Chromatography Forum

Dear All,
I am using DEAE sepharose FF Ion exchange resin as a first purification step in downstream processing (Large Scale). The column is packed with new resin, however now we are facing problem that column is not getting packed properly. We are getting very low number of plates (~500 for 16 cm Height). Also the bed height is continuously reducing at every packing. Can't figure out reasons for so low N value. As per slurry concentration (60%) we have not lost much of resin during repeated packing and unpacking of column. Now we are expecting as per new volume to be 15.5 cm but still wont get required hydraulic pressure at this height. We went further compressing the piston down to 14 cm to pack column at ~1.5 bar.

So my concern is
1) why we are getting so low N value, despite getting good As= 1.4.
2) why column height is continously reducing, does resin is getting degraded? Although resin is fairly new (only 20 cycles carried out).
3) What are reason for resin getting degraded? Does use of process water for long term storage have any effect?
4) What is oxidative degradation of ion exchange resin?

DEAE shrinks in pure aquaous invironment. You mitght like to ad some ethanol to your mobile phase - something like 15 - 20%
Regarding the decrease of N, material staying on the stationary phase "permanently".
Do you regenerate the stationary phase by very high salt concentration regularily?
Best tegards

in my experience a Sephadex DEAE phase will expand in pure aqueous environment, and shrink progressively more as stronger (more concentrated or more alkaline) buffer is added.

The material swells when water or any other liquid is added in comparison to the initial state – i.e. before start using it. Otherwise, adding organic modifiers will cause swelling, while in pure aqueous liquid it will shrink – again in comparison. pH changes etc. will cause swelling or shrinkage as well.

Best Regards

DEAE shrinks in pure aquaous invironment. You mitght like to ad some ethanol to your mobile phase - something like 15 - 20%
Regarding the decrease of N, material staying on the stationary phase "permanently".
Do you regenerate the stationary phase by very high salt concentration regularily?
Best tegards

Now we are using 0.2M NaCl as packing solution.
Yes, during trial batches we have loaded some material with slightly higher impurities than expected. Also we have run column with piston raised to higher height (19 cm) from actual 15 cm due to back pressure increase. Do it will have lead to insufficient regeneration of resin after use.

We use three regeneration: 1) 0.5M NaCl for three bed volumes
2) 1 M Sodium Acetate ph 3 for two bed volumes
3) 0.5M NaOH for two bed volumes

we regenerate column after every use.

Those impurities (that you know of as well as those you don’t know of) are contaminating the stationary phase with stuff that doesn’t elute or get rinsed under the utilized conditions – hence the pressure increase and N decrease.
I don’t know what the rationale for the 0.5M NaOH is and it seems to me a bit stronger - now thinking of the pH - but if at all, it should be applied right after end elution of your substance/s. I would reduce the concentration to 0.2M NaOH.
After application of the high salt concentration buffers the column should be brought to initial conditions (very low ion strength) and then the object of purification could be loaded.
Again, you may consider 10 – 15 – 20 % ethanol mixed into your eluents.

Best Regards