Low-pH taling of bases in RPC

[kaavie0801]

To confirm the silanol activity, I usually inject benzyltrimethyl ammonium chloride and see the peak shape for detecting silanol activity. If the peak shape is symmetrical and the elution time is not too high, it means there is little cation exchange going on. You can convince the reviewer by a similar experiment.

Thank you for the good suggestion. Actually, I found protonated histamine showed no retention and no notable peak tailing under the same conditions (see the figure below; sodium nitrate as void marker).
(The first peak represents histamine)
In my opinion, this may indicate that there was no significant silanol effects: If If any significant ion-exchange interactions had taken place, protonated histamine would have had some silanophilic retention on the column.
Any comments?
Thanks

[kaavie0801]

As the name of your stationary phase implies, it has large steric groups (probably isopropyl) which protect the silica suface. Even if there are silanols, they must be shielded.

Thank you. A brochure from Agilent said：
“Agilent ZORBAX StableBond columns use patented, unique, nonfunctional silanes with bulky diisobutyl (SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN, and SB-Aq) side chain groups that sterically protect the key siloxane bond to the silica surface from hydrolytic attack at low pH.”

[M Farooq]

To confirm the silanol activity, I usually inject benzyltrimethyl ammonium chloride and see the peak shape for detecting silanol activity. If the peak shape is symmetrical and the elution time is not too high, it means there is little cation exchange going on. You can convince the reviewer by a similar experiment.

Thank you for the good suggestion. Actually, I found protonated histamine showed no retention and no notable peak tailing under the same conditions (see the figure below; sodium nitrate as void marker).
(The first peak represents histamine)
In my opinion, this may indicate that there was no significant silanol effects: If If any significant ion-exchange interactions had taken place, protonated histamine would have had some silanophilic retention on the column.
Any comments?
Thanks

This experiment along with the concept of the bulky silane will be a good educational addition to your manuscript. Note that when bulky silanes are bonded, the surface coverage is usually lower, so you actually have more free silanols; it is the bulky group which acts like an umbrella to protect the analytes and the stationary phase being exposed to bare silica surface. It is not clear whether the manufacturer further end-caps after bonding with a small silane or not. By chromatographic characterization, it is pretty clear that the analyte cannot see silanols very well. The reviewer, like any scientist, must have made an educated guess, which in your specific case turned out to be incorrect. Good luck!