Making an UV inactive salt UV active during purification of charged anion with HILIC purification?

Question

In the context of a purification setup I am asking myself the question: Can a UV inactive charged substance made be UV active by a counter ion that absorbs UV light during a column run?

If so, when no other UV active material is present, than would it be a legitimate statement for me to say in a publication, that this UV signal of the counter ion is directly proportional to my product during the elution run and every product fraction would absorb (Or any anion that might present as an impurity?) Because a sole anion could not travel on a normal or hilic phase, right?

For example imagine a mixture of two components impurity $A^-$ and product $B^-$ which are very polar and do not show absorption because the cation $C^+$ (same for both) is inactive. I would assume that you see nothing on the UV/Vis detector. However, I was able to exchange recently the counter ion to one that absorbs at 254 nm: $C^+_{UV}$, resulting in overall stoichiometry of the impurity $AC_{UV}$ and product $BC_{2,UV}$. However, if I would add buffer salts to the mobile phase, the stoichiometry of the different components might change during the run, and product could elute which is not paired with the UV active counter ion? To avoid this, can I run the column completely without any additional buffer salts or is this not advisable?

Answer 1

You case study is an interesting scenario but in the question there are implicit misconceptions associated with hydrophilic interaction liquid chromatography (HILIC). The late inventor of this technique was a good acquaintance and we often discussed HILIC phases in conferences. The punch line is that HILIC mode does not work without buffers. As you have studied about HILIC, the role of buffer is to promote the formation of water layer on the surface of the stationary phase. The "thicker" the water layer, higher is the retention of a polar molecule. He used to emphasize that you must have high concentration of buffer in the mobile phase in order to use the HILIC mode (even on bare silica). The typical concentration should be 25 mM salts; usually ammonium salts adjusted to desired pH. Otherwise, one is basically using mixed-mode retention and ion-exchange properties of the stationary phase/silica surface.

You are right, in the presence of buffers one cannot guarantee that the ion-pair AB or BC are still intact.

Suppose you do not add any buffer to ACN and water, and use bare silica as a stationary phase. Your analytes, A and B are anions, silica at neutral pH has a negative zeta potential due to ionized silanols. Both ions will be repelled, at times, and they may elute even before the dead time and C may show some retention. Note that there are ions from everywhere, including ambient dissolved CO2, so there might not be a very strict charge balance condition for AC and BC to travel together. Ions can also come from glass and metals in the chromatograph. If your molecules are bigger peptides etc., they will show a horrible peak shape on bare silica. In short, salts are needed, with the right pH to ensure a good peak shape of analyte in HILIC mode. I do not recall seeing any HILIC separation without salts. Also, bare silica is not a good HILIC phase for biological compounds.