Chromatography Forum

Basic compounds would be better analyzed at high pH (above pKa). Why some better methods for basic compounds use low pH?

A wide range of reversed-phase columns is available for low-pH operation (down to about 2.x). Only a handful of columns are commercially available for operation above about 7.x

Basic compounds would be better analyzed at high pH (above pKa).

is this statement accurate? I don't remember reading the prerequisite of HPLC is a neutral molecule. Could anyone please explain.

Tom
Thank u for ur comment, which explain the choice of the pH based on the availability of suitable columns.
Yet, I would like to understand why low pH also give good results for the analysis of basic compounds on RP chromatography. I thought that the lower the pH, the higher the ionisation of basic compounds and thus less good RP chromatography.

Richardinio,

At high pH, basic solutes are neutral and thus unable to interact in an adverse manner with residual silanols while at the same time providing maximum retention. At low pH, basic solutes are fully ionized and exhibit the minimum in retention (in general) but at low pH most, if not all, silanols are protonated so the effects of residual silanols are eliminated.

Thank u Chris,
Since the retention of basic compounds is minimun at low pH, the selectivity is also minimun. Right?

Richardinio,

Often, it's true that working at low pH will minimize selectivity differences between basic solutes but not always. This is especially true when the basic functionality is not identical for a pair of analytes to be resolved or when the basic functionality is aromatic. The main problem with working at low pH is that for highly polar basic solutes it may be difficult to achieve adequate retention in order to accomplish resolution. And this case, addition of an ion pair reagent, operating in cation-exchange mode or selecting a reversed phase column compatible with high pH operation may be advisable.

I currently use a method at low pH to separate 3 analytes of which one is a basic cpd with high pkas. Basically at low pH, the basic cpd ionises which is essential but addition of ion-pair reagent into mobile phase(anionic e.g SDS ) leads to interaction between SDS & basic cpd thus basic cpd is retained. You can then use your organic to vary retention of the other two neutral cpds.

The key is addition of ion-pairing reagent so that column surface is coated, once this is achieved, you can then have ion-exchange interaction between SDS & basic compound, and thus retaining your basic cpd.

I thought that the lower the pH, the higher the ionisation of basic compounds and thus less good RP chromatography.

Here is a link to an excellent introductory textbook. Check out the section on pH.

http://hplc.chem.shu.edu/HPLC/index.html