Chromatography Forum

I want to ask a question that pertains to a reversed phase analysis we have been working on. So this question does not pertain to a hilic separation. The reason I mention hilic in the title is that I think people with hilic knowledge are the ones who can answer this.

We have an analysis that we run in our lab for minocycline. We have been using it for a while and it works well. But recently we were asked to analyze samples that have minocycline in the salt form instead of the base form. When we inject these samples we get a smeared peak, because it converts from the salt form to the base when injected into the mobile phase.

So we have been trying to convert it to the base form in the sample prep (if we can accomplish the conversion in the sample prep we can eliminate the smeared peak). The diluent is 90/10 ACN/Water and we tried adding some basic species into the diluent to accomplish the conversion to the base form. We tried triethylamine, and we tried K2HPO4. But so far we have had no success.

The reason I'm directing this question to hilic folks is because I think you have experience with accomplishing acid/base reactions in non-aqueous (or low aqueous content) solutions.

So can anyone suggest why our approach did not work, and how we may be able to make it work. I know that it's possible to accomplish conversion from ionized form to unionized form in 90% ACN, but thus far we can't get it to work.

Well, 90%ACN as diluent is not really good for RP chromatography, to start with. Do you really need to use such high% organic in your diluent?
What's your mobile phase, do you use a buffer in it? I hope so, since your analyte is ionizable, so you should control the eluents' pH. If you don't use a buffer, I'd strongly recommend to do so, this might solve your problem at once and will make your method more robust.
Generally, rather than adding stuff to your diluent I'd rather try to bring the diluent as close as possible to the mobile phase. That is, use the mobile phase as diluent, if possible, if not, at least use the same buffer as in the mobile phase and use the least amount of organics as possible.

What is the goal of your experiment? If you are looking to quantify basic drug and acidic counter-ion you do mixed-mode chromatography to retain both:
http://www.sielc.com/upload/file/pdf/SI ... y_2009.pdf

if you want to isolate your compound as a base, then use anion-exchange resin, load your salt on the resin and wash is off.

Your smeared peak can be related to solubility of your drug salt in high ACN, to overloading of the column or to mismatch of diluent and mobile phase.

Vlad:

We know that the smearing, in our case, is due to the molecule converting from one form into another, upon injection. We have done experiments to show this.

Does anyone have any feedback regarding how to accomplish acid/base conversion in a high organic diluent. I know that it can be done. In fact I know that most HILIC analysis are done with the goal of getting the molecule in an ionized form (in the initial conditions, which are usually high organic). But I'm wondering if higher levels of acid or buffer are needed, or heating.

At least for this application, our approach has not worked.

salt form exists only in solid state, if it is in solution then it is protonated base and acidic counter-ion in "vicinity" when you separate it you still have protonated base unless you went up in pH above pKa of the base.
your problem can also be related to ket-enol isomerization of your molecule

We had a similar situation with an application in our lab (but in our case we were doing a HILIC separation).

We never were able to convert the molecule to the un-ionized form. We eventually gave up and put in a retention gap to let the molecule convert once in the mobile phase.