An Introduction to Lipids and Gas Chromatography

Chapter 2 - Sections E and F

E.  Extraction of Lipids from Tissues and Other Preliminaries to Analysis

1.  Solvent extraction procedures

Before analysis of lipid samples can be commenced by any chromatographic procedure, it is first necessary to extract them from their tissue matrices in a relatively pure state. This should always be carried out as soon as possible after the removal of the tissue from the living organism. The author has dealt with these aspects in some detail in previous texts [163,168], so essential details only are covered here.

Lipids occur in tissues in a variety of physical forms. The simple lipids are often part of large aggregates in storage tissues, from which they are extracted with relative ease. On the other hand, the 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. Generally, lipids are linked to other cellular components by weak hydrophobic or van der Waals forces, by hydrogen bonds and by ionic bonds. For example, the hydrophobic aliphatic moieties of lipids interact with the nonpolar 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, in contrast, can interact more strongly with biopolymers via hydrogen bonds.

In order to extract lipids from tissues, it is necessary to find solvents which will not only dissolve the lipids readily but will overcome the interactions between the lipids and the tissue matrix. Various solvents or solvent combinations have been suggested as extractants for lipids, and currently some interest is being shown in isopropanol-hexane (2:3 by volume), because its toxicity is relatively low [355,743], but it does not yet appear to have been tested with a sufficiently wide range of tissues. It does not extract gangliosides quantitatively. Most lipid analysts use chloroform-methanol (2:1 by volume), with the endogenous water in the tissue as a ternary component of the system, to extract lipids from animal, plant and bacterial tissues. Usually, the tissue is homogenized in the presence of both solvents, but better results may be obtained if the tissue is first extracted with methanol alone before the chloroform is added to the mixture. With difficult samples, more than one extraction may be needed, and with lyophilised tissues, it may be necessary to rehydrate prior to carrying out the extraction. The homogenization and extraction should be performed in a Waring blender, or better in equipment in which the drive to the blades is from above, so that the solvent does not come into contact with any lubricated bearings. Generally, there is no need to heat the solvent to facilitate the extraction, although there may be times when this is necessary.

The extractability of tissues and of particular lipids is variable, and there are many instances when alternative or modified procedures must be used. Butanol saturated with water appears to be the most useful solvent mixture to disrupt the inclusion complexes of lipids in starch and gives the best recoveries of lipids from cereals [188,606,627]. This solvent combination has also been recommended for the quantitative recovery of lysophospholipids, which are more soluble in water than are many other common phospholipids [103].

Plant tissues should be pre-extracted with isopropanol to minimize artefactual degradation of lipids by tissue enzymes.

Lipid extracts from tissues, obtained in the above manner, tend to contain appreciable amounts of nonlipid contaminants, such as sugars, amino acids, urea and salts. These must be removed before the lipids are analysed. Most workers use a simple washing procedure, devised originally by Folch, Lees and Stanley [259], in which a chloroform-methanol (2:1, v/v) extract is shaken and equilibrated with one-fourth its volume of a saline solution (i.e. 0.88% potassium chloride in water). The mixture partitions into two layers, of which the lower phase is composed of chloroform-methanol-water in the proportions 86:14:1 (by volume) and contains virtually all of the lipids, while the upper phase consists of the same solvents in the proportions of 3:48:47, respectively, and contains much of the nonlipid contaminants. It is important that the proportions of chloroform, methanol and water in the combined phases should be as close as possible to 8:4:3 (by volume), otherwise selective losses of lipids may occur. If a second wash of the lower phase is needed to remove any remaining contaminants, a mixture of roughly similar composition to that of the upper phase should be used, i.e. methanol-saline solution (1:1, v/v).

Any gangliosides present in the sample partition into the upper layer, together with varying amounts of oligoglycosphingolipids. They can be recovered from this layer by dialysing out most of the impurities of low molecular weight, and then lyophilising the residue [459].

Many modifications of the basic extraction procedure have been devised for use in particular circumstances, and the analyst must decide what he requires of a method. One which extracts all of the more minor lipid classes exhaustively is obviously desirable for many applications, but may be too tedious and time consuming for routine use. On the other hand, a method which is suited to the quantitative extraction of the main lipid classes in large numbers of samples in a routine manner by relatively inexperienced staff may not give complete recoveries of certain trace components of biological importance. The modified "Folch" procedure [967], which follows, probably falls somewhere between these extremes.

"The tissue (1 g) is homogenized with methanol (10 mL) for 1 minute in a blender, then chloroform (20 mL) is added and the homogenization is continued for 2 minutes more. The mixture is filtered, when the solid remaining is resuspended in chloroform-methanol (2:1 by volume, 30 mL) and homogenized for 3 minutes. It is filtered again and re-washed with fresh solvent. The combined filtrates are transferred to a measuring cylinder, one-fourth of the total volume of 0.88 % potassium chloride in water is added, and the mixture is shaken thoroughly before being allowed to settle. The aqueous (upper) layer is drawn off by aspiration, one-fourth the volume of the lower layer of methanol-saline solution (1:1, v/v) is added and the washing procedure is repeated. The bottom layer containing the purified lipid is filtered before the solvent is removed on a rotary film evaporator. The lipid is stored in a small volume of chloroform at -20°C, until it can be analysed."

With plant tissues as cautioned above, it is necessary to extract first with isopropanol, in order to deactivate the enzymes, as follows [664,665].

"The plant tissues are homogenized with a 100-fold excess (by weight) of isopropanol. The mixture is filtered, the residue is re-extracted with fresh isopropanol, and finally is shaken overnight with isopropanol-chloroform (1:1, v/v). The filtrates are then combined, most of the solvent is removed on a rotary evaporator, and the lipid residue is taken up in chloroform- methanol (2:1, v/v) and is given a "Folch" wash as above."

In any extraction procedure, it is important that the weight of fresh tissue extracted is recorded, together with the weight of lipid obtained from it. For some purposes, it may be desirable to determine the amount of dry matter in the tissue, so that the weight of lipid relative to that of dry matter can be calculated. A more comprehensive review of extraction methodology is available on this web site here...

 

2.  Minimising autoxidation

If they are not protected, polyunsaturated fatty acids will autoxidize very rapidly in air, and it may not be possible to obtain an accurate analysis by chromatographic means. The mechanism of autoxidation involves attack by free radicals and is exacerbated by strong light and by metal ions. Once it has been initiated, the reaction proceeds autocatalytically. Linoleic acid is autoxidized twenty times as rapidly as oleic acid, and each additional double bond in a fatty acid can increase the rate of destruction by two- to threefold. Natural tissue antioxidants, such as the tocopherols, afford some protection to lipid extracts, but it is usually advisable to add further synthetic antioxidants, such as BHT ("butylated hydroxy toluene" or 2,6-di-tert-butyl-p-cresol) to solvents at a level of 10 to 100 mg/litre, depending on lipid concentration. This compound need not interfere with chromatography, as it is relatively volatile and can be removed (sometimes inadvertently!) together with solvents when they are evaporated in a stream of nitrogen; it is also rather nonpolar and tends to elute at the solvent front, ahead of most lipids, in many liquid chromatography systems. In contrast, excessive amounts of added antioxidants can sometimes act as pro-oxidants!

Wherever possible, lipids should be handled in an atmosphere of nitrogen. On the other hand, it is rarely necessary to go to the length of constructing a special nitrogen box to contain all the equipment used in the handling of lipids. Usually it is sufficient to ensure that nitrogen lines are freely available, so that the air can be flushed out of glass containers or reaction vessels.

When it is necessary to concentrate lipid extracts, large volumes of solvents are best removed by means of a rotary film evaporator at a temperature, which in general should not exceed about 40°C. The flask containing the sample should not be too large, otherwise the lipid can spread out over a large area of glass and so be more accessible to oxygen. At the start of evaporation, it may be advisable to flush out the equipment with nitrogen, but the solvent vapours eventually will displace any air. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent. This should not be done too vigorously or at too high a temperature, since the more volatile fatty acid derivatives, including methyl esters of fatty acids, may also be lost by evaporation or by physical transport as an aerosol.

PLEASE NOTE!

As constant repetition is tedious, it will be assumed in all the subsequent discussion of methodology in this book that precautions will be taken at all times to minimise autoxidation.

 

3.  Hazards

It is inevitable that lipid analysts will make use of large volumes of solvents in their work, whether in extracting lipids from tissues, in the chromatographic separation of lipid classes or in the preparation of derivatives for chromatography. Most solvents exhibit some degree of toxicity if inhaled in large amounts. Benzene, in particular, is frequently mentioned in the older literature as a solvent with valuable properties in the analysis of lipids, but it is now known to be extremely toxic and is best avoided entirely; toluene has comparable chromatographic properties and is much less hazardous. Similarly, it was once thought that chloroform was relatively safe, but it is now known that there are real hazards. Acetonitrile also has toxic properties, and indeed analysts should not view any solvent with complacency.

All solvents should be used with care in well-ventilated areas, or in fume cupboards if at all possible. Solvents should never be evaporated or distilled on an open bench. Some operations generate more vapour than others, and filtration is probably the procedure which produces most. When not in use, all solvents should be stored in well-stoppered bottles, made of dark glass, and in flame-proof cabinets. No more solvent than is required for immediate needs should be stored in the laboratory. In general, care should be taken to prevent spillages, to keep storage vessels closed, and generally to minimise any exposure of the laboratory personnel to solvent vapours.

Nor should it be forgotten that many solvents, especially low molecular weight hydrocarbons, ethers and alcohols, are highly inflammable. Ethers develop peroxides on storage, especially in bright light, and many explosions have resulted as these were concentrated when large volumes of ethers were distilled. It may seem self evident, but there should be no naked flames in laboratories in which solvents are handled. All electrical equipment, especially that in chromatographic systems, should be correctly wired and grounded to reduce the risk of sparks.

The concentrations of chloroform vapour permitted in the laboratory atmosphere have steadily been reduced in recent years as regulations have been revised. Unfortunately, there appears to be no proven substitute in many of the problems that face lipid analysts. As supplied, chloroform usually contains 0.25 to 2% of added ethanol, which acts as a stabiliser, but also has a considerable effect on the chromatographic properties of the solvent. It can be removed, if there is a need, by a simple washing procedure [163], but photochemical formation of the highly poisonous substance, phosgene, can then occur and the destabilised solvent should not be stored for any length of time. Chloroform-methanol mixtures are powerful irritants when they come in contact with the skin.

Many other reagents to be found in laboratories are known to have toxic properties, some of which may take some time to manifest themselves, and the catalogues of suppliers are often informative on the subject as should be the labels on containers. The toxicity of numerous other reagents has yet to be investigated, and it is best to err on the safe side and assume that there is some unknown hazard associated with all chemicals. They should then be handled accordingly. Similarly, the dangers associated with strong mineral acids should be well known to analysts. They should not be stored in the same cupboard as solvents.

 

F.  Separation of Lipid Classes

Gas chromatography is of most value for the separation of the aliphatic components of lipids, and some pre-fractionation into simpler groups or individual classes, generally by adsorption chromatography (HPLC or TLC), is often desirable. Again, the author has described these in great detail elsewhere [163,168], and the topic has been dealt with by Kuksis and Myher [507,516,517], but the brief summary which follows may be of value to some readers. Small-scale procedures only are described as they generally give sufficient material for GC analysis.

 

1.  Simple group separations

The complexity of natural lipid extracts is such that it is rarely possible to claim that all the lipid classes of a sample can be separated in one operation. It is therefore often worthwhile to be able to isolate distinct simple lipid, phospholipid or glycolipid fractions for further analysis. For example, it is frequently easier technically to isolate small amounts of pure lipid classes preparatively by means of HPLC (or other methods), after a preliminary fractionation has been carried out. Unfortunately, no procedure appears yet to have been described that is satisfactory in all respects, although some useful methods are available provided that their limitations are recognised.

The simplest small-scale procedure for isolating groups of lipids consists of making a short column of silica gel (about 1 g), in a glass disposable Pasteur pipette, say, and applying about 30 mg of lipid to this. Elution with chloroform or diethyl ether (10 mL) yields the simple lipids, acetone (10 mL) gives a glycolipid fraction, and methanol (10 mL) yields the phospholipids. Different brands or batches of silica gel tend to vary somewhat in their properties and some cross- contamination of fractions may be found. For example, the acetone fraction may contain some of the less polar phospholipids, especially phosphatidic acid and diphosphatidylglycerol but occasionally phosphatidylethanolamine even; if this is observed to occur, it can be minimised by adding some chloroform to the acetone prior to elution. Indeed for many purposes, there may be no need to include an elution step with acetone, as some tissues contain negligible amounts of glycolipids. On the other hand, it is possible to insert an additional elution step with methyl formate before the acetone wash, to obtain a fraction that contains most of the prostaglandins in the extract (together with some of the glycolipids) [799].

It may now be more convenient to use small proprietary pre-packed cartridges of silica gel or ion-exchange media for these small-scale group separations [101,102,453,486,777,1007]. In one application, milk fat samples (100 mg), high proportions of which consist of triacylglycerols, were applied to Sep-Pak™ cartridges of silica gel (Waters Associates, Milford, U.S.A.); nonpolar lipids were recovered by elution with hexane-diethyl ether (1:1, v/v; 40 mL), while the complex lipids were recovered by elution first with methanol (20 mL), and then with chloroform-methanol-water (3:5:2 by volume, 20 mL) [102]. In other work with these cartridges, chloroform (40 mL) was used to elute the simple lipids, acetone-methanol (9:1, v/v; 160 mL) gave the neutral glycosphingolipids, and chloroform-methanol (1:1, v/v; 80 mL) eluted the phospholipids [1007]. One further procedure, which appears to have much to commend it, is for the isolation of glycolipids and consists in using boric acid bound to a polymeric matrix in a column; carbohydrate moieties of glycolipids are retained much more strongly than are other lipids so facilitating separation [496].

 

2.  High-performance liquid chromatography

Lipid analysts were relatively slow to adapt HPLC to the separation of lipid classes, largely because of limitations in the availability of a suitable detector. In spite of this, some excellent separations have now been achieved [168], and the technique is rapidly supplanting TLC in many laboratories. In comparison to the latter, it offers superior resolution, easier quantification together with a degree of automation, cleaner fractions, and a more hygienic working environment.

In discussing lipid class separations by means of HPLC, it is not possible to use a "recipe" treatment, as the approach will depend largely on the nature of the detection system available to the analyst. For example, this determines the nature of the solvents used in the mobile phase and whether gradient elution is possible. Some relevant separations are therefore described below in terms of specific detectors, as examples of what is possible.

Most lipids lack chromophores of value in spectrophotometric detection, but the absorbance of isolated double bonds (and some other functional groups) at about 205 nm in the UV range can be used successfully if care is taken in the choice of the solvents for the mobile phase. UV detection at low wavelengths has its limitations, however. For example, only a few solvents are transparent and can be used in the appropriate range (e.g. hexane, methanol, acetonitrile, isopropanol and water), and the molar extinction coefficient is so low that traces of impurities in the solvents or in the samples (e.g. hydroperoxides) can swamp the signals from the compounds of interest. It is possible to convert lipids or their component parts into derivatives with a high UV absorptivity for HPLC separation in some circumstances, and procedures of this kind are generally favoured for the separation of individual glycolipids.

One of the most popular approaches to the separation of phospholipid classes consists in the use of UV-transparent mobile phases, such as hexane-isopropanol-water or acetonitrile-methanol-water mixtures, with detection at about 200 nm. Strong acids have been incorporated into the mobile phase in some laboratories as ion suppressants, but this can lead to degradation of plasmalogens and superior alternatives are available. Of the large number of procedures of this kind described, that of Patton and co-workers [694] appears particularly convincing, and has been adopted by a number of others. It has the additional merit of employing an isocratic elution scheme, so reducing the requirements in terms of costly equipment. Hexane-isopropanol-25 mM phosphate buffer-ethanol-acetic acid (367:490:62:100:0.6 by volume) (see the original paper for the method of mixing) is the mobile phase, at a flow-rate of 0.5 mL/min for the first hour when it is increased to 1 mL/min. The column (4.6 × 250 mm) used originally contained LiChrospher™ Si-100 silica gel, and detection was at 205 nm. Figure 2.4 illustrates the nature of the separation achieved with a rat liver extract. Phosphatidylethanolamine eluted just after the neutral lipids, and was followed by each of the acidic lipids, i.e. phosphatidic acid, phosphatidyl-inositol and phosphatidylserine, then by diphosphatidylglycerol and by the individual choline-containing phospholipids. Only the phosphatidyl-choline and sphingomyelin overlapped slightly. As each component was eluted, it was collected, washed to remove the buffer, and determined by phosphorus analysis. In addition, the fatty acid composition of each lipid class was obtained with relative ease, by GLC analysis after transmethylation of the fractions.

HPLC separation of rat liver phospholipids

Figure 2.4. Isocratic elution of rat liver phospholipids from a column of silica gel with hexane-isopropanol-25 mM phosphate buffer-ethanol-acetic acid (367:490:62:100:0.6 by volume) as mobile phase at a flow-rate of 0.5 mL/min for the first 60 minutes then of 1 mL/min, and with spectrophotometric detection at 205 nm [694]. (Reproduced by kind permission of the authors and of the Journal of Lipid Research, and redrawn from the original publication). Abbreviations; NL, neutral lipids; PE, phosphatidylethanolamine; PA, phosphatidic acid; PI, phosphatidylinositol; PS, phosphatidylserine; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; SPH, sphingomyelin; LPC, lyso- phosphatidylcholine; x1, x2, x3 and x4, unidentified lipids.

 

One of several bonded stationary phases to have been used in phospholipid separations with isocratic elution had a benzene sulfonate residue as the functional group. A column (4.6 × 250 mm) of Partisil™-SCX and elution with acetonitrile-methanol-water (400:100:34 by volume) at a flow-rate of 2.5 mL/min were used to effect separation of the main ethanolamine- and choline-containing phospholipids of animal tissues [199,316]. While spectrophotometry at 203 nm was used to detect the components, phosphorus assay was preferred for quantification purposes.

Refractive index detectors also have several applications in lipid analysis. They are "universal" detectors, but lack sensitivity, require isocratic elution conditions and are sensitive to minor fluctuations in temperature. Their main value is probably in small-scale preparative applications, say with 1-2 mg of a lipid extract. For example, a refractive index detector was utilized with a column (4.6 × 250 mm) of Ultrasil™ Si (5 micron silica gel) and isocratic elution with isooctane-tetrahydrofuran-formic acid (90:10:0.5 by volume) to separate most of the common simple lipid classes encountered in animal tissue extracts, such as those of liver [304]. Cholesterol esters, triacylglycerols and cholesterol were each resolved and gave symmetrical peaks.

Transport-flame ionisation detectors probably represent the future in HPLC analysis. With such systems, the eluent is entrained on a moving belt and carried through an oven where the solvent is removed, and then in essence into a flame ionization detector, where the separated components are combusted and detected as in a gas chromatograph. A wide range of solvents can be used, and the response is highly rectilinear with respect to mass. Unfortunately, the existing commercial instruments are rather too costly for most analysts, and their reliability has still to be ascertained.

The author has been using the "mass detector", also known as the "light-scattering detector" or "evaporative analyser", for some years now. It is an optical device, marketed by Applied Chromatography Systems (Macclesfield, UK). With this detector, the eluent from the column passes into a heated chimney where the solvent is evaporated in a stream of compressed air; the solute does not evaporate, if it is a lipid, and passes as a "fog" through a light beam, which is reflected and refracted. The amount of scattered light can be measured and bears a relationship to the amount of material eluting. It can therefore be termed a universal detector as it is not dependent on particular chromophores, and it can be used with gradients and a wide variety of different mobile phases. It is relatively inexpensive and rugged, but has limitations in quantitative analysis. Although the sample is lost during detection, it is possible to insert a stream-splitter between the end of the HPLC column and the detector so that a high proportion of the eluent is diverted to a fraction collector.

In order to separate and quantify the more abundant lipid classes in animal tissues, ideally on the 0.2 to 0.4 mg scale and in as short a time as could conveniently be managed, the author made use of the ACS mass detector with a ternary solvent delivery system and a short (5 × 100 mm) column, packed with Spherisorb™ silica gel (3 μm particles) [165]. In selecting a mobile phase, the choice of solvents was constrained by the need for sufficient volatility for evaporation in the detector under conditions that did not cause evaporation of the solute, and by the necessity to avoid inorganic ions, which would not evaporate. Similar restrictions apply to detectors operating on the transport-flame ionization principle. It was necessary to use a complicated ternary-gradient elution system with eight programmed steps, starting with isooctane to separate the lipids of low polarity and ending with a solvent containing water to elute the phospholipids; a solvent of medium polarity was then needed to mediate the transfer from one extreme to the other, and mixtures based on isopropanol gave satisfactory results. The three solvent mixtures selected by trial and error were isooctane-tetrahydrofuran (99:1, v/v)(A), isopropanol- chloroform (4:1, v/v)(B) and isopropanol-water (1:1, v/v)(C). In essence, a gradient of B into A was created to separate each of the simple lipids, then a gradient of C into A plus B was produced to separate each of the complex lipids; finally, a gradient in the reverse direction was generated to remove most of the bound water and to re-equilibrate the column prior to the next analysis. A relatively high flow-rate (2 mL/min) appeared to assist the separation greatly, perhaps compensating for the absence of strong acid or inorganic ions, which others have found necessary for the separation of phospholipids.

In later work [166], it was observed that much better resolution of the minor acidic components was obtained by adding small amounts of organic ions to the aqueous component of the eluent. The lifetime of the column was also greatly extended by this simple step. In practice, the optimum results were obtained with 0.5 to 1 mM serine buffered to pH 7.5 with triethylamine. In addition, hexane replaced isooctane in the mobile phase, in order to reduce the maximum operating pressure required.

The nature of the separation achieved with a lipid extract from rat kidney is shown in Figure 2.5 [166]. In spite of the abrupt changes in solvent composition at various points, little base-line disturbance is apparent, and each of the main simple lipid and phospholipid classes is clearly resolved in only 20 minutes. Only the highly acidic lipids, phosphatidic acid and to a lesser extent phosphatidylserine, do not give satisfactory peaks. There is no "solvent peak" at the start of the analysis, as is often seen with other detectors, and BHT added as an antioxidant evaporates with the solvent so does not interfere. After a further 10 minutes of elution to regenerate the column, the next sample can be analysed.

HPLC separation of rat kidney lipids Figure 2.5. Separation of rat kidney lipids (0.35 mg) by HPLC on a column (5 × 100 mm) of Spherisorb™ silica gel (3 μm particles) with mass detection; the elution conditions are described in the text [166]. (Reproduced by kind permission of the Journal of Chromatography). The legend to Figure 2.4 contains a list of abbreviations (or see below). In addition: CE, cholesterol esters; TG, triacylglycerols; C, cholesterol; DG, diacylglycerols; MG, monoacylglycerols; FFA, free fatty acids.

There are of course many other applications of HPLC in the separation of lipids that complement or otherwise assist GC analyses, and some of these are discussed in later chapters and elsewhere [168].

 

3.  Thin-layer chromatography

TLC has been much used by lipid analysts over the last 30 years or so, and it has served them well. The equipment required is inexpensive and flexible, in that in can be used both analytically and preparatively with many different types of layers. While HPLC is supplanting it in several areas, it is likely to continue to be of value in many circumstances.

TLC procedures with silica gel G layers (containing calcium sulfate as binder) have been employed most frequently for lipid class separations. Commonly, the solvent elution system used is hexane-diethyl ether-formic acid (80:20:2 by volume), and this gives the separations shown in Figure 2.6. Cholesterol esters migrate to the solvent front, and they are followed by triacylglycerols, free fatty acids, cholesterol, diacylglycerols, monoacylglycerols and phospholipids (with other polar lipids). For small-scale preparative purposes (2 to 20 mg), the author prefers glass plates (20 × 20 cm) coated with a layer 0.5 mm thick of silica gel G. Bands are then conveniently detected by spraying with an 0.1% (w/v) solution of 2',7'-dichlorofluorescein in 95% methanol and viewing the dried plate under ultraviolet light; the lipids appear as yellow spots against a dark background, and they can be recovered from the adsorbent by elution with solvents for further analysis. Diethyl ether or chloroform will elute simple lipids quantitatively, while chloroform-methanol-water (5:5:1, by volume) is required for phospholipids. It is possible to add an internal standard, such as the methyl ester of an odd-chain fatty acid not present naturally in the sample, to transesterify, and to determine both the fatty acid composition and the amount of lipid (relative to the standard) simultaneously by GLC analysis [182]. Suitable procedures are discussed in subsequent chapters.

 

Figure 2.6. Schematic TLC separation of simple lipids on a silica gel G layer. Hexane-diethyl ether-formic acid (80:20:2 by volume) was the developing solvent.  TLC separation of simple lipids

One-dimensional TLC procedures can be recommended for the separation of natural mixtures of phospholipids with relatively simple compositions for the most abundant components, for rapid group separations, and for small-scale preparative purposes. When acidic phospholipids are present in a sample at low levels only, the common components may be separated on layers of silica gel G, by using chloroform-methanol-water (25:10:1 by volume) as the mobile phase for development. The nature of the separation is shown in Figure 2.7 (plate A). The minor acidic lipids, such as diphosphatidylglycerol, migrate ahead of phosphatidylethanolamine and the choline-containing phospholipids; phosphatidylserine tends to elute with phosphatidylethanolamine while phosphatidylinositol co-chromatographs with phosphatidylcholine.

TLC separation of phospholipids

Figure 2.7. Schematic TLC separations of phospholipids. Plate A, silica gel G layer and development with chloroform-methanol-water (25:10:1 by volume); Plate B, silica gel H layer and development with chloroform-methanol-acetic acid-water (25:15:4:2 by volume) [847]; Plate C, plant complex lipids on silica gel G and development with diisobutyl ketone-acetic acid-water (40:25:3.7 by volume) [664]. The legends to Figures 2.4 and 2.5 contain a list of abbreviations (or see the end of this document); St, sterols; SG, sterol glycosides; MGDG, monogalactosyldiacylglycerols; DGDG, digalactosyldiacylglycerols.

 

In most circumstances, it is preferable to employ layers of silica gel H (without a binder) as sharper separations of most of the individual phospholipid classes, but especially of the minor acidic components, are obtained in the absence of metal ions. Unfortunately, silica gel H layers tend to be more fragile than those prepared from silica gel G, and different brands can vary greatly in their elution characteristics. The most popular separation system of this kind (especially for the lipids from animal tissues) consists of a layer of silica gel H, made in a slurry with 1 mM sodium carbonate solution to render it slightly basic, and developed in chloroform-methanol-water-acetic acid (25:15:4:2 by volume); the separation is shown in Figure 2.7 (plate B) [847]. Diphosphatidylglycerol and phosphatidic acid move to the solvent front, and phosphatidylserine and phosphatidylinositol migrate between the most abundant phospholipid constituents. If simple lipids are present in the sample, they can be run to the top of the plate by elution with acetone-hexane (1:3, v/v), prior to separation of the phospholipids [846]. Many modifications of this system have been described, often to compensate for local conditions of temperature and humidity, or for changes in the properties of particular makes of silica gel. Problems are most often manifested in the separation of phosphatidylserine and phosphatidylinositol from the other lipids, and some commercial brands of silica gel H appear to be better than others for the purpose. Other workers have reported improved separations by incorporating either ammonium sulfate [471] or EDTA [40] into the silica gel H layer.

One recently described procedure, which does appear to afford distinctive separations, makes use of silica gel layers containing boric acid, which presumably forms complexes with hydroxyl groups such as those in phosphatidylinositol, retarding the rate of migration of this lipid class; the order of elution is thus very different from those obtained with other systems, and both phosphatidylserine and phosphatidylinositol especially are separated with relative ease from the other phospholipids [256].

One-dimensional TLC systems have been used less often with plant lipid extracts, as glycolipids tend to co-chromatograph with phospholipids when many of the common elution systems are used. Nonetheless, some valuable separations have been described [627,664,722] and one is illustrated in Figure 2.7 (plate C). Mono- and digalactosyldiacylglycerols each elute as distinct bands.

In most instances, many more distinct phospholipid classes can be separated by two- than by one-dimensional TLC procedures, and this is of special value with some of the biologically important phospholipids, which are often present in tissues at rather low levels. As innumerable examples of such separations have been published, the reader is referred to the author's previous book for further details [163].

 

4.  Ion-exchange chromatography

Column chromatography on diethylaminoethyl-(DEAE)-cellulose is a valuable but somewhat under-used method for the isolation of particular groups of complex lipids in comparatively large amounts [163,778]. The principle of the separation process is complex, involving partly ionic interactions between the packing material and the polar head groups of complex lipids, and partly adsorption effects with the polar regions of the molecules. In practice as a rough guide, about 300 mg of complex lipids can be applied to a 30 × 2.5 cm column to yield fractions with distinctive compositions and little cross-contamination. Although it has been used mainly on this scale, it is also possible to use the technique with much smaller columns and proportionately less lipid, e.g. for the isolation of plant lipids [777].

In the typical elution conditions, chloroform is employed first to obtain the simple lipids. All the choline-containing phospholipids are eluted with a chloroform-methanol mixture of relatively low polarity, while a much higher proportion of methanol is required to recover the ethanolamine-containing phospholipids; phosphatidylserine is eluted with glacial acetic acid, and a solvent of high ionic strength is required to recover the more acidic phospholipids (the salts can be removed later by a "Folch" washing step). Further separation of the individual components of particular fractions can later be achieved by means of HPLC or TLC, more easily than with the unfractionated extract. With plant lipid extracts, monogalactosyldiacylglycerols tend to elute with the chloroform fraction, but digalactosyldiacylglycerols can be recovered on their own if care is taken [666]. At the end of the analysis, it is an easy matter to regenerate the column for re-use, though it is important not to move too abruptly from solvents of high to those of low polarity.

An ion-exchange material consisting of quaternary triethylammonium (QAE) groups covalently bound to controlled-pore glass (Glycophase™) appears to afford similar separations to those obtained with DEAE-cellulose, but the former has better packing and compressibility properties in low-pressure column chromatography applications, and will probably be more widely used in future [76].

 

Abbreviations

The following abbreviations are employed at various points in the text of these chapters:

amu, atomic mass units; BDMS, tert-butyldimethylsilyl; BHT, 2,6-di-tert-butyl-p-cresol; CI, chemical ionisation; DNP, dinitrophenyl; ECL, equivalent chain length; ECN, equivalent carbon number; EI, electron-impact ionisation; FCL, fractional chain length; GC, gas chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; IR, infrared; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAF, platelet-activating factor; ODS, octadecylsilyl; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, ultraviolet.

 

This document is part of the book Gas Chromatography and Lipids by William W. Christie and published in 1989 by P.J. Barnes & Associates (The Oily Press Ltd), who retain the copyright.

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Updated June 27, 2011