Chromatography Forum

I'm interested in performing RP-HPLC of a protein at neutral pH in order to detect and quantitate a bound ligand that has an increased extinction coefficient in pH range of 6-8. Since I've never done RP-HPLC at elevated pH's, I would like to get some advice on appropriate columns, mobile phases, and ion pairing agents. Some questions:

What is the best way to achieve neutral pH? Presumably this can be achieved by simply adding a buffer (phosphate?) to both mobile phases or using mixed ion pairing agents.

What is the best way to minimize silanol interactions? Presumably one can include an additive (sodium perchorate?), use end-capped silica-based columns or simply avoid silica-based columns entirely.

Thanks for any information.

Jon

6-8 is well within the buffering range of phosphate. In general, you buffer the aqueous part of mobile phases and then add organic as appropriate.

The best way to avoid silanols is, indeed, to use non-silica-based columns (no silica = no silanol), but these come with their own baggage of problems. If you use silica-based columns, newer ("Type B") columns made with high-purity silica have a lot fewer tailing problems than older ("TypeA") columns. For any given type of column, an end-capped column will tend to have fewer tailing problems than a non-endcapped column.