NOT if the sample is in hexane. Under most conditions pure hexane is not soluble in Acetonitrile. *Samples must be fully soluble in the mobile phase. You might try IPA, not methanol, as an intermediate solvent

With perhaps one or two very rare exceptions, pure ACN should not be your "mobile phase". I think you meant to say ACN and Water (or buffer) for a RP application.

Proper chromatography fundamentals include these guidelines: You must always use either the mobile phase OR a weaker, but FULLY soluble solution as the injection solution for all chromatography. Samples must be fully soluble. If you inject a sample which is not fully soluble in the mobile phase, then you are basically using the expensive HPLC column as a tubular filter and not performing chromatography at all.

To find the appropriate column and method to analyze your sample with you will first need to research your sample's properties and then follow some basic method development guidelines (HPLC or GC; If HPLC, NP or RP, Solubility?, Which detector(s) is/are appropriate What is your goal and what are you looking for?)'

Some basics can be found at the webpage linked below:

"Modern HPLC Method Development Tips (PART I & II)"

http://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html

http://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html

NO!  You would have to substitute a Hexane soluble solvent for Acetonitrile like Methanol.

Even Methanol is to polar a solvent.  Have you thought about 'Normal Phase' using a silica column and water saturated Hexane as the mobile phase.

Normal phase HPLC is to separate or detect polar compounds. I have non polar compounds dissolved in hexane. So I can't run normal phase HPLC.

Try SPE or dry your Hexane off and reconstitute with Methanol

No, hexane do not dissolve in ACN (moreover be careful because ACN tends to polymerize mainly in the check valves). Maybe you can dissolve your samples/extracts in isopropanol: this solvent is able to dissolve apolar compounds (e.g. lipids) and it is miscible with ACN. 

If you give us more information about the analysis we can give you a more accurate answer.  

Good luck and work!.

Also put a small amount of IPA in your mobile phase.

Normal phase HPLC is used to separate non-polar or low polarity compounds not polar compounds. If you need to use reverse phase, solvent exchange to acetonitrile or preferably mobile phase would be recommended. If your compound has some polarity, this solvent exchange could be performed by liquid/liquid partition with acetonitrile.

I am facing the same problem, my samples are from hexane fractions but I already redissolved them in methanol. my compounds for sure are non polar, so what mobile phases and column I should use for reverse phase HPLC

Faisal,

Reverse phase solvents are; Methanol, Acetonitrile, IPA, Water, THF (sometimes). Hexane (~1% water saturated), Chloroform, and Methylene Chloride are 'normal phase' solvents.

In your case since your compounds are non polar use IPA and a C8 or C4 or amino column.