We decided to develop a method for separation and determination of fatty acids from different tissues by high-performance liquid chromatography. That method allowed us to determine cholesteryl ester composition and content in umbilical cord arteries. Studies were performed on the umbilical cord arteries taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Cholesteryl esters were isolated by thin layer chromatography. Fatty acids were liberated by basic hydrolysis and analyzed by HPLC of their p-bromophenacyl derivatives using detection at 254 nm. It was found that saturated fatty acids were the main group of fatty acids incorporated to cholesteryl esters in all control and preeclamptic umbilical cord arteries. Preeclampsia caused a significant increase in cholesteryl ester content in the umbilical cord arteries. An increase of neutral lipid content in vessel walls of newborns delivered by mothers with preeclampsia may be one of the factors that evoke the initiation of hypertension in utero and its amplification throughout childhood and adult life. The described method reduces time and cost consumption and allows us to determine almost all fatty acids forming cholesteryl esters contained in the tissue sample.

Article Separation and Determination of Fatty Acids from Lipid Fract...

Try to use the protocol of the PLFA analysis. I'm not sure that it is work but it is an idea.

What column you use?

Think about GPC/SEC.

regards

G

We decided to develop a method for separation and determination of fatty acids from different tissues by high-performance liquid chromatography. That method allowed us to determine cholesteryl ester composition and content in umbilical cord arteries. Studies were performed on the umbilical cord arteries taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Cholesteryl esters were isolated by thin layer chromatography. Fatty acids were liberated by basic hydrolysis and analyzed by HPLC of their p-bromophenacyl derivatives using detection at 254 nm. It was found that saturated fatty acids were the main group of fatty acids incorporated to cholesteryl esters in all control and preeclamptic umbilical cord arteries. Preeclampsia caused a significant increase in cholesteryl ester content in the umbilical cord arteries. An increase of neutral lipid content in vessel walls of newborns delivered by mothers with preeclampsia may be one of the factors that evoke the initiation of hypertension in utero and its amplification throughout childhood and adult life. The described method reduces time and cost consumption and allows us to determine almost all fatty acids forming cholesteryl esters contained in the tissue sample.

Article Separation and Determination of Fatty Acids from Lipid Fract...

Dear Grzegorz Boczkaj and Mohammed H. Musleh

The problem with going to HPLC is I need to find the solvent system. But my compound doesn't show any movement in TLC to choose the solvent system. Anyhow I get my compound of interest as major constituent when I treat the extract with Acetone. But purification is merely not possible at all since I am not able to find the solvent system. My compound of interest is Elaidic acid. But it comes with elaidamide and Isopropyl Myrsitate as minor compounds.

You will use THF for SEC. The Stat. Phase will be SDVBa.

Check the papers of A. Stolyhwo (Stołyhwo).

Regards

G

One can form free fatty acid methyl esters derivitives using ethyl ether in a rotery evaporator. Flame ionization gas chromatography on a C18 column can seperate them.

May be after lipid extraction by an organic solvents mix, you should form fatty acid methyl esters considering that after about 6 minutes of derivatization at 70°C you will have methyl esters from free fatty acids, after about 12 minutes of derivatization at 70°C you will obtain methyl esters from triglicerides and after about 18 minutes of derivatization at 70°C you will obtain methyl esters from polar lipids. You can extract the obtained methyl esters from the derivatization mixture by using exane and analyze them by GC. A 5%-phenyl-methylpolysiloxane column of 30 m (at least) lenght is usually right for the separation.

I presume you are going to use a fraction collector to isolate the fatty acids and lipids which means you can't use a derivatization agent. Yes, you can! But you will have to use an ELSD detector. There are many HPLC methods on the internet. Check the Waters, Agilent, Thermo Scientific, and Phenomenex websites...

Bruce Neagle The actual problem is the compounds in the extract are not getting separated in liquid phase. I need to optimize the solvent system.

Absul in which mode you use HPLC?

Abdul,

What are your chromatographic conditions? Use the same mobile phase(s), columns... as the manufacturers above. SPE (a form of liquid chromatography) can separate lipids and fatty acids.

Look up "partitioning" in old books on "organic qualitative analysis". The idea is to "shake up" your mixture dissolved in ether plus aqueous sodium bicarbonate. The fatty acids prefer to dissolve in the water fraction. You can then freeze-dry the fraction (now as sodium salts) derivitize all of them to make esters for HPLC or GC. This used to be a Junior year Chemistry course!

@David Arthur Carlson Thank you, Sir. Will try that

I actually worked on lipids analysis, and on algal oil using a C18 and the Corona Charged Aerosol Detector. The lowest acid that can be measured is Lauric Acid, and sensitivity increases with acids of higher boiling points. No derivatisation is needed.

Article The Use of Charged Aerosol Detection with HPLC for the Measu...

1. TLC pre-fractioning followed by methylation and GC analysis is probably the most accurate way.

2. Short-chain fatty acids can be directly quantified by GC but long-chain ones are not volatile.

3. LC-MS is capable of quantifying fatty acids but peak shape and/or linearity are not so good for some fatty acids.

4. Currently, there are no simple and accurate methods for selectively determining free fatty acids.

@Zhiqian Liu That is exactly my problem. Thanks for addressing it right. I will try with LC-MS too. I performed LC, but not MS. Will try with LC-MS once and get back to you. Thanks again.

I Think this article help you, Hexane iso propyl alcohol method is more useful given inthe article. You can find exraction method inthe article

Contribution of Vitamin C and α-Lipoic Acid and Their Combination on the Products Level of Desaturase Enzymes in the Lung and Muscle Tissues of Poorly Controlled Experimental Diabetic Rats

Most lipid separations are based upon physical property differences among lipid, mainly boiling point [or freezing point] and molecular properties such as chain length.   This science has been worked out /solved/ is well established.  More complicated lipid separations are required when lipids are polyunsaturated; so a-linolenic acid and y-linolenic acid have precisely the identical molecular weight, the same number of double bonds, except the first is an Omega-3 and the second is an Omega-6 fatty acid.  Also some PUFA H-C=C-H bonds are cis whereas others can be trans.   Indeed, "proof" of a  natural lipid structure analytically is quite complicated.  Two structural analogs of a lipid could easily have very similar LC or LC-MS retention time and fragmentation patterns.   A limitation of LC-MS in principle is because lipids structure is elastic. Molecular rearrangements are possible [that is why they fragment differently] depending upon which specific molecular site is ionized.  If one H- is lost from [or added to] H-C=C-H,   the fragmentation pattern will be very different if a different  C=C bond loses [or gains] an H atom.  We have used spectroscopy to differentiate lipid structural fingerprints  to distinguish N-3 DPA from N-6 DPA.

Underivatized organic acids don't behave well on standard GC columns. That's why methylation is often used prior to GC analysis. For GC analysis without derivatization have you looked at fatty acid GC columns? For example:

https://www.agilent.com/en/products/gas-chromatography/gc-columns/capillary/hp-ffap

or

http://www.phenomenex.com/Products/GCDetail/Zebron/ZB-FFAP

or

http://www.perkinelmer.com/product/col-elite-ffap-30m-25-m-25-mm-n9316352

Here are some references:

Article Development and validation of a GC-FID method for quantitati...

This one discusses fatty acids from marine organisms:

https://academic.oup.com/chromsci/article/51/7/599/471782

Thank you everyone for such discussion. It was quite informative.

may be hexane isopropile alchol(3/2) be better, we studied in past, you can describe fatty acids have more than 6 carbons. I think more of your result amount will be less than % 10

Another method has been used with HPLC-CAD for omega fatty acids. A hydrolysed fish oil showed 35 different peaks.

Technology for identifying natural lipids such as fish oils has been complicated by their high mass often requiring hydrolyzing the lipids to form free fatty acids.

A recently published manuscript identifies fish oils by its Raman spectral signature collected during a temperature gradient (e.g. -100 C to 20 C). No chemical treatment is required to obtain a spectral fingerprint.