Ion Exchange chromatography of high salt sample??

[rick1112]

hi

I would like to know what would be the effect of analysis of High salt sample (protein) by cation exchange chromatography ?? (lets say the amt of salt is approx same as that in elution buffer)

thanks a lot

[tom jupille]

Isocratic or gradient elution?

[rick1112]

its gradient elution...

[Kostas Petritis]

You will have some undesirable effects such as peak splitting and/or deformed peak etc... The magnitude of the effect will depend on your injection volume relative to your column volume... You could minimize the effect by minimizing the injection volume (in detriment of your sensitivity). Your best bet would be though some kind of buffer exchange before you inject...

[rick1112]

hi kostas petritis

i cant do a buffere exchange as i need to see the stability in the present matrix so an exchange of buffer will kill my purpose...

i went on to ask this as i remember reading somewhere about an RP method analyzing sample containing high % organic phase (i tried searching for the article i cant get it...)....

[Uwe Neue]

Retention in ion-exchange depends on two different things: pH, which can be used to change the charge of complex analytes such as proteins, and salt concentration. If you have a high salt concentration, you may still be able to retain your analyte if you start with an eluent pH that creates a lot of charge on your molecule, then run a step gradient to your elution pH, followed by the usual salt gradient to elute.

[HW Mueller]

rick, I don´t understand what you are doing. You are running a salt and/or pH gradient, but you are not "allowed" to change the matrix?