Chromatography Forum

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

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?

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