Chromatography Forum

This was discussed early: https://www.chromforum.org/viewtopic.ph ... ase#p33698

I like to revisit: what happens if you inject a 5 uL sample in hexane to RP HPLC (C18 or C8, M.P. 10% to 95% ACN)?

Benefit: when hexane is used as extracting solvent and injected directly you do not need to worry about the potential loss of unknown during evaporation and reconstitution

The questions: what happen to hexane? stay in the column, bleeding, or eluted out completely every injection(eluted up to 95% ACN)?

how analytes behavior in column? polar, medium, and non-polar analytes, are they doing the same or different?

When it is OK and when not? why?

I know it’s an old post but in case you’re still wondering ...
Since neither water nor acetonitrile is miscible with hexane the most likely outcome is hexane slug coating the particles, blending with the c18 layer and staying there until it is flushed out if this is at all possible.
My guess is that the column may actually still retain some efficiency, unless you keep injecting new plugs of hexane in which case you will have a trickle of nano bubbles of hexane physically pushed out by the momentum of the mobile phase leaving behind only a stable thin layer on the particles. In this case you will have analytes partitioning into moving layers and bubbles... possibly very cool physics but no chromatography.

"what happens if you inject a 5 uL sample in hexane to RP HPLC (C18 or C8, M.P. 10% to 95% ACN)?"

All you do is contaminate the column. It serves no purpose and such a tiny amount of liquid relative to the column volume (which we do not know what it is because you did not provide that key info, but an average column has 1 to 3 mls of volume inside) would result in nothing at all or some residual contamination. Worst case, the column would need to be flushed with some IPA to "chase" any remaining hexane out of the system.

Injection of hexane into RP HPLC is not terrible, in my opinion. Although I have never injected neat hexane, I did dilute hexane extract with IPA first prior to injection, no adverse effect was observed as long as the diluted sample is miscible with the initial mobile phase.

I don't believe hexane is much more hydrophobic than PAHs, it should elute from a c8 or c18 column under conditions with high organic composition.

As no signal for hexane can be observed with common LC detectors such as UV, CAD or ELSD, or MS, it would be interesting to see what happens when injecting 1ul of hexane into a 100% ACN with a c8 column and RI detector. I would expect it to elute at the void volume.