Chromatography Forum

I have developed a method using a 250 x 4.6 mm YMC ODS-AQ reverse phase column that yields a result that I cannot explain and I was hoping someone could.

My analyte is an unknown compound which appears to be a small highly polar molecule - very likely a COOH. All of my attempts to get it to retain at all on a reverse phase column had predictabley failed - the analyte eluted with the void volume. Then I tried a run in which I ran isocratic on the aforementioned column with 95% acetonitrile and 5 % water. The compound retained for ~12 minutes at 1 ml/min. The experiment was perfectly reproducible.

A collegue suggested that when the very polar analyte is in a system where both the mobile and stationary phases are nonpolar that secondary interactions become prominent and uncapped silyl groups are the mechanism of retention.

I would appreciate any responses which could definatively explain the behaviour.

Thanks,
benzech

Benzech, The idea that your collegue has certainly sounds plausible to me. Let me throw out another possible explanation. When you have an organic acid in a solution that has high content of organic modifier, the pKa of the acid is shifted to higher value. This shift could be several pH units at such a high conc of acetonitrile moving the pKa of your organic acid into basic range and hence causing the analyte to be neutral instead of anionic. Do you buffer the pH of your mobile phase?

And I'll throw out another possible/partial explanation: YMC ODS-AQ is a "polar endcapped" column (although YMC's literature is vague about the details), so you may well get secondary interactions (e.g. hydrogen bonding) with the endcapping moieties.

I would appreciate any responses which could definatively explain the behaviour.

I don't think you will ever get that. Many, if not most separations involve mixed mechanisms of various kinds. We can (and do) speculate, but getting enough information for a definitive answer is a PhD thesis project.

...and uncapped silyl groups are the mechanism of retention.

...that would mean that your retention mechanism is similar to HILIC mode and you should be able to get a similar result on a real HILIC-column.

Beside this, Tom's proposal convinces me even more, as I would expect only very few free silyl-groups on the surface of an endcapped column.

And I would also expect a very poor loadability (and peak shapes) for a HILIC separation on an endcapped ODS column.

Other question: Is your analyte still soluble under the proposed conditions of 95%ACN?

Hollow wrote:

...that would mean that your retention mechanism is similar to HILIC mode and you should be able to get a similar result on a real HILIC-column.

Exactly. I, personally, have observed this effect on several occasions.

Beside this, Tom's proposal convinces me even more, as I would expect only very few free silyl-groups on the surface of an endcapped column.

Actually, Tom’s clarification points towards the original assumption’s direction (i.e. HILIC mechanism).
One’s attention should be focused on the end-capping’s nature, which in this case is "polar endcapped". So, actually there is no need for free silyl-groups at all, in order to exploit the HILIC interactions.

Other question: Is your analyte still soluble under the proposed conditions of 95%ACN?

If it elutes, it’s soluble!

Best Regards

benzech, what is your evidence that the 12 min peak is your substance? What were the conditions under which it supposedly eluted at the tm? What evidence do you have that it diluted at tm under those conditions?