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#1 Posted : 15 February 2017 17:43:18(UTC)

Joined: 15/02/2017(UTC)
Posts: 1

I have a rather old Superdex 200 20x60 prep grade column. It has been stored in our refrigerated Chromatography cabinet, sealed on both ends and equilibrated in 20% EtOH. The column has been well maintained over the years and is dedictated to one protein purification protocol. Recently, I washed the column with several col. vol. of HOH & equilibrated the column in our equilibration buffer which contains 10% glycerol (temperature is about 6 C in the refrigerated chromatography cabinet). In the past, the pressure increased with this buffer, but I was able to run the column at 0.8 ml min-1 and pressure held between 0.25 to 0.28 MPa (AKTA reading). However, in this recent run, the pressure began increasing beyond 0.3 MPa. I re-equilibrated the column in HOH, inverted it and washed it with 0.65 M NaOH, followed by 1M PBS. The PBS wash is still going and the pressure is still above 0.3 MPa (~0.35 MPa). Once the NaOH is clear of the column, I will invert the column again and add equilibration buffer and check the pressure. Are there any steps that I can take to reduce this back pressure? Thank you.
GE Protein purification team  
#2 Posted : 20 February 2017 10:29:57(UTC)
GE'GE Protein purification team

Joined: 16/11/2015(UTC)
Posts: 106

  • GE Healthcare sells two Superdex™ 200 PG HiLoad ™ columns; 16/600 and 26/600. The 16x600 column has a maximum flow rate of 1.6 ml/min in water at room temperature whereas the 26/600 column has a maximum flow rate of 4.4 ml/min in water at room temperature. It should be possible to run the larger column at 0.8 ml/min at 6 C in buffers containing 10% glycerol but for the smaller column, this flow rate might be too high. Both the low temperature and the glycerol will cause increased backpressure.
  • Another explanation for the increased backpressure might be that it is caused by the ÄKTA™ system, e.g. the ÄKTA online filter or the flow restrictor. Our older ÄKTA systems such as ÄKTAexplorer do not allow direct measurement of the pressure over the packed bed. Bypass the column and determine the system pressure. The maximum system pressure running either of these column equals the maximum backpressure over the packed bed (0.3 MPa) + the system pressure without the column. See product instruction for a more detailed explanation
  • If the increased backpressure is caused by the column, more rigorous cleaning or changing the adaptor net ring can help. Further information about this is also explained in the product instruction.
Best regards, Therese Granér, Sr Research Engineer at GE Healthcare Life Sciences
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