Hi,
I am trying to separate by size exclusion some small molecules from bacterial lysate (100 - 500 Da). It works well by HPLC on SEC-60 column from Thermo, but the largest available is 7.8 mm diameter only. Does anybody use a higher scale solution with high resolution in this MW range ? Maybe a resin that we can use to pack a big FPLC column ?
Thanks.
Also, how many cycles are you willing to run with collection and assay of peaks? Since this is a crude lysate from E. coli, how long do you expect the column to last even with regeneration cycles and a guard column? Is your scale up going to involve 1% or less of the column volume at the same sample density, viscosity, residence time, etc.? I assume your scale up will be by column chemistry and diameter and not height in order to have linear alimetrics and the same alpha value, right? Depending on your column length at 5 micron particles you may really be pushing the pressure limits of an AKTA system at reasonable linear flow rates.
If you're set on keeping everything the same except for column diameter, because you're focused on scale up, then you might have to purchase bulk resin and pack your own column but it may be risky to run it on an AKTA which is reasonable at 8 micron 6% cross-linked soft gel particles at 30 cm column length and less than 0.1 CV/min residence time. With a hard and uniformly spherical bead of narrow particle size distribution, you might be able to circumvent the pressure limitations on the AKTA but then you will need an appropriate column that can handle the pressure such as a Tricorn at 100 bar for see though column or go to metal. You may also need to re-plumb the system with wider bore PEEK tubing. The swith valves also have narrow bores. Keep in mind that the pressure limit for the P920 pump is about 5 mP but the important pressure is for the flow cell (2mm) at about 4 mP.
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