Chromatography Forum

hello all,

i am setting up a method to use HPLC- MS to detect some metabolites and according to the protocol they used hexane in solvent A and B. so A has hexane/2propanol/formic acid/NH3 solution at 80/200/0.1/0.05 whereas B has 2 propanol/water and same ratio of formic acid and NH3. so following the protocol, we have no issues of detecting the molecules using LC MS.

however one colleague came to me and said he is worried hexane may damage LC autosampler and pump seals etc as we are not sure if hexane is compatible , as we usually use acetonitrile/methanol as solution A/B in other applications. my question is whether hexane is indeed a problem for pump seals ect.

our LC system is Ultimate 3000.

thank you.

Generally, it is preferable to have a dedicated LC system (at least a dedicated pump) for normal phase (NP) applications with NP pump seals and (for some pumps) some other parts.
Search for the words "normal phase" in the instruments manuals, for example:
https://assets.thermofisher.com/TFS-Ass ... ration.pdf
https://www.agilent.com/cs/library/user ... _ebook.pdf

To go along with what Vmu is saying, different solvents will affect different seals differently. High organic modifiers in RP-LC conditions have even been known to cause small issues so switching from a reverse-phase to a normal-phase analysis should be done carefully and with care. Personally, I'd be worried about the miscibility of your two phases. IPA is miscible with everything, but water and hexane will not mix at all and could cause you some issues down the line. The stationary phases are also made for specific types of analyses and could be affected differently in a RP vs NP separation. It just seems weird that your mobile phase A is more of a non-polar MP, while the mobile phase B is a "tougher" reverse-phase solvent mixture.