Chromatography Forum

I am a bit confused about the principles involved in ion-exchange.

For example, when working with an anion-exchange column, if I raise the pH, won't I get contradictory effects?

i.e.
the column resin is less positively charged (decreasing binding), but

the sample is more negatively charged (increasing binding)


I'm trying to separate a protein for further analysis using an anion exchange column (TSKgel DEAE 5-PW, 7.8mm x 7.5cm).

The pI of the protein is ~ 4.6, and I've tried binding buffer pHs of 6 - 9, but the protein is still not binding to the column, and is eluting close to the void volume. Can anybody suggest anything I can try?

Btw, thanks to all the selfless people who contribute to this forum, it's pretty great .

You can get contradictory (and confusing) effects with the weak base (or weak acid) exchangers, which is why I prefer to work with the strong exchangers if possible.

That said, the pKa of DEAE resins is somewhere around 9-10 (if memory serves), so changes in the ionization of the stationary phase should not be an issue until you get up above pH 7-8. That should give you plenty of usable pH range for a protein with a pI of 4.6.

You didn't specify the buffer concentration or the salt gradient used, but one possibility is a decrease in ionic strength. Another possibility is to look for a higher-capacity (and therefore presumably more retentive column).

I have tried using Tris-Hcl, Bis-tris, and Bis-tris propane, with a buffer strength of 10mM up to 50mM.

How low can I go? And should I match the ionic strength of the sample to the mobile phase?

The counter ion I have tried is chloride (up to 0.5M), but I didn't think it had any bearing, since I just realized that the protein is eluting even before the gradient starts, i.e. it is flowing right through the column.

TQ

you can try a scouting run by starting from zero salt (buffer only) followed by steep salt gradient. Then optimize your gradient on basis of your retention.

SSGG: If I'm reading your post correctly, you get no retention with 10 mM buffer at pH 7 and zero NaCl concentration. Assuming your sample does not contain a huge amount of salt, a number of possible explanations crop up:

• - your protein is very weakly retained, and you need a higher capacity column (actually, this is fairly unlikely; I would expect to see at least some retention).
  
  - your protein is a lot more basic (higher IP) than you think (in which case, try cation exchange)
  
  - your column has been seriously contaminated/deactivated (sulfate? phosphate?).

Dear Tom,

I feel worried now - are phosphates and sulphates not supposed to be used with a DEAE stationary phase?

The column is quite new, and I have followed the recommended cleaning procedure (i.e. one or more of the following : d.d. water, 0.1M NaOH, , 10% acetic acid, 10% MeOH) each time. I have also stayed away from any additive not recommended by the manufacturer.

That being said, I have used PO43- and SO42- as counter-ions with this column previously. Have I deactivated the column?

Can anyone suggest a common substance to do a column performance test with? I don't have any UMP/CMP with me at the moment.

In general, polyvalent ions are much more strongly bound to ion exchangers than are monovalent. Therefore, if you use an anion exchanger with sulfate, for example, you will convert the resin to the sulfate form. If you then try to use the column with chloride, the chloride will only slowly displace the sulfate.

If you've cleaned the column as described since using it with sulfate or phosphate, that should take care of the problem (remember, I was throwing out hypotheses there!).

You've already anticipated my next suggestion, which is to test the column. Check with Tosoh and see what test mix they use. Alternatively, try to duplicate one of their applications notes for that column.

Sulfate or phosphate won't ruin your column (as Tom has indicated) but it could take time to remove these anions with dilute NaCl. Try one of these methods first:

1. Pump 0.5M NaCl through the column for an hour or so.

2. Pump 0.1M NaOH through the column for an hour or so until the column effluent pH matches the eluent pH. Then pump 0.5M NaCl containing the intended buffer for at least 30 minutes (until the effluent pH matches the eluent pH).

In case it helps, I was browsing for some LC columns on ebay tonight and noticed several strong and weak cation and anion Dionex columns on ebay. Pretty good deal too but I already have my set and I aint selling.