Hello Tatiana,

Below I attached an article that might be useful to your requirement.

kindly,

Ricardo

Hi Tatiana, there are three different methods that might help you.

Folch J, Lees M, Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509

Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

J. Lipid Res. 2008 49:(5) 1137-1146.

Feng

Hi Tatiana,

here is a efficient procedure to separate neutral lipids and phospholipids which works well by using flash chromatography

http://www.cyberlipid.org/cyberlip/home0001.htm

Greetings

Alexis

Sorry, I couldn't copy the URL, so I paste the procedure here:

The procedure we used has proved applicable to most lipid mixtures before further fractionations by HPLC, another low pressure chromatography or thin-layer chromatography.

Apparatus:

Glass columns (inside diameter: 8-10 mm, length: 20 cm) with a stopcock (glass or Teflon) at the bottom to control the solvent flow. Solid phase extraction cartridges (SPE columns) available from various companies (Alltech, Waters, Supelco, Analytichem Int...) may be also used if filled with silica.

Pyrex glass wool.

Glass Pasteur pipettes

Small vials

Reagents:

Silica gel 60 230-400 mesh (Merck)

Chloroform, methanol, acetone

Procedure:

Prepare a slurry of 300 mg silica gel in 2 or 3 ml chloroform and transfer into the column (equipped with a glass wool plug) with the help of a long Pasteur pipette. Open the stopcock to drain slowly the solvent and rinse the column with a portion of chloroform. Allow the solvent level to drop to the top of the gel.

Transfer the lipid sample in chloroform (about 1 ml) to the top of the gel. The amount of lipid that can be practically applied to this type of column is about 10 mg, for higher amounts increase the column size accordingly. A critical study with details on the optimum ratio between lipid mass and sorbent mass has been published (Pernet F et al., J Chromatogr A 2006, 1137, 127).

1- After complete elution of the solvent, add carefully down the sides of the column 10 ml chloroform. This will elute (at about 1 ml/min) neutral lipids (hydrocarbons, pigments, sterols, triglycerides, waxes, fatty acids..).

2- After draining the first solvent, add on the column 15 ml of acetone/methanol (9/1). This will elute glycolipids (cerebrosides, sulfatides, mono- and digalactosyl diglycerides, sterol glycosides) and ceramides.

3- After draining the second solvent, add on the column 10 ml methanol. This will elute all the phospholipids.

Evaporate the three fractions and dissolve the dry lipids with a small volume of chloroform (for neutral lipids) and/or chloroform/methanol (2/1) for all three.

Comments:

If a quantitative recovery of the free fatty acids are required, it is recommended to elute the neutral lipid fraction with 10 ml chloroform containing 1% acetic acid instead of pure chloroform. All other neutral lipids are present in this fraction.

Greetings

Alexis

IMO neutral lipids and phospholipids are difficult to be "managed" during single run as they have very different retention in the same chromatographic conditions. Thus I suggest to analyse these compounds separately. For Phospholipids a ternary mixture of Hexane:isopropanol:water (see paper by Toshihiko Yamagishi (JAOCS, Vol. 66, no. 12; December 1989)) is convenient and able to distinguish phospholipids class on silica column. It is recommended to use only RI or ELSD detection but not UV.

We have written a number of papers focusing on the sample preprartion and analysis of phospholipids published in JAOCS and the prep isolation of lipids publishd in JLC. Finally, there is a paper by Bill Christie using ELDS and a ternary mobile phase that does an elegant job of separating lipid classes

I suggest looking at Application Notes or other technical briefs published by LC column manufacturers. These include Supelco, Phenomenex, Agilent, Thermo and coutless others. I would look for separations specific to your compounds of interest. There are also effective internet search engines which may be helpful, and also try PubMed if your application is conical/medical.

If you are trying to purify, you can use amino propyl columns. Kaluzny and Epps published an approach that has been modified several times by Salem and others to do this on solid phase extraction columns (PMID: 3973509). For analytical, I would suggest LC/MS approaches. Various methods are published in recent lipidomics literature. It will depend greatly on your instrumentation at hand and your research goals. Good Luck

Thank you for all great suggestions!

I am currently using two different protocols to separate neutral lipids and phospholipids by HPTLC, which actually work very well. For the extraction step, I do use modified Bligh and Dyer (1959) protocol, which also works.

I have started looking for lipid separation protocols by HPLC now as we have it 'in house'. Hope to find one with all great suggestions, listed above. Thanks again!

Alexis, thank you for the procedure!!

William, Malgorzata, John and Ricardo, thank you for suggesting these papers!

try with RP-C8 column

Thank you Rajendra.

Hi,

I would go for a RP-18 separation - have a look here:

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Volume 1821, Issue 11, November 2012, Pages 1415-1424

LPC, LPE, PC, PE, SM and the TG and CE are nicely separated, the paper is a cap method but it should be upscalable easily.

see also Anal. Chem., 2011, 83 (17), pp 6648–6657.

A HILIC based fractionation you can find here:

Journal of Chromatography A, Volume 1218, Issue 31, 5 August 2011, Pages 5146-5156

But of course Aminopropyl and Silica based procedures also work, especially when larger amounts are to be fractionated.

Best

You could also use this 2006 paper - LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures. J. Lipid Res. 47(4), 804-

14.

Thank you Ulf!

Thank you Martin!

Bill Christe years ago published a method that works quite well. My son set up that method in his PI"s lab at Univ. of Nebraska and it works well. This has been a major issue of how to separate both the NL and PL on a single run. I don't have Bill's paper reference in my mind, but Cameron Murphy has had this method up and running using ELSD detection. It was a bit tricky to get going, but once it work it was quite nice. Try contact Cam and see what he has to say about it.

check out this article:

Agren JJ, Julkunen A, Penttila I. 1992. Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column. J Lipid Res 33(12):1871–1876.

You cal also obtain valuable information by the review article,

Li et al, Mass Spectrometry Methodology in Lipid Analysis, Int. J. Mol. Sci. 2014, 15, 10492-10507 (http://www.mdpi.com/1422-0067/15/6/10492)

If you want to separate the lipid classes, I would concur that the Christie method on a normal phase support with a ternary gradient and ELSD is the way to go. It takes about an hour per injection. Should you wish to separate the various phospholipid fractions, then work that came out of my lab on an amino column could be attractive. We published that work in JLC. If you want additional info, feel free to contact me.