I understand that to separate molecules by HPLC (or SFC) you commonly adjust the pH of the mobile phase to get preferable charges (i.e. negative charges) on the molecules - so that they interact with the column and are therefore well separated and thus give a large distinct peak.

Im not completely sure I understand what happens to zwitterions like amino acids when you try to separate them. Is it correct to think that they randomly interact with the column and thus "spread out"resulting in a chromatogram with a lot of noise? Why is it that the connected MS also has problems detecting them (should it not continuously detect the molecule)?

Example of amino acids that could be difficult to detect:

enter image description here

  • 1
    $\begingroup$ Same as with other molecules, you adjust the pH of the mobile phase to get the anion or cation depending on what you are detecting. At neutral pH zwitter ions carry no net charge but are polar species so do not interact with the column like other neutral molecules. $\endgroup$ – Waylander Feb 13 '20 at 11:13
  • $\begingroup$ That is not the right structure for aspartate. You've drawn the side chain with an aldehyde group instead of a carboxylic acid. Also, the amino acid on the left, 2,4-diaminobutyric acid, is a bit unusual. It's perfectly similar to the "usual" proteinogenic amino acids, and the same methods would work OK with it, but you won't see it in most published methods. $\endgroup$ – Curt F. Feb 26 '20 at 0:06

Zwitterionic amino acids are routinely detected by HPLC and by MS. The only challenge is to find an appropriate stationary phase. The most common stationary phases for this type of separation would include octadecyl-derivatized silica (ODS, also known as C18) and a HILIC stationary phase. Many column vendors make columns explicitly marketed for separation of amino acids.


  1. Sciex markets an LC-MS method using a C18 column.

  2. Agilent markets an LC-MS method using a HILIC column.

  3. A decently-cited paper from 2016 describes a 3-minute LC-MS method for amino acid separation using HILIC columns.

What needs to happen is that analytes need to reversibly equilibrate between the stationary and the mobile phase via a single "mode". If there are multiple modes by which analytes can interact with the column, then peak shapes will be highly distorted and/or flattened into noise. An example of this would be if the analyte can both interact hydrophobically with the C18 moiety of ODS and via Lewis acid / Lewis base interactions with bare silanols (Si~OH) groups on the surface of exposed, underivatized silica. These days, column manufacturers go through great lengths to insure that very few if any exposed silanols remain exposed on derivatized silica resin used for analytical LC columns. Zwitterions are really no different than other analytes in this regard.

At the MS, zwitterions will need to be transferred to the gas phase as charged ions for detection. But this isn't really a problem either, at least for e.g. electrospray mass spectrometry.


Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.