Chromatography Forum

We are setting a high pH RP method for a mixture of tryptic peptides (9 proteins).

Column Gemini C18
Buffer A: 20mM Amm Fomate ph 10
Buffer B: 20mM Amm Formate 80% Acetonitrile pH 10
Flow rate 200ul/min
Column Temp 25 C
Sample buffer 20 mM Amm Formate in water
Gradient elution
Detection at 214 nm

We are getting binding of some peptide but there appears to be an inordinate amount of (hydrophillic?) peptide in the void.

Anyone have experience with high pH tryptic peptide separation and can suggest some modifications to improve peptide binding?

Thanks

At this high pH many peptides (if not all) are extensively charged thus predominantly hydrophilic.
Why not running the most conventional mode for this type of analysis i.e. TFA/water/ACN?
Best Regards

Thanks for reply. I would like to use high ph RP as an orthogonal separation method to low ph RP.

This is to reduce sample complexity for LCMS proteomics

I'm not sure you can call it orthogonal separation when both of them are reversed phase.

Anyway if the detection is MS (regardless of the description you provided in your original post where UV detection is mentioned) you have a few options regarding the mobile phase composition, thus retention control.

I will sugest a polar embedet RP column instead of a traditional C18 ditto.

Best Regards

Or, if you're looking for orthogonality, try HILIC.