Chromatography Forum

Hey

I'm doing a project about neurotransmitters (GABA, 5-HT and ACh) and phopholipids (DMPC). I want to separate them on a HPLC system, with a refractive index detector.

So far i haved tried with different eluents (ex. methanol, ethanol, 1-propanol) in different concentrations. I have a problem with a negative water peak, which interferes my other peak(s). I've tried using eluents that did not contain any water, but then i had a solubility problem with my neurotransmitter. As i see it, it "should" be relatively easy to separate the two molecule classes, when they are as different as neurotransmitter and phospholipid. So far i have only tried to measure with GABA, without any luck.

I use a C18 reversed phase column and varian HPLC equipment.

Does anyone have some good idea's i perhaps can use?

Sincerely
BoGa

have you tried doing a 2 step sample preparation?
the first one with some water like you do right now; to get your compounds dissolved and then to dilute the first solution in the mobile phase.

If i did a 2 step preparation like you mention, i can not see how i can remove the water peak. Since there will be more water in the sample, than in the mobile phase.

I have tried dissolving my sample in the mobile phase, i still get a large negative peak.

I've not used a RI detector, but i've done some phospholipid seperation, and my sample diluent is straight methanol.

Are you trying to measure the neurotransmitters in biologicals? I think the detection limits may be to low for RI. This is typically done using Electrochemical detectors. You may wanrt to check your LOD vs expected levels. Which lipids are you trying to separate?