Step 4

We have established that your compound is a hydrophilic zwitterion. Below is an example of a separation of a similar compound. This example can be used a starting condition in your method development.

Hydrophilic zwitterions have no or very low interaction with the stationary phase by reverse mechanism. Instead, the molecule’s charge can be used as a means of retention in ion-exchange mode. For zwitterions with carboxilic acid, lowering the pH of the mobile phase leads to suppression of the ionization of carboxilic acid and creating an excess of positive charge which can be used in cation exchange retention. For zwitterions with a sulfate acidic group, pH has no effect on ionization and only HILIC mode can be used for retention. For zwitterions with a phosphate group, both anion exchange and cation exchange mechanisms can be used to promote retention and separation. A special case is a -amino acids. They can be retained and separated by BLIS (buffer-less ion separation mechanism).