HPLC method dev strategies for Zwitterions

[Mike H.]

I've done some basic Method development, taking a couple classes and know the basic stuff, but need some help on how to deal with a separation where the main peak is a zwitterion and the majority of the impurities are acidic (-COOH).

Is it possible to suppress both species at the same time?

Are there LC techniques/columns where ionic interactions are not part of the overall mechanism?

Any tips would be helpful. thx

[Vlad Orlovsky]

Mike,

What is the nature of your zwitterions - quaternary amines and sulfate/phosphates, carboxylic acid and primary and secondary amines or else. Depending on the number of ionizable groups strategies can be different. With Quaternary amines and sulfates/phosphates it is almost impossible to suppress ionization of these strong groups. If you have basic and acidic groups ionized your compound becomes much more polar and you might need to use HILIC mode, if you molecule is hydrophobic enough it might be analyzed by RP. IN any case, mixed-mode chromatography (RP/IE and HILIC/IE offer nice retention control and separation for zwitterions. Here are few examples:

https://helixchrom.com/applications/hpl ... lc-column/ (DOTA and DO3A zwitterions)

https://helixchrom.com/applications/hpl ... de-column/ (isomers of aminobenzoic acids)

https://helixchrom.com/applications/hpl ... de-column/ (isomers of pyridinecarboxylic acids)

https://helixchrom.com/applications/hpl ... hd-column/ (retention of 18 underivatized amino acids)

https://helixchrom.com/applications/eff ... de-column/ (effect of mobile phase on retention of zwwitterions)

https://helixchrom.com/applications/hpl ... de-column/ (retention of zwitterion taurine)

Let me know nature of your zwitterions and I will try to advice you.

[Mike H.]

One primary amine and one carboxylic acid.

[Vlad Orlovsky]

For zwitterions with carboxylic acid you can go to pH 2 (TFA, sulfuric or phosphoric acid) and suppress ionization of carboxylic acid, amino group will be fully ionized. To suppress amine ionization you need to go to pH 12. Not too many columns will survive at such high pH. Between pH 2 and 12 you will have both of groups ionized to some extend. You can go to pH 2 and analyze your zwitterion as slightly hydrophobic amine. In most of the cases you will need to go to really low organic (0-5%). It is easy to analyze underivatized aminio acids with something like Coresep 100 (RP/cation-exchange) or Amaze TH (HILIC/cation-exchange/anion-exchange).
If you share structure with me I can advice you on what to do, alternatively you can send us a sample (5-10 mg) for fast free screening.

[Mike H.]

Thank you for the help.