Retention Problems FAQs
I have a problem retaining my compound on a reverse phase column. The compound is an amino acid and it has very little retention on all RP columns, even at zero organic concentration in the mobile phase. Can a Primesep column help in this situation?
Cation exchange Primesep columns provide additional electrostatic interaction to analytes with basic functional groups. Thus, they can retain molecules with very little or no hydrophobic properties, primarily by ion-exchange mechanism. As a result, the amount of organic modifier has little effect on retention, but it effects the selectivity of separation. The pH of the mobile phase should be selected below the pI point for the AA in order to create a positive charge on the AA molecule.
Most likely your compounds are retained by the ion-exchange (IE) mechanism, and your mobile phase cannot provide a sufficient concentration of ions to promote the IE process. Typical modifiers such as acetic acid and formic acids produce insufficient amount of ions in the mobile phase. Use a mobile phase with a higher ion strength to solve this problem.
Acetic acid alone is rarely used as a means to generate ions for mixed-mode chromatography. Ammonium acetate is often used instead, because it can provide the required ion strength much more easily.
For example, acetic acid in a 10 mM concentration produces about 0.1 mM of ions in a solution with a pH of aqueous solution around 4. In contrast, 10 mM solution of AmAc at a pH of 4 produces about a 20 mM concentration of ions which is 200 times more than acetic acid alone.
If your acidic compounds are carboxylic acids, then they can be converted to a non-ionized form by lowering the pH of the mobile phase below the pKa point. In a non-ionizsed state, the carboxylic acids are usually more retentative than in an ionized state. In this case, cation exchange columns such as Primesep 100, Primesep 200, or Primesep A can be used to provide both ion-exchange interaction for polar amines and RP interaction for carboxylic acids. If your acidic compounds are phosphates or sulfates, then they are ionized at all useful pHs. In this case, the Primesep AB column can be used to provide cation exchange and anion exchange at the same time.