Chromatography Forum

I was wondering if anyone had tried injecting hexane (or some other non polar solvent) into a reversed phase HPLC system.

The obvious problem is that it's not soluble in reversed phase mobile phases, but I wondering if it would be possible to do it with injection volumes of 3 - 5 uL.

(the reason I ask is that in some cases hexane would work better from a sample prep perspective)

I would dry down the hexane after the sample prep and redissolve in acetonitrile:water for injection.

Not deliberately, but I inadvertently do it reasonably often when I have samples I'm analysing by NP and RP.

It usually doesn't work in highly-aqueous mobile phases. The components that are soluble in hexane drop out in RP solvents, blocking guards, and the peaks that do appear are seriously deformed, telling me that I messed up.

If the hexane doesn't dissolve, you have a slow flush with a miscible solvent ahead of you to clean the system. I keep getting reminded that methanol, water, and acetonitrile aren't miscible with n-hexane.

I've never encountered a situation where samples in an immiscible NP solvent gave superior peak shape on RP columns to samples in RP solvents.

Some solvents that are miscible in bothe phases, eg IPA, work OK, but I still like to match the sample solvent to the mobile phase, as that means insoluble material usually remains in the sample vial.

Please keep having fun,

Bruce Hamilton

If the peak of interest elutes really late, then it might not hurt. If it's an early eluter, then you'll have the problems described by Bruce. Which brings up a point: why not do NP ? If hexane is the "only" solvent that dissolves the sample, maybe it's not a ood RP candidate. Also, have you tried DMSO, THF, etc ? Of couse with those you'll also have some troubles with early eluting analytes getting peak shape distortion.

What about the opposite? Has anyone ever tried injecting - let's say - 100% ACN into a normal phase system.

Again, it's obviously not the best solvent. But I'm wondering if people have been able to do this successfully by injecting small volumes (5 uL or less)

Depends on the normal phase system. The biggest issue with hexane -> reversed phase is miscibilty.

If your normal-phase system had a significant amount of something like methylene chloride (for example), then there shouldn't be much of a miscibility issue injecting acetonitrile. The "strong solvent" distortion of peak shape would still be a problem.