I wrote to this forum previously for advice, and now have a method which is working well, and at least I have happy customers. However, I am not sure I understand what is happening chromatographically. As I said before, I have more experience with GCMS than HPLC.

I am separating cetylpyridinum chloride from other components in mouthwash and toothpaste formulations. I am using a Restek Ultracyano column with a mobile phase of 67%MeOH / 33% pH2.5 PO4 buffer. Again, this seems to work well, I am able to separate the CPC from the benzoic acid, sodium benzoate,dyes etc.

So, my question is this. I can't visualize what is happening chromatographically. CPC is a quaternary ammonium salt. So by maintaining a low pH, I am stabilizing the ammonium ion, so I am chromatographing a cationic species, is this correct? But when I increase the aqueous component of my mobile phase, I increase retention. I would expect that if it is an ionic species, increasing the aqueous would reduce retention.

If anyone can help shed a little light here, I would appreciate it.

Mike

Mike,

In the first place, cetylpyridinium chloride is a "Quat" with a permanent positive charge. Changing the pH should have no effect on the retention process other than for dealing with side issues (see below). In essence, since cetylpyridinium chloride is a surfactant, it will tend to concentrate at hydrophobic surfaces so as to lower the surface tension. Even though cyano columns are typically not very hydrophobic, the nature of the analyte is such that it will still tend to concentrate at this surface. The partition equilibrium for the analyte between the mobile phase and the stationary phase is controlled by the concentration of solvent molecules which bind competitively at the surface. It is for this reason that you observed increasing attention when you decrease the solvent concentration. The reason for using low pH is connected to the tendency of silanols on the surface of the silica to bind cationic analytes. Binding to the silanols typically brings with it very poor peak shape. A simple solution to the problem is to lower the pH to the point where all the silanols are protonated. So lowering the pH is simply a means of masking any residual silanols which might otherwise adversely effect the chromatography. In addition, because the analyte is surface active, added electrolytes will also influence retention. For example, more polarizable anions such as perchlorate will greatly increase retention. Also, increasing the ionic strength will generally increase retention although the effect is greatest for polarizable anions and you might find the effect fairly minimal with phosphate buffer.