Hello!

I write to ask for help in developing a
RP HPLC method for analyzing a tricky
compound; I have little experience so don’t
be bashful with suggesting the obvious. I
would like to analyze a small chemical that
has pKa’s of 2.3 and 3.3; below 2.3 it carries
a positive charge and above 3.3 it is negative.
Most methods I’ve seen use a pH of 2.8 but
I think this is too close to the pKa’s to be
repeatable. I don’t have access to an ion
chromatograph. Have any of you analyzed a
chemical with a negative charge using RP?
Do I need an ion exchange column or can I
do this somehow with a carbon/silica column?
The chemical’s molecular weight is ~320.

thanks!

Jon

Jon,

In the first place, I suspect the decision to use pH 2.8 stems from the fact that retention is generally maximal when the sample charge is zero. A pH value of 2.8 should put the compound at a point of formal neutrality although, of course, a good portion of the molecule will be zwitterionic under these conditions. Even though operation under these conditions might render the separation of bit more sensitive to pH than normal, I wouldn't go so far as to say that it rules out repeatable chromatography. You'll just need to be a bit more careful about eluent pH control. I would say that weight based eluent preparation instead of pH meter based eluent preparation will give you more than enough accuracy for reproducible chromatography.

However, if you feel that the separation isn't good enough then you could use ion pair (I would use tetrabutylammonium ion), to increase retention of the anionic form of the analyte. Generally, I would start with 5 mM ion pair reagent concentration. The thing to remember is that once you use an ion pair reagent with a C-18 column, it's difficult to get back to "normal" operation so you really need to dedicate a column if you're going to use ion pair. Also, it's good to make sure you have a clean (in the UV) ion pair reagent. Otherwise you'll see a lot of artifacts in your chromatography. In my opinion, the best reagent comes from Fluka and/or Sachem.

thanks!