Lipid Analysis Andréina Laffargue, IRD CRYMCEPT Montpellier workshop, October 17th 2005 Introduction to lipid structures Fatty acids Acylglycerols Glycerophospholipids Sterols Strategies involved in lipid analysis General strategy Example of PL analysis in plant physiology Lipid analysis techniques Solid Phase Extraction Gas Chromatography High Performance Liquid Chromatography with ELSD detection
«Lipids» «Lipids are fatty acids and their derivatives substances related biosyntheticallly or functionally to these compounds», W.W. Christie. «We consider that lipids are compounds based on fatty acids or closely related compounds such as the corresponding alcohols or the sphingosine bases», F.Gunstone and B. Herslöf «soluble in «fat solvents» such as diethyl ether or etroleum ether» No exact and agreed definition of lipids. All definitions agreed over a wide range of compounds but there is still a small zone of compounds that are or not accepted as lipids. Main lipids Fatty acids (RCOOH) FA = carbon chain (R) + acid function (COOH) Number of carbon atoms The main fatty acids in plants palmitic 16 : 0 stearic 18 : 0 oleic 18 : 1 linoleic 18 : 2 linolenic 18 : 3 arachidic 20 : 0 18 : 1 Number of double bonds Oleic acid
Acylglycerols Triacylglycerols Diacylglycerols Monoacylglycerols R - CO = acyl group (FA) Glycerophospholipids Acyl groups Lysophosphatidylethanolamines Phosphatidylcholines Polar head group Main glycerophospholipids in plants PE phosphatidylethanolamine PC phospatidylcholine PI phosphatidylinositol
Sterols Sterol esters O R C - O Main sterols in plants Sitosterol Stigmasterol Campesterol H - O Free sterols Lipid matrix (total lipids) Classes Sub-classes Fatty Acids Pool Acylglycerols Phospholipids Sterols Tocopherols Free fatty acids X e.g. phospholipids PE, PC, PI, X Fatty acids Complex several analytical techniques involved
Extraction Analytical strategy Fractionation Analysis Thin Layer Chromatography (TLC) Gas Chromatography (GC) High Performance Liquid Chromatography (HPLC) Liquid-Liquid Extraction Solid Phase Extraction (SPE) Thin Layer Chromatography (TLC) Physico-chemical procedures Mass Spectrometry Physico-chemical analysis Analytical strategy Example : determination of the PL composition Extraction Fractionation Analysis Liquid-Liquid Extraction (Folch) Thin Layer Chromatography (TLC) + I.S Gas Chromatography (GC) «Old method» used for PL analysis in plant physiology
Analytical strategy Example : determination of the PL composition Extraction Fractionation Analysis Liquid-Liquid Extraction (Folch) Solid Phase Extraction (SPE) High Performance Liquid Chromatography (HPLC) «Our method» used in banana meristems, coffee and citrus seeds Analytical strategy Example : determination of the PL composition Extraction Fractionation Analysis Liquid-Liquid Extraction (Folch) Mass Spectrometry Very recent «Mass method»
Analytical strategy Strategy Cost Labour Sensitivity Old method + +++ + Our method ++ ++ ++ Mass method ++++ + +++ Mass spectrometry is not affordable to our lab «Our method» is particularly financely and technicaly well adapted to project such as CRYMCEPT Major progress in plant physiology Analytical techniques developped at IRD during the CRYMCEPT project Lipid content Folch s extraction + gravimetric determination Fatty acid composition Saponification + methylation + Gas Chromatography (GC) Sterol composition and content Unsaponifiable + thin layer chromatography (TLC) + GC Phospholipid composition Solid phase extraction (SPE) + high perf. liquid chromatography (HPLC) Fatty acid composition of the PL fraction SPE + TLC + saponification + methylation +GC Free fatty acid composition and content SPE + transmethylation + GC
SPE 2 main strategies 1. Elute the product of interest, retain interferences 2. Elute interference, retain the product PL NL FFA Ex. : PL separation (C8) Ex. : FFA separation (NH 2 ) Gas chromatography (GC) Injector Carrier gas Detector Separation technique Stationary phase : column packing Mobile phase : carrier gas Temperature control Oven Data acquisition Injector : split/splitless Detector : FID (Flame Ionisation Detection) 200 Fan Capillary column Signal intensity (pa) 150 100 50 0 0 2 4 6 8 10 12 14 16 Time (min) Ex. : GC analysis of Fatty Acid Methyl Ester from FFA fraction
HPLC pumps Autosampler Injector Column Guard column Mobile phase Detector Data acquisition Separation technique Stationary phase : column packing Mobile phase : solvent mixture Detector : ELSD sample standards Ex. : HPLC analysis of PL fraction Evaporative light scattering detector (ELSD) Universal detection of non volatile compounds Sensitive detection of weak chromophores (e.g lipids, phospholipids ) More sensitive than RI and UV detection Compatible with gradient elution
Protect your material from lipid degradation Oxygen Evaporate under nitrogen, use antioxidants Light Store the samples away from sunlight, in dark glass flasks Temperature Evaporate at less than 40 C, store at 5 C Merci!