There are lots of lipids, but they all share the trait of being at least partially hydrophobic meaning they won't mix with water. Water molecules are polar because they have positive and negative ends, rather like little magnets. Most lipids are non-polar having no charged areas or only slightly polar, with a very few charged areas. Water mixes with hydrophilic water-loving compounds by sticking to their charged groups. Since lipids lack charged groups, the water molecules have nothing to stick to and don't mix with them.
Think of trying to pick up a glass marble with a magnet. Bio L Index Page. Lipids: Melting Points. Whitson Home. Bio Home. Biology Department. NKU Home. Calculations : R f Values R f values are a numerical way of comparing the movement of various substances along a chromatography strip.
Return to top. They may be saturated or unsaturated. Most fatty acids are unbranched and contain an even number of carbon atoms. Unsaturated fatty acids have lower melting points than saturated fatty acids containing the same number of carbon atoms.
The hydrolysis of fats and oils in the presence of a base makes soap and is known as saponification. They are discussed in this and the next section respectively. Many different solvents will dissolve pure single lipid classes, but they are only suitable for extracting lipids from tissues if they can overcome the strong forces of association between tissue lipids and other cellular constituents, such as proteins and polysaccharides.
However, even polar complex lipids, which do not normally dissolve easily in non-polar solvents, can sometimes be extracted with these when they are in the presence of large amounts of simple lipids such as triacylglycerols. Therefore, the behaviour of a given solvent as a lipid extractant for a specific tissue cannot always be predicted. In order to release all lipids from their association with cell membranes or with lipoproteins, the ideal solvent or solvent mixture must be fairly polar.
Yet, it must not be so polar that it reacts chemically with the lipids nor that triacylglycerols and other non-polar simple lipids do not dissolve and are left adhering to the tissues. If chosen carefully, the extracting solvent may have a function in preventing any enzymatic hydrolysis, but vice versa it should not stimulate any side reactions. There is an increasing awareness of the potential toxicity of solvents to analysts, and this is another factor that must be taken into consideration when selecting a solvent mixture, especially if the laboratory is not adequately equipped with fume hoods or other ventilation.
No solvent is completely safe. Those factors affecting the extractability of lipids by solvents have been reviewed comprehensively by Zahler and Niggli []. The two main structural features of lipids controlling their solubility in organic solvents are the hydrophobic hydrocarbon chains of the fatty acid or other aliphatic moieties and any polar functional groups, such as phosphate or sugar residues, which are markedly hydrophilic.
Any lipids lacking polar groups, for example triacylglycerols or cholesterol esters, are very soluble in hydrocarbons such as hexane, toluene or cyclohexane and also in moderately polar solvents such as diethyl ether or chloroform.
In contrast, they are rather insoluble in a polar solvent such as methanol. The solubility of such lipids in alcohols increases with the chain length of the hydrocarbon moiety of the alcohol, so they tend to be more soluble in ethanol and completely soluble in butanol. Similarly, lipids with fatty acyl residues of shorter chain length tend to be more soluble in more polar solvents; tripalmitin is virtually insoluble in methanol but tributyrin dissolves readily.
Unless solubilized by the presence of other lipids, polar lipids, such as phospholipids and glycosphingolipids, are only slightly soluble in hydrocarbons, but they dissolve readily in more polar solvents like methanol, ethanol or chloroform. Such solvents with high dielectric constants and polarity are required to overcome ion-dipole interactions and hydrogen bonding. Tabulated data on the solubilities of a limited range of "typical" lipids are available [98].
Analysts should be aware that water is also a solvent for lipids, contrary to some definitions of the term, and water in tissues or that used to wash lipid extracts, for example, can alter the properties of organic solvents markedly.
Most complex lipids are slightly soluble in water and at least form micellar solutions, and lipids such as gangliosides, polyphosphoinositides, lysophospholipids, acyl-carnitines and coenzyme A esters are especially soluble see Section G. Lipids exist in tissues in many different physical forms. The simple lipids are often part of large aggregates in storage tissues, such as oil bodies or adipose tissue, from which they are extracted with relative ease.
In contrast, complex lipids are usually constituents of membranes, where they occur in a close association with such compounds as proteins and polysaccharides, with which they interact, and they are not extracted so readily.
These interactions are only very rarely through covalent bonds, and in general weak hydrophobic or van der Waals forces, hydrogen bonds and ionic bonds are involved. For example, the hydrophobic aliphatic moieties of lipids interact with the non-polar regions of the amino acid constituents of proteins, such as valine, leucine and isoleucine, to form weak associations.
Hydroxyl, carboxyl and amino groups in lipid molecules, on the other hand, can interact more strongly with biopolymers via hydrogen bonds. Lipids such as the polyphosphoinositides are most likely bound to other cellular biopolymers by ionic bonds, and these are not easily disrupted by simple solvation with organic solvents. It is usually necessary to adjust the pH of the extraction medium to effect quantitative extraction in this instance.
In addition, purely mechanical factors can limit the extractability of lipids. The helical starch amylose molecules in cereals form inclusion complexes with lysophosphatidylcholine, for example, limiting its accessibility to solvents. Also, cell walls in some microorganisms are rather impermeable to solvents, especially in the absence of water, which must be added to cause swelling of cellular polysaccharides.
Some fatty acid or other alkyl moieties may indeed be linked directly to proteins or polysaccharides by covalent bonds, and then the optimum isolation procedure is likely to be one more suited to the analysis of the biopolymer rather than of the lipid. In order to extract lipids from tissues, it is necessary to use solvents that not only dissolve the lipids readily but overcome the interactions between the lipids and the tissue matrix, and it is essential to perturb both the hydrophobic and polar interactions at the same time.
As with much other interesting work, the method was not extended to other tissues, and there is some danger of transesterification occurring as a side reaction under these conditions see Section E below. Since the publication of a classic paper on the subject by Folch, Lees and Stanley [26] in , this has become the standard against which other methods are judged although there are earlier applications of these solvents [25,].
The capacity of chloroform to associate with water molecules, presumably by weak hydrogen bonds, is a key property. Provided that the ratio of chloroform-methanol to tissue assumed to be mainly water is greater than , the equivalent of 5.
In contrast, mixtures of methanol with carbon tetrachloride or tetrachlorethylene, which lack the active proton of chloroform, were only able to solubilize relatively small amounts of water. Many practical methods have been developed for chloroform-methanol extraction and these are discussed in Section F below. There are disadvantages, however. In addition to the toxicity problem, which is controllable in a well-ordered laboratory, the mixture is a potent irritant to skin. Neither chloroform nor methanol is completely stable and both were found to generate acidic by-products, which could catalyse esterification of free fatty acids or transesterification of lipids see also Section E below [99].
Better recoveries of prostaglandins were reported with this mixture than with the Folch procedure [94], but it does not extract gangliosides quantitatively. Systematic studies of the solubilities of certain lipids in toluene-ethanol mixtures indicated that this combination might have superior properties to chloroform-methanol, but it does not appear to have been tested adequately with complex lipids or with samples of real biological interest [97].
Butanol saturated with water has been recommended for the extraction of cereals or wheat-flour [60,68], in which the lipids may be in close association with starch, some in the form of inclusion complexes. The structure of the starch granules appears to be the most important factor, however [67]. This solvent may have wider uses, for example for quantitative extraction of lipids that are relatively soluble in water, such as lysophospholipids [7,31] or acylcarnitines [56,69]; hexanol has even been recommended for the latter [69].
It is well known that diethyl ether or chloroform alone are good solvents for lipids, yet they are poor extractants of lipids from tissues. They can, however, have some practical value for the isolation of the non-polar lipids from triacylglycerol-rich tissues, such as oil seeds or adipose tissue, as they do not extract significant amounts of non-lipid contaminants at the same time. When they are used to extract plant tissues, these solvents also enhance the action of phospholipase D [42] unfortunately, as does butanol [20].
Propanol and propanol strongly inhibit this reaction and the latter, which has the lower boiling point, has been recommended for use with plant tissues, as a preliminary extractant especially [43,77,78].
While simple lipids and glycolipids dissolve readily in acetone, it will not dissolve phospholipids readily and indeed is often used to precipitate them from solution in other solvents, in effect as a crude preparative procedure. The lipid mixture is usually dissolved in diethyl ether and then four volumes of cold anhydrous acetone is added to precipitate the phospholipids [33].
On the other hand, endogenous water and the solubilizing effects of other lipid components may permit acetone to extract more phospholipids from animal or plant tissues than might be predicted from a knowledge of the solubility of lipid standards in the pure solvent. For example, ethyl acetate-acetone-water by volume has been recommended for extracting lipids from cultures of human cells [,]. Acetone has also been recommended as a preliminary extraction solvent, before conventional chloroform-methanol extraction [59].
A disadvantage is that it can react with certain lipids to produce artefacts see Section E below. As glycolipids are soluble in acetone, chromatographic solvents containing this solvent are frequently utilized in the separation of glycolipids from phospholipids.
In recent years, supercritical fluids have been evaluated as extractants for lipids reviewed elsewhere [6,50]. While these appear to hold promise for selected simple lipids, there appears to be little prospect for more general use at the moment. As an alternative to conventional solvent extraction, the technology of column chromatography has been adapted to the purpose in specific circumstances see Section G. When polar organic solvents are used to extract lipids from tissues, they tend to co-extract appreciable amounts of natural non-lipid materials, such as amino acids, carbohydrates, urea and even salts, as contaminants.
A variety of procedures have therefore been developed to eliminate these, ideally without causing losses of lipids. Such a clean-up is rarely complete so the procedure is little used, although it should not be overlooked when large numbers of similar samples have to be purified for routine analysis by less demanding techniques, such as thin-layer chromatography TLC.
Other procedures that have been tried, but with limited success only, include dialysis, adsorption and cellulose column chromatography, electrodialysis and electrophoresis. The solvents then partition into two layers or phases, the lower consisting of chloroform-methanol-water in the ratio by volume with an upper phase in which the proportions are respectively Unfortunately, any gangliosides that may have been present also partition into the upper layer.
These are minor compounds and their analysis is rather specialized, so a simple washing procedure of this kind yields satisfactory lipid samples for most purposes.
When they are required for further analysis, gangliosides can be recovered from the Folch upper phase by dialysis followed by lyophilization see Section G.
It is not always recognized how important it is that the proportions of chloroform, methanol and water in the combined phases should be as close to by volume as is practicable. In a successful adaptation of the above method that is especially suited to large samples with a high water content, Bligh and Dyer [8] took into account the water already present in the samples when adding further water in the washing step.
As this procedure uses smaller volumes of chloroform and methanol, it is both economical and convenient. While this type of lipid purification procedure was first developed by Wells and Dittmer [], a modification described by Wuthier [] is simpler and thus more suitable for large numbers of samples. While lipids were eluted rapidly by further lower phase, contaminants of low molecular weight remained on the column.
Gangliosides and non-lipids could be recovered from the column by washing with 'Folch' upper phase, and the column regenerated for further use. The technique can also be used to remove acid, alkali and salts from lipid samples.
An alternative simple procedure was described by others []. As various bile acids were obtained in distinct fractions from the conventional lipids, the method appeared to be particularly suited to the analysis of bile lipids [90]. However, all of these methods are very time-consuming, and as a result they have never achieved widespread use. Such column procedures are rather different in principle from a solid-phase extraction method described below Section G.
An alternative approach consisted in pre-extraction of tissues with 0. Although this procedure appears only to have been applied to brain tissue and soybeans to date, it might repay further investigation. The production of free fatty acids, diacylglycerols, phosphatidic acid or lysophospholipids, due to faulty storage of tissues prior to extraction is discussed in Section B above. Extraneous substances can be introduced into lipid extracts from innumerable sources, and these are discussed elsewhere in this web site see my review of methylation procedures.
Plasticisers are especially troublesome. When chloroform-methanol or, indeed, any alcoholic extracts that contain lipids are heated or stored for long periods in the presence of small amounts of sodium carbonate or bicarbonate of tissue origin, base-catalysed transesterification can occur and appreciable amounts of methyl esters may be found in the extracts [55]. By adjusting the pH of the extraction medium to 4 to 5, the problem can be circumvented [55,].
Similar findings are often described, and it is possible that both acidic and basic non-lipid contaminants may catalyse the same reaction. Tissue acyl-transferases can also catalyse ethyl ester formation [64]. However, small amounts of methyl and ethyl esters do appear to occur naturally in some tissues, and when they are detected confirmation should be obtained of their natural origin.
Artefacts of extraction or storage can be eliminated simply by extracting the tissues with solvents that do not contain any alcohol, such as diethyl ether [23], hexane [44], acetone [46] or acetone-chloroform [54], and repeating the analysis for methyl esters on this material.
While 6-O-acyl-galactosyldiacylglycerols are known to be natural components of some plant tissues, it appears that they may also be formed as artefacts by acyl transfer from other lipids when cells are disrupted; they are found in much smaller amounts when the tissues are homogenized in the presence of the extracting solvent [38].
Similar difficulties may be encountered with homogenates of bacteria []. Some rearrangement of plasmalogens may occur when they are stored for long periods in methanol []. Acetone should not be used in the analysis of tissues rich in polyphosphoinositides, such as brain lipids, as it causes rapid de-phosphorylation [22,].
An acetone derivative imine of phosphatidyl-ethanolamine was reported to be formed in vitro during extraction of freeze-dried tissue with acetone [3,39]. All solvents contain small amounts of lipid-like contaminants and only those of highest quality need not be distilled routinely before use. Organic solvents will extract substantial amounts of these compounds, and wet animal tissues alone in contact with plastic can extract small amounts.
Polyunsaturated fatty acids will autoxidize very rapidly if left unprotected in air. This need not interfere with later chromatographic analyses, as it tends to evaporate on removal of solvents or appears in conveniently empty regions of chromatograms [18].
It is helpful to de-aerate solvents by flushing them with nitrogen or helium before use. If a blender is used to homogenize tissues, it should be one in which the drive to the knives or grinders is from above, so that there is no contact between solvent and any greased seals or bearings.
Lyophilized tissues are particularly difficult to extract and it may be necessary to re-hydrate them before extraction to ensure quantitative recovery of lipids.
When the aqueous and organic phases are partitioned in an extraction procedure, it should be noted that centrifugation may be necessary or of assistance in ensuring complete separation of the layers.
This also serves to compact the interfacial layer, rendering it easier to recover for further extraction if need be. When a large amount of solvent must be evaporated, the extract should be concentrated to a small volume and then transferred to as small a flask as is convenient so that the lipids do not dry out as a thin film over a large area of glass. While there is no need to bleed nitrogen in continuously during the evaporation process, as the solvent vapours effectively displace any air, the vacuum should be broken eventually with nitrogen.
Lipids should not be left in the dry state, but should be taken up in or covered by an inert non-alcoholic solvent such as hexane. The last traces of water may be removed by co-distillation with ethanol or toluene. As cautioned earlier, it should never be forgotten that chloroform and methanol are highly toxic and should only be used in well-ventilated areas. The single operation most likely to introduce appreciable amounts of solvent vapour into the atmosphere is filtration.
Mixtures of chloroform and methanol, especially, are powerful irritants when they come into contact with the skin. A colleague of the author was taught a painful lesson when he spilt a large volume down his trousers! A large number of different extraction procedures, varying in the nature of the solvents, the method of homogenizing, removal of contaminants and many other aspects, have been described in the literature.
Unfortunately, a high proportion of these have been tried in only one laboratory or with a limited range of tissues or organisms. Only rarely have they been tested exhaustively and compared rigorously with a widely used procedure, such as that of Folch, Lees and Stanley [26] or Bligh and Dyer [8]. Until this is done, whatever the virtues of alternatives, the latter two procedures are likely to continue in their position of pre-eminence.
An extraction procedure must be selected to give the optimum yield of representative lipids in as practical a manner as possible. While a method chosen for routine analysis of the cholesterol content of large numbers of clinical samples, say, must be accurate and reproducible, it need not incorporate modifications to recover every trace of such complex lipids as gangliosides or inositides.
If the latter compounds are required for analysis, however, there is no alternative but to use complex and exhaustive extraction procedures. For consistent results with any method, a strict protocol must be adopted that follows the principles laid down by the originators of the method.
With the procedure of Folch et al. On the other hand, some variation in the approach to attaining the optimum concentrations may be possible. More than one extraction may be needed, but with most tissues the lipids are removed almost completely after two or three treatments.
Generally, there is no need to heat the solvents with the tissue homogenates, but this may sometimes be necessary, for example with wet bacterial cells []. Treatment with acid [79] or proteases [86] may also be of benefit in this instance.
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