Operating Recommendations for SEC Separations

Background on SEC Use

The PolyHYDROXYETHYL Aspartamide column was created specifically to perform HILIC and retain solutes solely through hydrophilic interactions. However, when used with a mobile phase which does not contain enough organic solvent to induce hydrophilic interaction, then it functions in the size exclusion chromatography mode. Swelling the coating with a suitable mobile phase causes the effective pore diameter to become the spacing between polymer chains of the coating (~15Å), allowing solutes as small as water to be separated by size.

The HEA column is available in three pore sizes, 200Å, 300Å and 1000Å. A column with 200Å pores has a fractionation range of 20-10,000 MW allowing resolution of the smallest of bioorganic molecules. The 300Å pore size accommodates a range of 20-80,000, and the 1000Å pore material can fractionate over a size range of 1,000 - 2,000,000, (over 5 orders of magnitude). The HEA columns may also be used with volatile mobile phases.

Start-up

Flush new columns (4.6 mm ID) with 25 ml water, then condition with at least 60 ml of a buffer solution with salt concentration of 0.2 M and a pH in the range of 3-6 (exact figures are not important here). Flush with another 20 ml water, then equilibrate for six hours (flow rate 0.5 ml/min) using one of the mobile phases recommended below before injecting samples. (For 9.4 mm ID columns, the above volumes should be multiplied by a factor of 4, and the flow rate for equilibration is 2 ml/min.) It is not necessary to repeat this conditioning step thereafter unless the column is flushed with organic solvent for long-term storage or used under HILIC conditions.

HEA Columns will exhibit two different fractionation ranges, depending on the mobile phase used. For a mobile phase of 0.2 M (Na)2SO₄ + 5 mM K-PO₄, pH 3.0, containing 25% acetonitrile, the fractionation range will be approximately Mol. Wt. 400-10,000 for columns with 200Å pores (P1020 209). For HEA columns with 300Å pores (P1030 209, P1030 204), the fractionation range is approximately MW 1000-200,000.

For a mobile phase of 50 mM formic acid, the fractionation range will be approximately MW 20 - 1000 for columns with 200Å pores. For 300Å columns, MW 20 - 80,000. The same column can be used for both fractionation ranges, simply by switching between these two mobile phases. The formic acid mobile phase is volatile, but precludes detection below 240 nm. The use of volatile mobile phases which are transparent at 215 nm (e.g. hexafluoro-isopropanol, HFIP) is experimental and hazardous, although it has the same effect on the fractionation range as formic acid.

Sample Composition

The sample solvent should not differ greatly from the mobile phase in ionic strength or organic solvent content, in order to prevent a significant difference in viscosity of the two. With high viscosity, solute molecules might not diffuse from the mobile phase to the stationary phase before the sample passes through the column. The loading capacity of a 4.6 mm I.D. column is roughly 0.4 - 0.8 mg peptide with no significant loss of resolution, but this number depends on the composition of the sample.

See PolyHYDROXYETHYL for Part Numbers and Prices.