HPLC retention question

[chemgc779]

could someone please explain the reason behind an increase in
retention as a result of an increase in the proportion of acetonitrile
part of mobile phase in reverse-phase HPLC?

Thank you.

[HPLC chemist]

I will give a shot, though only experimentation can give you 'good' peak separation.

Usually, in reverse phase as you increase the amount of organic in the mobile phase (in this case Acetonitrile), the molecule of interest will run toward the injection/solvent front. However, Acetonitrile is aprotic (unlike Methanol) and has no hydrogens to donate. A protonated molecule is retained by the HPLC column and elutes later.

You can test my hypothesis by using an acidic pH modifier (like formic acid) that is soluble in Acetonitrile. It should elute later.

[chemgc779]

Thank you for your kind explanation but would you be able
to explain a bit further as it is still difficult to understand it..

e.g. why would the analyte of interest would run toward the injection/solvent front..

Thank you.

[HPLC chemist]

Basically, what I am saying is that there is another, more prominent, retention mechanism. While, the analyte is soluble in Acetonitrile mobile phase (as it should be), its electron structure (pH) and in particular ability to accept electrons, determines its retention time.

Most molecules have amino and carboxyl acid sidegroups that can be protonated and increase its solublity in water. This thing is closer to Vitamin A which has a long phytol chain and is responsible for electron transport. Most HPLC methods for it use 95% Methanol and a C18 column.

I would try IPA and a mobile phase (IPA and Acetonitrile) closer to 'normal phase' and maybe an amino column.

[tom jupille]

You didn't specify the analyte or column but I would speculate that you are actually seeing "HILIC" behavior. Here's an example where you can see a the transition (this slide is from our HPLC Method Development course):


Note that the x-axis here is increasing H2O (i.e. *decreasing* ACN).

Granted, this was on a bare silica column, but if you have a lightly-loaded reversed-phase column you might see the same sort of thing.

[chemgc779]

HPLC Chemist: thank you for your explanations and it is easier to understand the reason now

tom jupille: thank you for your explanation