Separation of Lipids

University Campus Bio-Medico, Rome, Italy University of Messina, Messina, Italy 

Recent Advances in Liquid Chromatography-Mass Spectrometry Techniques 

Recently, evidence has emerged of a relationship between abnormalities in lipid structure or function and the pathogenesis of a number of diseases, including Diabetes mellitus [5] and obesity [6], as well as atherosclerosis [7], cancer [8], and Alzheimer s disease [9]. 

---

PTLC was also used for the separation of lipid components in pathogenic bacteria. Mycobacterium avium has a requirement for fatty acids, which can be fulfilled by palmitic or oleic acid, and these fatty acids are then incorporated into triagylglycerols [80]. PTLC was used for the separation of fatty acids and triacylglycerols in the extracts of these bacterial cells to study the lipid classes in the bacterial cells cultured under different growth conditions. 

In matrix solid-phase dispersion (MSPD) the sample is mixed with a suitable powdered solid-phase until a homogeneous dry, free flowing powder is obtained with the sample dispersed over the entire material. A wide variety of solid-phase materials can be used, but for the non-ionic surfactants usually a reversed-phase C18 type of sorbent is applied. The mixture is subsequently (usually dry) packed into a glass column. Next, the analytes of interest are eluted with a suitable solvent or solvent mixture. The competition between reversed-phase hydrophobic chains in the dispersed solid-phase and the solvents results in separation of lipids from analytes. Separation of analytes and interfering substances can also be achieved if polarity differences are present. The MSPD technique has been proven to be successful for a variety of matrices and a wide range of compounds [43], thanks to its sequential extraction matrices analysed include fish tissues [44,45] as well as other diverse materials [46,47]. 

A general chromatographic system for separation of lipids does not exist. Whereas sophisticated systems for groups of lipids are described in literature, do not hesitate to try other solvent systems. 

Four aspects of research involving the use of SFE for the improvement of quality of muscle food products are briefly discussed. These include supercritical CO2 extraction of lipids fi om fresh ground beef and from dried muscle foods the extraction and separation of lipid and cholesterol from beef tallow supercritical CO2 extraction of flavor volatiles from beef and pork lipids for use as additives in synthetic meat flavors and identification and quantitation of flavor volatiles extracted with SC-CO2. 

Extraction and Separation of Lipid and Cholesterol from Beef Tallow with SC-CO2. The cumulative yields of the fractions for various operating conditions used to extract 100 g of edible beef tallow are shown in Figure 2. The SC-CO2 used to extract all lipids charged in the extractor at 345 bar and 241 bar were 10 and 22 kg, respectively. At 138 bar, 22% of the total beef tallow was extracted after 20 kg CO2 was passed. These results indicate the high dependency of triglyceride solubility on the applied pressure and temperature and confirm the greater extraction efficiencies at the higher pressures. 

This review emphasizes an intriguing and potentially useful aspect of the polymerization of lipid assemblies, i.e. polymerization and domain formation within an ensemble of molecules that is usually composed of more than one amphiphile. General aspects of domain formation in binary lipid mixtures and the polymerization of lipid bilayers are discussed in Sects. 1.1 and 1.2, respectively. More detailed reviews of these topics are available as noted. The mutual interactions of lipid domains and lipid polymerization are described in the subsequent sections. Given the proper circumstances the polymerization of lipid monolayers or bilayers can lock in the phase separation of lipids, i.e. pre-existing lipid domains within the ensemble as described in Sect. 2. Section 3 reviews the evidence for the polymerization-initiated phase separation of polymeric domains from the unpolymerized lipids. 

Chromatographic Separation of Lipids A mixture of lipids is applied to a silica gel column, and the column is then washed with increasingly polar solvents. The mixture consists of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, cholesteryl palmitate (a sterol ester), sphingomyelin, palmitate, -re-tetradecanol, triacylglycerol, and cholesterol. In what order do you expect the lipids to elute from the column Explain your reasoning. 

Separation of crude lipid extracts into individual lipid classes is difficult and time-consuming. In some cases a crude separation of lipids can be attained by selective solvent extraction. For more extensive purification of lipids, the researcher must turn to chromatography. Chromatographic methods can both resolve a complex lipid mixture into the various classes of lipids and separate the individual components of a single class of lipids. 

Figure D1.6.2 TLC-FID separation of lipids recovered from the gastric contents of a hooded seal pup. The mobile phase was 91 6 3 1 (v/v/v/v) hexane/ethyl acetate/diethyl ether/formic acid. Time refers to scanning time of the Chromarod. Abbreviations DG, 1,2-diglyceride FFA, free fatty acid MG, monoglyceride IS, internal standard TG, triglyceride. Reproduced from Ackman and Heras (1997) with permission from AOCS Press.

MN Vaghela, A Kilara. A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction. J Am Oil Chem Soc 72 1117-1121, 1995. 

MA Kaluzny, LA Duncan, MV Merritt, DE Epps. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res 26 135-140, 1985. 

WW Christie. Separation of lipid classes by high-performance liquid chromatography with the mass detector. J Chromatogr 361 396-399, 1986. 

CA Demopoulos, S Antonopoulou, NK Andrikopoulos, VM Kapoulas. Isolation and complete separation of lipids from natural sources. J Liq Chrom Rel Technol 19 521-535, 1996. 

Another type of precoated thin-layer chromatographic plate that is particularly effective for a separation of lipids is Whatman K6. With this plate, a suitable solvent would be chloroform-acetone-methanol-acetic acid-water (4.5 2/1 1.3 0.5, v/v). However, a word of caution in the use of these plates is that it is not feasible to heat activate them prior to use due to a darkening effect under elevated temperatures. Hence, sulfuric acid charring is not recommended. Nonetheless, the TNS and phosphorus sprays are satisfactory detection agents. 

Florisil columns are used for the separation of lipids and the target compounds [1, 4, 7]. Zook et al. [9] used a dialysis technique with a polyethylene film for the removal of lipids, followed by gel permeation chromatography (GPC), using S-X3 Bio beads with dichloromethane/n-hexane (50 50, v/v), carbon column chromatography, and florisil columns. De Boer et al. [8] have tried to avoid the use of florisil, because of the extensive pre-treatment and its relative instability. They used GPC, Bio beads S-X3 with dichloromethane/ m-hexane (50 50, v/v) for the separation of lipids and TCPM and TCPMe. The GPC elution was carried out twice and was followed by a silica gel column fractionation to separate TCPM and TCPMe from the PCBs. The recovery of a TCPM spike in a seal blubber extract in this method was 90%. 

For examples of separation of lipids see general references. For tabulated examples, see Ref. B1 for separation of molecular species of phospholipids by HPLC, Ref. I for separation of lipids in food by HPLC, Ref. H for HPLC of phosphatidic acid, and Ref. B2 for preparative HPLC of lipids. 

W. W. Christie and R. Anne Urwin, Separation of lipid classes from plant tissues by HPLC on chemically bonded stationary phases,/. High Resolut. Chromatogr. 18 97 (1995). 

S. E. Ebeler and T. Shibamoto, Overview and recent developments in solid-phase extraction for separation of lipid classes, in Lipid Chromatographic Analysis (T. Shibamoto, ed.), Marcel Dekker, Inc., New York, 1994, pp. 1-49. 

HPTLC using TC-CCC. Calcium plays an important role in a biomembrane. It is interesting to note that calcium improves the separation of lipids by the HPTLC.In this system, it is possible to see the effect of either calcium or pH on the elution profiles of lipids. 

---