I'm interested in finding any methods that are available on the web, or if anyone has, for the analysis of DPPE, DPPC, DPPG, and DPePC. Particularly being able to seperate the lipids based on the polar head group. Thanks
Daren

The following gradient method will separate all of these and their lyso analogs all in one run. However, it's a gradient method, requires a mass evaporative detector (also called ELSD) and uses three mobile phases. There may be newer methods out there but I know this works because I used it for hundreds of samples over a three-year period. If you need to detect only one at a time you can try the isocratic method. If you don't have a mass evap detector, using the isocratic method you might be able to use an RI detector but I've never tried it.

HPLC System: Quat system
Mobile phases:
A) 1:99 (v/v) THF: hexane (or iso-octane)
B) 80:20 (v/v) IPA:chloroform
C) 50:50 (v/v) IPA:water
Column: Spherisorb 3µ silica, 4.6 x 100mm, or equivalent
Run Conditions: flow rate - 1.5 mL/min
Column temp - 50°C
Detector: ACS Mass Evap from Polymer Labs (there are newer and more sensitive models from Polymer Labs or Varex--try the Alltech catalog)
Detector conditions: Photomultiplier Tube (PMT) - 3; Time constant - 5; Evaporator Temp. - 40°C; Nebulizer Pressure - 28 psi; Nebulizer gas - GC grade nitrogen
Sample diluent: 3:1 v/v chloroform:methanol
Sample conc: 1.0mg/mL
Sample Injection Vol - 100µL
Gradient:

Time
0 min (A,%)100
1 min (A,%)100
5 min (A,%)80 (B,%)20
5.1 min (A,%)42 (B,%)52 (C,%)6
26 min (A,%)30 (B,%)50 (C,%)20
26.1 min (A,%)30 (B,%)70
31 min (A,%)100
36 min (A,%)100

DPPG will tail badly which can be somewhat avoided by conditioning the column with DPPG.

Good Luck!

Thank you for your method. I will try it. I have another question. I have developed a reverse phase method that separates DPPC, DSPC, their Lyso-analogs, stearic, and palmitic acid (w/ELSD). However I have been having inconsistent recoveries with the fatty acids, when analyzing degradation of the phospholipid should I expect to see the lyso and fatty acid peaks grow proportoinally. Could it be a stability issue with the fatty acids, I normally prepare my samples in pure methanol. I tried preparing pure stearic and palmitic acid in a methanol/acid mixture thinking I would be keeping the carboxylic group protonated and the structure more stable, however by 24 hours the peak was about one 10th the size. Any input would be great, Thanks alot
Daren

I do not know this particular case, but we have encountered esterification of acids in methanol. I suspect that this might be happening in your case as well.

Go to the technical section of www.avantilipids.com for an HPLC analysis of phospholipids. I wouldn't waste any time trying an isocratic method with an RI detector.

Who said anything about isocratic methods with an RI detector?

Please read carefully the post by Anonymous on Dec 7.

Wow! I did not know that an evaporative light scattering detector is the same as an RI detector...

The ternary system given in the reply of Dec. 7 is useful if you have neutral lipids in your sample as well (fatty acids, wax esters, mono, di, & triglycerides, etc.). For samples of just phospholipids, a binary system can be employed.

Column = Si or diol (Kromasil or Lichrosphere work well). I have also used a YMC PVA Si column (polymeric, polyvinylalcohol based)
A = 80% CHCl3, 19.9% MeOH, 0.1%Aq.
B = 80% MeOH, 20% Aq.
Aq. = 0.005% (50uL/L) Heptylamine (pH 9.4)
Gradient = 0%B to 25%B in 14 minutes (0%B to 7%B for the PVA column), hold for 9 minutes. Return to 0%B over 2 to 3 minutes, not in a step
Flow rate = 1 mL/min
Low mixing volume (500-600 uL)is also required for this method to work,
The heptylamine minimizes the tailing of the PG. LPC will be the last peak around 18-20 minutes, and may be a doublet, separating the two isomers.

Question ... You say you normally dissolve your samples in MeOH. Is it also an acidic solution like you prepared the standards in?

Uwe is correct. A carbox. acid in an acidic MeOH solution will form the Me ester (the reaction can be driven further toward the ester by including a water scavenger such as triethylorthoformate in the mixture). The Me ester will be more volatile, and it will evaporate appreciably even at 40C. In addition, some of the smaller acids (lauric, myristic, and possibly palmitic have appreciable volatility, and may give reduced response at elevated temperature).

Addition of heptylamine (same amount as in previous post) will form a stable ion-pair with the fatty acid (doesn't dissociate upon evaporation), reducing it's volatility and making it more detectable.

Hi,

I work with preparation of liposomes along with incorporating pure enzymes, to study kinetics. I have to quantify the total lipid in the liposomes.
Since these liposomes are stored in a potassium phosphate buffer I haven't been able to use the total phosphorus analysis method. Can I use HPLC to quantify the lipid in liposomes, will the phosphate buffer separate from the lipid? Can someone suggest the solvent system and kind of column to use and at what wavelength should I be able to detect the phophatidyl choline. I use 12:0 phosphatidyl choline. Any help is greatly appreciated.


Thanks

Is DLPC the only lipid in your liposome? If yes, you should be able to develop an RP method as Daren (above) did with DPPC. An MeCN/water system should work which would allow you to work at <210 nm. An ELS detector though, would be the detector of choice here. It will be much more sensitive than UV.

One thing to add which may be obvious to others but took me a little while to discover. If you are using an ELSD detector the drift tube temperature may be too high for some fatty acids. Stearic and palmitic have a melting point in the 60-70 C range. I had initially developed a method to look at the degradation levels of DSPC and DPPC, with a 85 C drift tube temp, and was seeing inconsistencies with these peaks. I have since altered the detector settings, the altech ELSD which I use allows you to block the majority of elluent passing through the drift tube and on to the detector (impactor on mode, they call it) This allowed me to drop the tube temp down to 35. The acids and even their methyl esters can be accurately quantified. I use methanol instead of ACN in my mobile phase but, either way you should be able to develop a RP method with out much difficulty. Hope this helps.
Daren

In response to "Anonymous" on April 5:

If you need only to measure total PC, you may do well with a simple colorimetric test. It only will measure lipids that contain choline and it will not descriminate between PC's, but we use it all the time as a QC check on our commercial grade liposomes. The kit is made by Wako, I believe. It's pretty quick and very easy to do. 20 samples can be done in 30-40 minutes, but you will only know total PC. If you're interested in further details, call me at 631-689-0200 ext 2807 and I'll dig up further details.

Best of luck,

Chris