Chromatography Forum

I am currently working on a method for separating glycerol, methanol, and acetic acid from an aqueous phase (all water). I have a Waters SymmetryShield Column and a whole range of solvents I can use and a DAD detector.

So far I've tried using a gradient from 100% water to 60% acetonitrile over the course of 30 minutes. That seems to sort of do the job (no water peak) but the rest of the peaks don't come out too well. I have never worked with HPLC before (I'm a GC guy) and am sort of shooting in the dark when it comes to this stuff, so any help whatsoever will be greatly appreciated! Thanks

Your DAD detector is a UV detector I think. You should only be able to detect acetic acid with that since it's the only one with a chromophore. You'll need a MALLS or ELSD. A refractive index will work, also.

These are very polar compounds, and you do not need a gradient on the SymmetryShield column.

Use water with a 20 mM pH 2.5 phosphate buffer, and use 210 nm with your DAD, in an isocratic run.

Since you saw peaks in your gradient runs, these maybe other components of your sample, and you may need to wash the column on occasion. Or it could be dirty water. But you should get good and decent peaks with your standards dissolved in water or mobile phase with the condition given above.