Chromatography Forum

Hello everyone

Ive inherited a titer assay using protein A hplc. In the binding and elution buffers is 150mM salt and I am trying to figure out why the salt is even there. Can someone explain it to me?? Please be detailed as I am new to HPLC and protein chemistry

Thanks so much!!

It may turn out to be a longer story, depending on which salt we are talking about.
Is it Sodium Chloride? Also, which type of column or rather stationary phase support do you use?

I think you need to describe the mobile phase/s more specifically and the stationary phase support as well.

The salt could be a chaotropic agent (depending on the chemistry) and it could be needed for it´s ion strength.
Either way it's not a short story that could be explained in a few words.

Best Regards

Since you're working with protein A, I'm assuming that you're binding and eluting some type of antibody. In this case the salt is most likely there to keep the antibody soluble. Many antibodies will precipitate at extremely high or extremely low ionic strengths. IgM isotypes are particularly unhappy at low ionic strengths.

The binding buffer is 50mm glycine 150mm nacl ph8 the elation buffer is the same as the binding buffer only ph 2.5? The method is a titer assay for igg quantitation.

The column is a poros protein a cartridge

Sorry for the delayed answer. I had some urgent matters to attend.
The elution is facilitated by a pH shift, so the NaCl is added for solubility purposes.
As for the stationary phase support, you don't seam to have a knowledge of it, but I suppose it's some organic polymer.

Best Regards