stability of hplc columns
Posted: Sat Jan 19, 2008 2:26 am
by olivia
HI, I have read that HPLC C18 columns should only be operated in the ph range 2-8. Is that when measured in the aqueous component of the mobile phase.
Does addition of organic increase the pH of the mobile phase.
Has anyone got a simple explanation for this
Thanks
Liv
Posted: Sat Jan 19, 2008 4:30 pm
by Uwe Neue
What you can do with a C18 column depends on its design. Most C18 columns can be operated in the pH range of 2 to 8. Some packings can be used at lower pH, some at much higher pH. This should be stated in the C/U manual of a column.
At low pH, standard monofunctional bonding is inferior to di-or trifunctional bonding or to bondings based on bulky side chains. At high pH, hybrid packings give you an improved stability. The best stability at alkaline pH is achieved with the XBridge particle.
The measurement of the pH is best done in water to avoid ambiguities. The pH of acid-based buffers go to a higher pH when organic solvent is added. The pH of base-based buffers (such as ammonia) will decrease when you add organic solvent. I am not sure that anybody has studied carefully, how the stability of a bonded phase will change with pH and organic solvent. You have multiple variables in this game.
I am not aware of any "official" explanation why the buffer pK is changing as described. My simple home-cooked explanation is that when there is less water around, the dissociation of acids and bases is less preferred...