandes@sial.com

Gel filtration chromatography (GFC) (or size exclusion chromatography) is a common technique for resolution of biological macromolecules based on size and/or shape. Analytes that are large relative to the pore size of the stationary phase are excluded from the pores and therefore elute relatively early. Conversely, analytes that are small relative to the pore size, are well-retained and therefore elute later. Shape also affects retention: Molecules with a more elongated structure are not as retained as much as those that are more spherical or globular.

The Discovery BIO GFC product line for gel filtration of macromolecules is based on uniform, spherical, high-purity silica with a hydrophilic surface. To date, Discovery BIO GFC has been available as a 5 μm product line; now, the 3 μm particles have been added. While the 3 μm will generate higher backpressures, the relatively low flow rates that are generally optimal for gel filtration chromatography only generate modest back pressures. However, the tremendous advantage of the 3 μm columns is their higher efficiency. This will directly translate to higher resolution and peak capacity. Request Application Note 187 (T309187) for further discussion of peak capacity.

Figure 1 compares the elution profiles of a protein mixture on Discovery BIO GFC, 3 μm versus 5 μm, with the same y-axis scaling. The improvement in the 3 μm packing is evident in the narrower and taller peaks. Also evident is improvements in fine detail of the chromatogram, which reflects the higher resolution. A comparison of the calculated peak efficiencies, however, is particularly striking. This is shown in Table 1 with a sampling of analytes. The greatest gains are evident with moderately retained analytes, with peak efficiencies improving on the order of 100%. Even for uracil, representative of a fully retained analyte, efficiency has improved 30%, which itself is significant.


Figure 1. Elution Profiles of 3 μm Discovery BIO GFC 300 (A) and 5 μm Discovery BIO GFC 300 (B) (567337-U)


Table 1. Peak Efficiencies (N) of Discovery BIO GFC, 3 μm versus 5 μm

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