Help: seperation problems on some lipids by HPLC

[zhzhengyuan]

I want to seperate some different kinds of lipids, including EPC,DSPE-PEG2000,cholesterol,DSPE-PEG-Mal,DSPE-PEG-D4 and D4(D4 is a peptide). I choose ELSD as the detector. The problem is that I am not sure elution condition. I need some suggestion.thx

[TimB]

I've done some phospholipid separation with HPLC and cCAD detection. (I am no longer with this company) From what I remember my mobile phases were Water + 0.05% Formic acid and Methanol + 0.05% formic acid around 1 or 1.5 mL/min.

Unfortunately i cannot remember the gradient or the column I used.

[ksharp]

Are you using PEG2000 tethers to link peptides to liposomes?

I use a high carbon load C18 (Phenomenex Gemini-NX, but almost anything will work) with mobile phases consisting of 85 to 95% methanol, remainder 0.1% TFA or Formic Acid.

I would start experimenting with a gradient of 0.05% FA in MeOH / 0.05% FA in H2O. I use UV detection @ 205, but ELSD is probably a better choice for detection if you have it. Good luck.

[jonathonswq]

zhzhengyuan and ksharp,hi!

Sound like a good idea.
i concern about it, too.

[Bruce Hamilton]

As always, when performing lipid analysis, start with the on-line site of one of the world's best lipid analysts. W.W.Christie.

If necessary, pay the money and purchase his books. He has a lot of free information on his site as well.

http://www.lipidlibrary.co.uk/

Bruce Hamilton

[zhzhengyuan]

Thanks for TimB and Ksharp, I'll try your mobile phase and gradient. yes,i use DSPE-PEG2000-mal to link peptides to liposomes. I want to know the proportion of how many peptides have been linked to liposomes.(i put the liposomes and DSPE-PEG-peptide togather, and incubate at 60C for an hour).
And thank you very much,Bruce Hamilton, it is very useful website, I have put it into my favorite, i'll scrutinize it later,thank you!
Maybe next week, I could find the answer and May the force be with me!!

[zhzhengyuan]

It is so difficult to seperate DSPE-PEG and DSPE-PEG-peptide.....
I have tried many mobile phase and gradient. but just single peak..

[Uwe Neue]

This is an interesting problem...

I have never done this kind of work, but I am wondering if RP is the right tool. Your reversed-phase retention is overshadowed by the long DSPE chains and the PEG, and the contribution from the peptides will be very small, making it very difficult to find differences.

I would try HILIC on a silica column, with a gradient from 95% acetonitrile to maybe 50% acetonitrile to start, with then some finetuning of the gradient. The reason is that now the hydrophobic chains will disappear, and you will get the interaction of the stationary phase / water layer with the peptide.

(PS: if it works, we should write a publication together...)

[juddc]

If you're determined to use RP or if Uwe's HILIC idea doesn't do the trick (tho' it sounds at least quite sensible to me), how about trying some of Kostas' perfluorinated carboxylic acids as volatile ion pairing agents? I'm assuming that DSPE is distearoyl phosphatidylethanolamine and my thinking is that between that and any 1' or 2' amines that may be on your small peptide, you might be able to alter selectivity enough to resolve the two components.

[zhzhengyuan]

The first gradient I've tried
ZOBAX 300SB-C18 4.6*150mm 35C 1ml/min ELSD
A: H2O; B:CH3OH;
0--15min:50%B-- 90%B;
15--25min:90%B；
25 --30min:90%B --100%B；
30-- 35min:100%B;
35-- 38min:100%B--50%B.


the DSPE-MAL means DPSE-PEG2000; the DPSE-MAL-D4 means DSPE-PEG-peptide.
Although this gradient could seperate the two things(I don't know if the two lipid has been seperated entirely ),but the width of the DSPE-PEG-peptide's peak is too wide and ugly.....any suggestion?

[zhzhengyuan]

[kanryu]

So this topic is a bit older but I came here with google while searching for some methods.

I could use some advice on a separation of two compounds we put in liposomes: PEG-DSPE and DPPC. I am using a 1260 from Agilent and got two brand new columns (MN: Isis, 250mm*3mm, 3µm and a SPHINX 100mm*3mm, 3µm) and try to separate forementioned lipids. I am using 80% MeOH + 0,1% Et3N at the moment in isocratic mode (I am using a RID-detector) but can't get them separated. I get a big peak from the methanol and a small peak afterwards (2,2 min MeOH, 3,8 other substance) on the Sphinx-column @ 0,5mL/min but when I coinject the substances there are no more peaks - I thought the PEG on the DSPE should have a big impact on the retention or is 80% maybe even too low and it gets stuck on the column?

What I wanted to try tomorrow is: 90%MeOH with TFA maybe and also dissolving of the lipids in this mixture (at 80% it gets totally slurry). It would be really cool if I could separate this with my equipment ..