anion exchange problem

[MK]

I'm looking at recovery of a production step (diafiltration to remove CsCl). I'm using Resource Q which is a strong anion-exchange column. Gradient is from 300mM NaCl to 1500mM NaCl in 50mM Hepes pH 7.5.

My problem is I'm getting too high recovery of about 150%.

A) samples before diafiltration are in 5 mM Hepes pH 7.8 and high in CsCl. I have diluted these to 50mM Hepes pH 7.5 to reduce the salt content.

B) samples after diafiltration are in 5mM Hepes-20% glycerol pH 7.8 and without CsCl. These have been diluted to starting buffer: 300mM NaCl-50mM Hepes pH 7.5 and injected.

I thought that this was Cl- interference in A) samples and did some spikes to confirm correct dilution factor. Strangely, the spike recovery looks fine, around 100%?

Any ideas, what this could be?

What is the role of analyte counter ion? Can Cs+ for A) samples act differently as a counter ion, compared to Na+ for B) samples? What about glycerol?

Many thanks

[Chris Pohl]

MK,

Cesium can definitely effect chromatographic properties. Anionic species will generally have significantly more retention in the presence of cesium if there is a component of hydrophobic retention in the retention process. However, I'm not quite sure I understand how such properties would result in high recovery. Is the effect analyte concentration dependent? Is your analyte a protein? What is its pI?

[MK]

Chris Pohl,

The analyte is a bit unusual, it's a virus with a negatively charged protein coat. pI is around 6. I've got the best response (peak area*dil factor) by 9x sample dilution, for samples high in CsCl. Response increases a lot from 1x to 4x and then sligthly from 5x to 9x. I haven't seen shift in RT. For B samples I get the same results with all dilutions.

Many thanks