Hi everybody,
I am trying to pack a Sephadex G25 column in a HiScale 16 column. I have been reproducing my runs and I consistently get the asymmetry to fall within the allowable threshold of 0.8-1.8 (as recommended by Cytiva). However my reduced plate height (h) is fluctuating quite heavily with random columns having h >=3 and so a fail.
Could someone shed some light as to why my reduced plate height might be failing?
As mentioned previously the setup is as follows:
I make a 75% slurry of the media and leave it for 3h at room temperature in 20% EtOH.
After the resin has been swollen I pour it gently into the column and allow it to settle down by itself for 10 minutes.
Then I top it up with 20% EtOH and start packing at 300cm/h for 10min.
After the bed has consolidated I push the plunger down to the height of the bed and run it for another 10min.
After the bed has fully consolidated I push the plunger down further 3mm as recommended by Cytiva.
Here's some more parameters.
Packing flowrate 300cm/h
Column equilibration flow rate 150cm/h
Column performance test 20cm/h
Column diameter 16mm
Spikes
Spike 1 Acetone spike in MQ water as a mobile phase
Spike 2 0.8M NaCl in PBS pH 7.2
Could anyone help me with figuring out how to improve my reduced plate height?
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