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October 26, 2017

Asked and answered: your questions about new-generation SEC columns

By Pia Liljedahl, Global Product Manager for SEC columns at GE Healthcare Life Sciences

During a webinar, Pia Liljedahl presented the new generation of agarose-based SEC columns from GE.
These columns—Superdex 30 Increase, Superdex 75 Increase, Superdex 200 Increase, and Superose 6 Increase—are designed for small-scale preparative purification and analysis.
During the session, Pia described how these columns impact positively on the resolution and the run time of your experiment.

This article is a recap of the questions raised during the live questions and answers (Q&A) session.

A: As the fractionation range for Superose 12 will be covered by Superose 6 Increase, Superdex 200 Increase, Superdex 75 Increase, and Superdex 30 Increase together, there will not be a Superose 12 Increase resin. Instead we give guidance on which of the above-mentioned SEC Increase columns to choose as a replacement for Superose 12 columns.
A: For SEC Increase columns:

These columns are packed to a flow rate specification. Small differences in bead size and packing will result in differences in pressure drop over the packed bed between columns. A typical pressure drop over the packed bed is 3 MPa (check in the Instructions for use for your specific column). For maximum protection of the column, the pressure drop over the packed bed should be measured for each column to set the pressure limit. This enables use of the columns at the highest flow rate limit. Do not exceed the flow rate limits, especially when running viscous solvents and at lower temperature.

For Superdex Prep Grade columns (HiLoad 16/600, 26/600):
The maximum pressure drop over the packed bed is 0.3 MPa (3 bar, 43.5 psi)

For Sephacryl columns (HiPrep 16/60, 26/60):
The maximum pressure drop over the packed bed is 0.15 MPa (1.5 bar, 21.8 psi)
A: In general, the interactions with our new-generation SEC resins called “Increase” are very low but there will always be molecules and conditions were interaction can occur. The interactions with these resins are comparable to the old resins.
A: We are always looking for how to improve our product offering. Currently, it is possible to order larger columns packed with our new-generation SEC resins via our Custom Column Packing services. The largest column available is 10 × 600 mm. Please contact your local sales rep for more information.
A: A general recommendation when choosing fractionation range is that the molecular weight of the target molecule should fall into the middle of the separation range of the resin. Given that LDH is an Mr 140 000 enzyme, the optimal column for analysis would be Superdex 200 Increase 10/300 GL (recommended sample volume 25 to 500 µL). If the sample amount is limited, the 3.2/300 column (sample volume 4 to 50 µL) is recommended.
A: Superose 6 Increase is designed for separation of protein complexes and other very large molecules and provides separation of large molecules that Superdex 200 Increase cannot separate. Superdex 200 Increase gives higher resolution of proteins with molecular weights lower than approximately Mr 400 000.
A: A general recommendation when choosing fractionation range is that the molecular weight of the target molecule should fall into the middle of the separation range of the resin. The resolution of proteins > Mr 70 000 is higher on Superdex 75 Increase compared to the resolution on Superose 6 Increase in the same separation range.

When choosing fractionation range for a column, it is important to think about what molecular weights the POI should be separated from. If the molecular weight of the POI is Mr 70 000 and the POI needs to be separated from smaller molecular weights, then Superdex 75 Increase would be the optimal choice. If the POI also needs to be separated from its dimer or higher molecular weights, Superdex 200 Increase would be the column of choice. Superose 6 Increase is designed for separation of protein complexes and other very large molecules and would be the choice if the POI needs to be separated from such molecules.