Chromatography Forum

Any experience out there with RP, HILIC LC-ELSD (or UV, if possible w/o deriv) of phosphatidylcholine (lecithin). Prefer to stay away from normal phase buffers.

Thanks alot...

I developed a method a while ago for DPPC and potential breakdown products. It was a RP method, C8 Xterra RP8, 250x4.6, 5 uM. Mobile phase A=0.05% TFA in water, B=methanol. Went from 70%B to 100% B in 25 minutes, then re-equilibrated, 1ml/min flow, using ELSD detection no derivitization necessary. Samples were in 75/25/1 meth/chloroform/water. Injection volume was 30uL, and LOD will be around 0.004 mg/ml. This should work pretty well depending on how long your fatty acid chains get, you may be limited with separation capabilities if the chains get too long. Hope this helps.

Daren

I don't no what are the other components of your matrix and if you have other phospholipid species in the same mixture, but I have used a RPC method to separate DMPC from DMTAP, DMPS and cholesterol and the resolution was satisfactory.
I used a C18 column
A: H2O + 0,15%TFA
B: Isopropanol + 0,05%TFA

You can start with a gradient of 50% to 100%B in 20 minutes. PC is well retained.
My goal was to quantifiy phospholipids in liposomes, so quantity was not an issue and it was possible for me to detect PC at 220nm. I don't know what kind of amount you are expecting.

Good luck

Martin

There were a couple of threads on this subject a while back, and there were a lot of good posts.

When I did a bit of this, I found the ELSD to be very good, and actually has better detection limits for saturated phospholipids than UV. Furthermore, the response factors for ELSD are all about the same, so you can easily get weight percents even if you don't know what every peak is. Natural sources have a pretty good spread of fatty acids.

One other chemist pointed out that UV is useful for detecting oxidized lipids.

When I did it I used a Dionex PolarAdvantage column with isocratic MeOH:MeCN:H2O:formic acid 47:47:6:0.25.

FastLC can you give more details about the goals of your method and the source of your lecithine (soy, egg, synthetic...)?

Dear Klaus,

The substrate is soy lecithin and the reaction looks at the decrease of the amount of lecithin or the apperance of the fatty acid residue.

Thanks

For soy lecithine the mentioned RP methods will give you a lot of different peaks, which is good to see the fatty acid distributon in your raw material. In your case, a method which gives single peaks for PC, lyso-PC and free fatty acids would be a better choice. In the past I used a amino-column with a MeOH/MeCN/NH4Acetate-buffer mobile phase (60/30/10 if I remember correctly) for that. It is surely not a perfect method but hopefully a good starting point for your method development. (For the choice of the detector, I might refer to my post under the topic "phosphatidyl serine")