Chromatography Forum

Hi folks,

turning to this forum again in desperate need for some inspiration.

The setup: FPLC, AIEX, gradient elution. Binding conditions:pH 7.5 ~2.5mS/cm. Gradient elution at pH 5.5 with a citric acid buffer of increasing molarity (starting as low as possible).

The problem: when I change from the 50mM Tris pH 7.5 buffer to a 10mM citric acid buffer at pH 5.5 (which is supposed to the beginning of my gradietn), the pH dips down to about pH 4 after ~1CV, eluting everything without any specificity.

Does anybody have a clue why this happens? How could I still achieve my gradient? What I want to find out is which citric acid concentration at this pH is the most effective for my separation, hence the gradient in buffer strength.

Thanks you guys for any help,

S

Hi Stefan,

I’m confused! How could it happen: Mixing 2 solutions – the one at pH 7.5 and the other at 5.5 and yet end up with pH of the mixture at 4?

Anyway, I’m not sure the setup you describe is optimal. Maybe you should drop the pH gradient and just utilize the ion strength part of the setup.
So if you just prepare a very low ion strength buffer at the relevant pH (for instance 2 pH units above the pI of your analyte) and use it as eluent A. Then prepare much more concentrated (10 – 50 times) buffer from the same salt and at the same pH and use it as eluent B. The preparation procedure can be reversed (i.e. prepare eluent B and then prepare eluent A by diluting a small fraction of eluent B)

With these eluents try to run a gradient starting from 0% B to 100% in about 20 – 30 min. The rest is small adjustment of the start conditions and the gradient profile.

Best Regards

Where are you monitoring pH (pre- or post-column)? If post-column, what I suspect is that your column is getting into the act to cause the wild fluctuation in pH.

Hi there, thanks for the answer. Yes it is either the column or the buffers themselves (more likely the coulmns) that cause this effect. Nontheless, as my product is eluted at too low a pH, I cannot work the gradient this way...I am thus looking for a way around that pH dip.

Yes it is either the column or the buffers themselves (more likely the coulmns) that cause this effect.

I guess that was a "blinding flash of the obvious", wasn't it?

What I was driving at was that the citrate is apparently somewhat retained on your column, and is displacing the counterion from your tris buffer. If that counterion is something like Cl-, it's no wonder the pH drops. One possible remedy is to pH adjust your tris with citric acid. Another is to keep the citrate in your elution gradient at 10 mM as a buffer, and increase the ionic strength using a neutral salt (maybe sodium sulfate, since your citrate would be mostly divalent at pH 5.5).

If that counterion is something like Cl-, it's no wonder the pH drops. One possible remedy is to pH adjust your tris with citric acid. Another is to keep the citrate in your elution gradient at 10 mM as a buffer, and increase the ionic strength using a neutral salt (maybe sodium sulfate, since your citrate would be mostly divalent at pH 5.5).

Cool, thanks a lot, that helped me out explaining that phenomenom, had not thought about that. I will try titrating my Tris buffer with citrate.
What would the neutral salt do though? Wouldn"t the citrate still displace them Cl-?

Again thanks for the answer,

S

What would the neutral salt do though? Wouldn"t the citrate still displace them Cl-?

Actually, that's a good point! The idea is to keep the column in the same ionic form throughout in order to avoid the pH swings.