Phospholipid seperation

[TimB]

Hi, this is my first post here so forvgive me if i'm asking things that have already been asked. I did a search for "Phospholipid" but the topic didn't turn up anything that really helped me out, so here goes...

I am working on a method for a Phospholipid that is ~95% Phosphatidylcholine (PC) and ~4.0% Lysophosphatidylcholine (LPC).

The method I am using was obtained from the vendor, and is a normal phase method that uses ELSD. However, after following the method carefully, my chromatagrams are coming out horrible. With ELSD, I understand reproduceability goes down, but is there anything I can do to get a somewhat normal baseline?

Second, I also tried cCAD with the same conditions as I did for ELSD, but my baseline starts off high, and i end up getting what almost looks like an Infrared spectrum.

In all of my chormatagrams, I also have a huge drop in signal at about 20 min which I think may be because of the gradient.

Any suggestions on how to improve this chromatography for a relatively new chemist? Method conditions are listed below:
-------------------------------------

Column: Phenomenex Luna HILIC 200A 5.0um 150 x 3.0mm with HILIC 4.0 x 2.0mm guard cartridge

Column Temp: 55 deg C
Flow Rate: 1.0 mL/min
Injection: 20.0 uL

ELSD Settings:
Temp: 50 deg C
Gain: 1
Gas Flow: 2.0 L/min

Mobile Phase A:
n-hexane 81.42%
2-propanol 17.00%
acetic acid 1.50%
Triethylamine 0.08%

Mobile Phase B:
2-propanol 84.42%
purified water 14.00%
acetic acid 1.50%
triethylamine 0.08%

Gradient (%B)

0.0min (5%)
5.0min (20%)
8.5min (40%)
15.0min (100%)
17.5min (100%)
17.6min (5%)
21.0min (5%)
22.0min (5%)
27.0min (5%)
29.0min (5%)

Sample Diluent:
50:50 n-hexane/2-propanol

Sample concentration is 2.1 mg/mL

Thanks in advance.

[Kostas Petritis]

What happens sometimes with normal phase mobile phases and ELSD is that the temperature at the evaporation tube decreases significantly making your mobile phase non-miscible so you start detecting non volatiles. This is supported by your gradient info where when you reach at 100% B where you only have proponol and water you experience a huge decrease in your base line (it will still be probably kind of noisy due to non-missible leftover in the evaporation tube. I do not know which ELSD you use but increasing gas temperature, drift tube temperature, evaporation tube temperature (or change completely to more miscible -in low temperatures- solvent combination can help with your problem.

[JSAS]

How about something like this instead;

http://www.jlr.org/cgi/reprint/30/4/607.pdf

[TimB]

Thanks for the replies. After talking to a few co-workers, I'm thinking of changing the mobile phase to what you suggested. Oh by the way, the ELSD I'm using is an Alltech ELSD 2000

JSAS- thanks for the article.

[Carl Sanchez]

These compounds can be nicely separated in HILIC mode. Since you are using a HILIC column I suggest the following conditions which I have used for lipids. You can adjust the gradient or go to isocratic conditons if you need more or less resolution.

A = weak mobile phase = 95/5 ACN/100mM ammonium formate pH 3.2
B = strong mobile phase = 50/45/5 ACN/H2O/100mM ammonium formate pH 3.2
time %A %B
0 100 0
1.5 100 0
11.5 0 100
13 0 100
20 100 0 (10CV requil)

The sample injection solvent organic should match the initial mobile phase, i.e. 95% ACN. If your system uses a wash solvent then the wash solvent must also contain 95% ACN.

The phosphatidyl cholines will elute first followed by the lysos later.

[mbicking]

These classic systems for the phospholipids can be very difficult to work with. I once tried to follow a "standard" method that suggested the silica column required three days for equilibration! (That's right three DAYS, not three hours!) Although I did not believe this was accurate, my experience indicated that the system was in fact not very stable over the first two days. There are some reports of better mobile phase systems that are more stable. I can provide the link if you are interested.

But I would try the HILIC suggestion. The mobile phase is much simpler.

[JGK]

I have validated this method for PC quantification in the range 100 – 600 µg/mL.

It was a quadratic calibration, and as mentioned previousley is a complete "bugger" to equilibrate. Our procedure was:

"Overnight, prior to performing an analysis sequence, perform multiple injections of DIluent (between 30 – 50 injections) to “accustomâ€

[mbicking]

I found the following conditions in an older Agilent application note. The ammonium acetate apparently helped with colum equilibration and stability, but was also volatile.

The application note ID is: 5989-2848EN

Mobile phase:
A = 95:2.5:2.5% hexane-isopropanolmethanol
B = 40:60% isopropanol-methanol
Both solvents contain 10-mM ammonium acetate
Flow rate: 1.5 mL/min (prep column)
Gradient: Time, min %B
0 0
20 18.7
20.2 100
25 100
25.1 0

Have not tried it, but discussed with the author, and he thought this was a better approach than the other methods. The ammonium salt probably adsorbs strongly to the silica surface, and somehow helps with equilibration.

[juddc]

I've had some luck with phospholipids, however I did things a bit differently than one usually sees.

I used a hybrid particle RP column (Xterra) at about pH 9-ish (Phase A = ~0.1% triethylamine, I think) along with acetone as my phase B and was able to quite nicely separate phospholipids from lysolipds from fatty acids. Using the acetone in concert with the ELSD gave me quite good sensitivity and the resolution and peak shapes were quite good.

There are a few caveats with this one, though:

1. Standards needed to to be dissolved in a solvent that was a good deal more hydrophobic than the starting MP, so injection volume is limited. I believe I used an IPA-Hexane mix for first std and acetone for second dilution. Still, I got away with 5-10-uL using a 4.6x100 column on a Waters 2695.

2. pH. I didn't see any problems with sample or standard stabilityfor my a with the MP at ~pH 9, but you would do well to check

[TimB]

Again thanks for all the comments. This forum is the greatest thing since sliced bread.

Anyway, I should give a little bit of an update on this project. At my boss's request I've canned the ELSD method. We have decided to go with a corona detector and a Phenomenex Hilic 3.0um 100A 75x4.6mm Column.

The method was based off a previous Phosphatidylcholine method someone developed. So far we've had great success with it. Not only can we use the corona detector, the method is easily transferrable to Mass spec.

While i would love to develop the ELSD method alongside the CCAD method, I don't have the time, and I am actually leaving my current company next week. (Such is the life of a temp i guess).

This whole project has been a great learning experience though for chromatography. (Especially that the vendor method we received was horrible)