Discovery BIO GFC Separation Volume and Peak Capacity

By: Hillel Brandes, Reporter US Volume 27.2

Hillel Brandes

hillel.brandes@sial.com

Two measures of the effectiveness of a gel filtration HPLC (GFC) column are separation volume and peak capacity. Separation volume (Vs) is the difference in the retention volume between a fully retained analyte and a completely excluded analyte. It is, in effect, the total accessible pore volume of the column. A larger separation volume provides a greater volume in which chromatographic separation takes place and potentially greater peak resolution. Separation volume is calculated using the following equation:

Vs = Vt - V0                           Eq. 1

Where:

Vs = separation volume

Vt = total column volume; Vt = elution volume (Ve) of fully retained analyte (analyte not adsorbed)

V0 = void volume of column; V0 = Ve of excluded analyte

Peak capacity (Pc) is the number of peaks that are resolved within a given retention time window. There are both isocratic and gradient forms of the equation. Isocratic peak capacity is determined using this equation:

Pc = (trn -tr1)/W                          Eq. 2

Where:

Pc = peak capacity

trn and tr1 = retention time of last and first eluting peaks, respectively

W = average peak width at baseline

Peak capacity is directly proportional to trn - tr1, which is related to Vt - V0 in equation 1 by the flow rate, and inversely proportional to peak width (W). Peak width is, in turn, related to column efficiency in that the higher the efficiency, the narrower the peaks. Therefore, the ideal GFC column exhibits high separation volume and high efficiency, which together provide high peak capacity for maximum analyte resolution.

These properties are demonstrated on Discovery BIO GFC columns in Figures 1 and 2. In this study, we compared Discovery BIO GFC columns to competitive silica-based GFC columns of comparable particle and column dimensions in the isocratic separation of two sets of peptides. In both examples, Discovery BIO GFC was shown to have ~0.5 mL higher separation volume than the competitive column. In Figure 2, Discovery BIO GFC also provided much narrower peaks and significantly higher resolution of the peptides than the competitive GFC column. Both of these attributes, combined with excellent column stability, long lifetime and the wide molecular weight separation range of individual columns and the entire line, make Discovery BIO GFC the columns of choice for gel filtration HPLC separations.

Figure 1. Peptide Mix 1 - Separation Volume Comparison on Discovery BIO GFC and Competitive GFC Column


Figure 2. Peptide Mix 2 - Separation Volume and Peak Capacity Improvements on Discovery BIO GFC Compared to Competitive GFC Column


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