Alternative or Complementary Methods for the Analysis of Molecular Species of Lipids
The complexity of natural lipid samples is such that it is unrealistic to expect to be able to separate all lipid classes into molecular species in a comprehensive way by GC alone. A preliminary separation by some other chromatographic method is often essential if sense is to be made of complicated GC traces. In addition, it must be recognised that there are many circumstances in which GC may not be the best method to resolve particular molecular fractions, although sometimes it may be the only one available to a particular analyst. The three most important alternative or complementary techniques are adsorption, reversed-phase partition and silver ion chromatography. They may be used in conjunction with either TLC or HPLC, although the latter is greatly to be preferred when the required equipment is available. Adsorption and silver ion chromatography can be regarded as valuable adjuncts to GC, since they provide a form of separation that may not always be attainable by the last. HPLC in the reversed-phase mode is probably used more as an alternative to GC, especially with lipids of high molecular weight and with polar complex lipids in intact form.
All of these procedures have one distinct advantage over GC in that they operate at ambient temperature, so there is less opportunity for thermal degradation. They can also be utilised for small-scale preparative purposes as well as for analysis per se. On the other hand, HPLC has a higher initial capital cost and higher running costs than GC. The author has reviewed these methods elsewhere in some detail [163,168], and the following is intended merely as an introduction to and brief summary of the subject so that the reader will be aware of the broader analytical strategies available. Whatever the analytical approach, it is frequently necessary to have recourse to GC at some stage, if only to analyse the alkyl and acyl constituents of fractions separated by the alternative means.
B. Adsorption Chromatography
Adsorption chromatography is of most value when some of the molecular species in a lipid class contain a polar moiety, such as a free hydroxyl group, or when the fatty acids have a much wider spread of chain lengths than normal. For example, the triacylglycerols of the Beluga whale contain isovaleric acid, and species with zero, one and two molecules of this acid have been isolated by TLC on silica gel G with a solvent system of hexane-diethyl ether-acetic acid (87:12:1 by volume) as illustrated in Figure 9.1(A); the presence of a short-chain acid retards the migration of the molecule relative to that of triacylglycerols with only long-chain fatty acids . Similarly, the triacylglycerols of ruminant milks can be separated by related TLC systems into two rough fractions, one containing three fatty acids of normal chain length and one containing two long-chain fatty acids and one butyrate or hexanoate residue. It is not uncommon to see individual glycosphingolipids and sphingomyelin migrating as double bands on TLC or HPLC in the adsorption mode, because of the presence of fatty acids of longer than usual chain length in some molecular species.
Figure 9.1. Schematic TLC separation of triacylglycerols containing short-chain or polar fatty acids on layers of silica gel. Plate A: triacylglycerols from the Beluga whale; the numbers refer to the isovaleroyl residues in each molecule . Plate B: seed oil containing vernolic acid; the numbers refer to the epoxy fatty acid residues in each molecule . The compositions of the mobile phases are given in the text.
Fatty acids with polar substituents also retard the migration of triacylglycerols on TLC adsorbents, and those from seed oils that contain epoxy fatty acids can be separated into simpler species that contain from zero to three epoxide residues per mole. For example, silica gel layers and a solvent system of hexane-diethyl ether (3:1, v/v) were used to isolate such fractions from seed oils (Fig. 9.1(B)) . Similar separations have been achieved with triacylglycerols that contain hydroxyl, estolide and other functional groups in the fatty acids.
As HPLC is a relatively new technique in lipid methodology, fewer separations of this kind have been described, but sufficient experience is available to leave little doubt as to the potential of the technique . HPLC in the adsorption mode is also of great value for the separation of peroxidized lipids, including both triacylglycerols and glycerophospholipids, from unoxidized material (c.f. [690,761,910]). As an example, a separation of a monohydroperoxide fraction prepared from autoxidised trilinolein is illustrated in Figure 9.2 . An HPLC column containing silica gel was eluted with 1% isopropanol in hexane, and the six fractions obtained represent different positional and configurational isomers of the hydroperoxyl group within the fatty acids of the triacylglycerol molecules.
Figure 9.2. Separation of a monohydroperoxide fraction prepared by autoxidation of trilinolein by HPLC . A column (250 × 4 mm) of Zorbax SIL™ was eluted with hexane-isopropanol (99:1, v/v) at a flow-rate of 1 mL/min with spectrophotometric detection at 235 nm. The peaks represent different positional and configurational isomers of the hydroperoxide group and the associated conjugated double bond systems. (Reproduced by kind permission of the authors and of Agricultural and Biological Chemistry (Japan), and redrawn from the original paper.)
C. Silver Ion Chromatography
The principles of silver ion chromatography and its value for the simplification of complex fatty acid mixtures have already been discussed in Chapter 6 (and see our specialist web pages on this topic here..). As the nature of the separation involves resolution according to degree of unsaturation of the combined fatty acid residues, the technique is perhaps of even greater value when applied to the fractionation of molecular species of intact lipids. High-temperature GC is at its best for separation simply by chain length, in spite of the recent developments discussed in Chapter 8. When fractions obtained by silver ion chromatography are subsequently resolved further by GC, there is an enormous gain in the amount of information acquired. For example, eight or more fractions can be obtained by silver ion chromatography from many natural triacylglycerols; if these each give four peaks when separated by high-temperature GC, 32 different molecular species are seen.
Because of the historical development of the technique most research has been carried out using TLC with silver nitrate incorporated into the silica gel layer (10 to 15%, w/w). For example, the normal range of triacylglycerols found in seed oils or animal fats contains zero to three double bonds per fatty acid, so they can contain species with up to nine double bonds in each molecule. Components migrate in the order:
SSS > SSM > SMM > SSD > MMM > MMD > SMD > MMD > SDD > SST > MDD > SMT > MMT > DDD > SDT > MDT > DDT > STT > MTT > DTT > TTT
- where S, M, D and T denote saturated, mono-, di- and trienoic acids, respectively (they do not indicate the positions of the fatty acids on the glycerol moiety), although there may be some changes in this order depending on the nature of the solvent mixtures used for development [330,775]. It is noteworthy that one linoleate moiety is more strongly retained than two oleates, and that species containing one linolenate are retarded more than those containing two linoleates. Some simplification of even more highly unsaturated triacylglycerols, such as in fish oils, has also been attained by silver nitrate TLC , but HPLC systems give much better results (see below). The solvent systems generally employed for development consist of hexane-diethyl ether, toluene-diethyl ether or chloroform-methanol mixtures. (Note that when chloroform is utilised in a mobile phase in this way, it is necessary to wash it with water to remove any ethanol present as stabiliser, then to dry it, to ensure that the composition of the mobile phase is that required ).
As all the fractions listed above cannot be separated on one plate, it is common practice to separate the least polar fractions first with hexane-diethyl ether (80:20,v/v) or chloroform-methanol (197:3, v/v) as the mobile phase, as illustrated in Figure 9.3, plate A, and then to re-chromatograph the remaining fractions with more polar solvents such as diethyl ether alone or chloroform-methanol (96:4, v/v) as illustrated in the same figure but plate B. Bands are detected under UV light after spraying with 2',7'-dichlorofluorescein solution, the components are recovered (see Chapter 6) and they are identified and determined by GC of the fatty acid constituents with an added internal standard after trans-methylation (Chapter 4). As an alternative, tridecanoin can be added as the internal standard for the analysis of intact fractions by high-temperature GC. For analytical purposes only, it is possible to estimate fractions by densitometry following bromination and charring . Approximately 10 mg of triacylglycerols can be fractionated on a 20 × 20 cm plate (0.5 mm thick layer), and excellent separations of large numbers of components have been obtained with 20 × 40 cm plates.
|Figure 9.3. Schematic TLC separation of maize oil triacylglycerols on layers of silica gel G, impregnated with 10% silver nitrate. Mobile phases: Plate A, chloroform-methanol (99:1, v/v); Plate B, chloroform-methanol (96:4, v/v). Abbreviations: S, M, D and T denote saturated, mono-, di- and trienoic fatty acyl residues respectively, esterified to glycerol.||
In addition, some separation of isomeric compounds is possible. For example, triacylglycerols of the type S2M, in which the monoenoic component is in position 2, are separable with care from the related compounds in which the monoenoic component is in position 1(3). Presumably, the presence of long-chain fatty acids on either side of the monoenoic acid weaken the pi-complex with the silver ion, permitting the component with the monoenoic fatty acid in position 2 to migrate ahead of isomers in which this residue is in position 1, since separation of this kind cannot be obtained with diacylglycerol acetates where one side of the molecule is comparatively open. When the fatty acid constituents of triacylglycerols contain trans-double bonds, the elution pattern is much more complicated as components with trans-acids migrate ahead of analogous compounds with fatty acids containing cis-double bonds, producing a complex elution pattern. Molecular species of triacylglycerols with polar fatty acid constituents, separated by adsorption chromatography as described above, may be further fractionated by silver nitrate TLC . One disadvantage of silver ion TLC is that it is messy; purple-stained fingers are an occupational hazard for the analyst.
Although column chromatography (low pressure) with silver nitrate-impregnated adsorbents has been used to separate triacylglycerols, it lacks the resolving power of TLC. HPLC with silica gel impregnated with silver salts has so far been little used for the purpose because of problems with silver ions eluting from the adsorbent, although some impressive separations have been recorded (reviewed elsewhere ). On the other hand, the column prepared by binding silver salts to an adsorbent containing chemically bonded sulfonic acid residues, and discussed already in Chapter 6, has shown considerable promise for triacylglycerol separations . It is necessary to employ mobile phases that do not contain alcohols, otherwise residual free sulfonic acid groups on the stationary phase catalyse transesterification of the solute.
As an example, a separation of the triacylglycerols palm oil was illustrated in the original paper, in which a linear gradient of acetone into 1,2-dichloroethane was employed as the mobile phase with evaporative light-scattering detection, and fractions with up to three double bonds were resolved, i.e. up to SMD. More recently, the author (W.W. Christie, J. Chromatogr., 454, 273-284 (1988)) has shown that species of a higher degree of unsaturation may be eluted by incorporating acetonitrile into the mobile phase at a later stage of the gradient. With the same palm oil sample, species up to SDD can then be analysed, as illustrated in Figure 9.4, and indeed trilinolenin elutes as a sharp single peak from linseed oil if the gradient is extended. The order of elution differs slightly from that obtained with the TLC system, one linoleate residue having an effect on retention time equivalent to just over two monoenes, while one α-linolenate is equivalent to two linoleates, i.e.
SSS > SSM > SMM > SSD > MMM > SMD > MMD > SDD = SST > SMT = MDD > MMT > SDT = DDD > MDT > STT ≥ DDT > MTT > DTT > TTT
Figure 9.4. Silver ion HPLC separation of molecular species of triacylglycerols from palm oil on a column of Nucleosil 5SA™ (250 × 4.6 mm) loaded with silver ions. The mobile phase was a linear gradient of 1,2-dichloroethane-dichloromethane (1:1, v/v) to this solvent with 50% acetone over 15 min, when acetonitrile was introduced to give a final mixture of acetone-acetonitrile (9:1, v/v) after a further 30 min, at a flow-rate of 0.75 mL/min and with evaporative light-scattering detection (see W.W. Christie, J. Chromatogr., 454, 273-284 (1988)). Abbreviations - as in the legend to Figure 9.3.
A major advantage of using HPLC as opposed to TLC in this way is that silver ions are not eluted with the mobile phase, so that fractions collected for analysis are particularly clean. In addition, the column is long lived. This methodology will undoubtedly be used more often in future.
2. Phospholipids and diacylglycerol derivatives
It is not easy to subject intact phospholipids to silver ion TLC, because the polar head group masks the comparatively small changes in polarity produced by the formation of pi-complexes between silver ions and the double bonds of the unsaturated fatty acids. Nonetheless, Arvidson [60,61] has achieved some valuable separations of phosphatidylcholines by using highly active TLC plates. TLC layers (0.35 mm thick) were prepared with silica gel H (without a binder) and silver nitrate in the proportions 10:3 (w/w), and were air-dried at room temperature (in the dark) for 24 hours initially, then either at 175°C for 5 hours or at 180°C for 24 hours; plates prepared under the latter conditions were much more active than those dried at the lower temperature. By utilising a mobile phase of chloroform-methanol-water (60:35:4 by volume) and with the more active plates, species with one to six double bonds per molecule were separable from each other. Only the saturated and monoenoic fractions were not completely resolved, but they could be separated on the less active plates with the same solvent system. Up to 20 mg of phospholipids could be separated on a 20 × 20 cm plate, and bands were recovered from the adsorbent after detection (see above) by elution with chloroform-methanol-acetic acid-water (50:39:1:10 by volume), washed subsequently with one-third of the volume of 4 M ammonia to remove the dye and excess silver ions.
Native phosphatidylethanolamines have been separated under similar conditions to these , although other workers have preferred to prepare nonpolar derivatives (retaining both the phosphorus and ethanolamine moieties), such as the N-dinitrophenyl-O-methyl- , N-acetyl- or N-benzoyl-O-methyl- , and trifluoroacetamide  compounds. Similarly, phosphatidylinositol was rendered nonpolar by acetylating the inositol moiety and reacting the phosphate with diazomethane, prior to silver ion TLC .
Chromatography of intact phospholipids in this way can be invaluable in biochemical studies, but the number of fractions obtainable is limited and subsequent analysis by high-temperature GC is not possible. If a more detailed analysis is required, it is therefore necessary to hydrolyse phospholipids with phospholipase C, as described in Chapter 8, for analysis as the diacylglycerol acetate or BDMS ether derivatives. The former have been used most for separation by silver ion TLC, and they migrate in the order -
SS > SM > MM > SD > MD > DD > ST > MT > STe > MTe > DTe > SP > SH
- where S, M, D, T, Te, P and H denote saturated, mono-, di-, tri-, tetra-, penta- and hexaenoic fatty acid residues, respectively (they do not indicate the relative positions of the fatty acids on glycerol as positional isomers are eluted together) [512,739]. Figure 9.5 illustrates the separation of diacylglycerol acetates prepared from the phosphatidylcholines of pig liver on layers of silica gel G impregnated with 10% silver nitrate, developed in either chloroform-methanol (99:1, v/v) or hexane-diethyl ether (95:15, v/v). When the sample contains high proportions of polyunsaturated fatty acids, it may be necessary to repeat the elution step with a solvent system containing an increased concentration of the more polar component. Fractions are eluted from the adsorbent for further analysis as described above for triacylglycerols. Diacylglycerol acetates with up to twelve double bonds in the combined fatty acid moieties have been separated on similar plates with chloroform-methanol-water (65:25:4, by volume) as the solvent system . With care, it is possible to separate molecular species of positional isomers of polyunsaturated fatty acids, and fractions containing 18:3(n-6) migrate ahead of those containing 18:3(n-3), while those containing 20:4(n-6) migrate ahead of 20:4(n-3) . Alkylacyl and alk-1-enylacyl derivatives can be analysed in a similar manner .
Figure 9.5. Schematic TLC separation of diacylglycerol acetates prepared from the phosphatidylcholine of pig liver, on layers of silica gel G impregnated with 10% (w/w) silver nitrate. Mobile phase: chloroform-methanol (99:1, v/v).
Abbreviations: S, M, D, T, Te, P and H denote saturated, mono-, di-, tri-, tetra-, penta- and hexaenoic fatty acyl residues, respectively, esterified to glycerol.
Although HPLC in the silver ion mode does not appear to have been used with diacylglycerol acetates, there is no reason why the column described above and utilised in triacylglycerol separations should not be employed for the purpose with very little modification to the elution schemes.
D. Reversed-Phase Partition Chromatography
1. Some practical considerations
While many practical TLC systems have been devised that utilise the principle of reversed-phase partition in the separation process, they have generally proved inconvenient and of doubtful reproducibility in use. The newer pre-coated high-performance TLC plates of this kind are much better but they are very costly. When reversed-phase chromatography is used in conjunction with HPLC, there are no such drawbacks and this approach is now generally favoured. It has been reviewed in detail by the author  and in relation to phospholipids specifically by others [661,696,779]. The technique is a form of partition chromatography, the term "reversed-phase" implying that the stationary phase is a nonpolar liquid and the mobile phase is more polar. Separations depend on differences in the equilibrium distribution coefficients of solutes between the two phases, and in the case of lipids, this is a function of both the chain lengths of the fatty acid residues and the number of double bonds, one cis-double bond reducing the retention volume by the equivalent of about two methylene groups. The principles and applications of the technique to analyses of simple fatty acids are described briefly in Chapter 6 above. When it is applied to molecular species of intact lipids, such as triacylglycerols, the separation is dependent on the combined chain lengths of the three fatty acyl residues and the total number of double bonds.
One of the major problems facing lipid analysts who wish to make use of HPLC is the choice of a suitable detection system, since lipids in general lack chromophores which can be detected spectrophotometrically. This problem has been discussed in relation to the separation of specific lipid classes in Chapter 2. It is frequently the availability of a particular detection system that is the principal factor in determining the strategy to be adopted for particular separations of molecular species. There is little doubt that detectors operating on the transport-flame ionisation principle are ideal for the purpose, although the commercial instruments are relatively costly and are not yet in widespread use. In essence, there are no restrictions on the range of solvents that can be employed in the mobile phase, and the response is quantitative.
Similarly, evaporative light-scattering detection permits most solvent combinations to be employed, but quantification is more problematical. UV detection at wavelengths around 205 nm, where isolated double bonds absorb, has been used more often, but the range of solvents transparent in the required range is limited and the detector response is highly dependent on the nature of each lipid fraction. Some better results in triacylglycerol separations have been obtained by detection at 220 nm by a careful choice of mobile phase (see below). Good linearity of response is possible with differential refractometry with the better-quality instruments, but it is not possible to use gradient elution. Of course, mass spectrometry coupled to HPLC is a splendid research tool, but it is available to a relatively few analysts only (reviewed elsewhere [168,515]). The discussion that follows therefore treats the subject in terms of specific detection systems.
The relative retention time of a given component has been defined in terms of an "equivalent carbon number" (ECN) or "partition number" value, defined as the actual number of carbon atoms in the aliphatic residues (CN) less twice the number of double bonds (n) per molecule (the carbons of the glycerol moiety are not counted for this purpose), i.e.
ECN = CN - 2n
Two components having the same ECN value are said to be "critical pairs". For example, triacylglycerol species containing the fatty acid combinations 16:0-16:0-16:0, 16:0-16:0-18:1, 16:0-18:1-18:1 and 18:1-18:1-18:1 have the same ECN value and tend to elute close together. (The positions of the fatty acids within the triacylglycerol molecules again have no effect on the nature of the separation). The ECN concept was useful in the early days of the technique, when the resolving power was relatively limited. On the other hand, the formula is now only of utility as a rough rule of thumb, by way of a guide to what may elute in a given area of a chromatogram, since the greatly increased resolving power of modern HPLC phases means that the factor for each double bond has to be defined much more precisely. Also, this factor can no longer be treated as a constant, as a second double bond in a molecule has a slightly different effect from the first. Accordingly, more complex formulae are necessary to define the order of elution of triacylglycerols from modern reversed-phase columns, which in essence means from octadecylsilyl (ODS) stationary phases, as these have been used almost exclusively for the purpose.
Under steady state conditions, i.e. with a given column and isocratic elution at a constant temperature and flow-rate, it would be expected from theoretical considerations that there should be a rectilinear relationship between the logarithm of the capacity factor (k'), or retention time or volume, and the carbon number of each member of a homologous series of triacylglycerols containing saturated fatty acids only. This was first shown to be true in practice by Plattner et al. , and it also holds for homologous series of triacylglycerols containing particular unsaturated fatty acids.
From such evidence, it was suggested that triacylglycerol species might be identified from a theoretical carbon number (TCN), defined as
TCN = ECN - Ui
- where Ui is a factor determined experimentally for different fatty acids (zero for saturated fatty acids, and roughly 0.2 for elaidic, 0.6 to 0.65 for oleic, and 0.7 to 0.8 for linoleic acid residues) . Thus, the TCN value for a triacylglycerol containing 18:1-18:1-18:1 (triolein) is calculated as -
TCN = (3 x 16) - (3 x 0.6) = 46.2
- while for 18:1-18:1-16:0, 18:1-16:0-16:0 and 16:0-16:0-16:0, which have the same ECN value, the TCN values are 46.8, 47.4 and 48.0 respectively. The values of Ui will vary with the elution conditions and have to be determined independently by each analyst for his or her own system. Although alternative identification methods have been suggested by others, the concept of the TCN has been found to be useful by many workers.
Virtually all the work on the reversed-phase separation of triacylglycerols has been carried out with ODS phases. Most commercial brands of ODS phase have been used by one analyst or another for the purpose, and while it is evident from direct comparisons that some are better than others, the reasons for this are not clear.
Perhaps the single most important factor in the separation of triacylglycerols is the choice of the mobile phase. Early in the development of the methodology, it became apparent that solvent combinations based on acetonitrile gave much better resolution than any others tested. These are now used almost universally, although propionitrile is preferred by some analysts. In order to obtain the optimum separations of triacylglycerols, it is necessary to add some other solvent to the acetonitrile to increase the solubility of the solute, to change the polarity of the mixture and to modify the selectivity. It is well established that the relative proportion of acetonitrile to the modifier solvent has a marked effect on the elution time of a given triacylglycerol species, i.e. the lower the polarity of the mobile phase, the lower the retention volume. No consensus has emerged as to which combination is best, partly because there appears to be no objective criterion that can be used to assess relative merits, and partly because the nature of the columns and other equipment used in different laboratories may impose constraints.
The separations that are described below are examples selected from the wide body of literature reviewed elsewhere  to illustrate the use of particular detection systems. Acetonitrile-acetone mixtures have been used more often than any other solvent combination as the mobile phase in the reversed-phase separation of triacylglycerol species, in an isocratic manner with refractive index detection and in gradients with the evaporative light-scattering detector. It has certainly permitted some fine separations, especially with seed oils, and an application to palm oil is illustrated in Figure 9.6 . Here, the mobile phase was acetone-acetonitrile (62.5:37.5 by volume) at 30°C. Five main groups of peaks, distinguishable by their ECN values, were recognized. Group 1 (ECN = 44) consisted of the combinations 18:1-18:2-18:2, 16:0-18:2-18:2 with 14:0-18:1-18:2, and 14:0-18:2-16:0 with 14:0-18:1-14:0. Group 2 (ECN = 46) consisted of 18:1-18:1-18:2, 16:0-18:1-18:2, 16:0-18:2-16:0 with 14:0-18:1-16:0, and 14:0-16:0-16:0. Group 3 (ECN = 48) is a particularly important one in confectionery fats and comprised predominantly 18:1-18:1-18:1, 16:0-18:1-18:1, 16:0-18:1-16:0 and 16:0-16:0-16:0. In group 4 (ECN = 50), only three main components could be recognized, i.e. 18:0-18:1-18:1, 16:0-18:1-18:0, and 16:0-16:0-18:0, while the fifth group (ECN = 52) contained 18:0-18:1-18:0 and 16:0-18:0-18:0. Although gradient elution is not possible with differential refractometry, temperature-programming can be utilised with a detector of sufficient quality in order to speed up separations and improve resolution with complex mixtures, and some excellent analyses of triacylglycerols from seed oils and from milk fat have been obtained in this way [264,265].
Figure 9.6. HPLC (reversed-phase) separation of triacylglycerols from palm oil . Two ODS columns in series were maintained at 30°C, with acetone-acetonitrile (62.5:37.5, v/v) as the mobile phase at a flow-rate of 1.1 mL/min, and with refractive index detection. The abbreviations refer to fatty acyl residues: M, 14:0; P, 16:0; S, 18:0; A, 20:0; O, 18:1; L, 18:2. The ECN values are listed above the appropriate groups of peaks. (Reproduced by kind permission of the author, and of Revue Française des Corps Gras, and redrawn from the original paper.)
The main difficulty associated with acetonitrile-acetone as the mobile phase, other than its unsuitability with UV detection, is that trisaturated species of high molecular weight, i.e. above C48, tend to be insoluble and can crystallize out in the column. Other solvent combinations have therefore been sought. Dichloromethane-acetonitrile has been used to good effect as a mobile phase, especially by Privett's group, with a transport-flame ionization detector of their own design [711,712]. The eluent was tested first with model mixtures, and so good was the resolution that it was possible to distinguish molecular species containing petroselinoyl (i.e. with the cis-double bond in position 6) from those with oleoyl (position 9) residues. It also gave good results with natural samples such as vegetable oils. Only one application of the commercial Tracor™ transport-flame ionisation detector to triacylglycerols has so far been published, and it confirms the potential of the instrument (unfortunately no longer manufactured) .
By a careful choice of solvents, it has been possible to use UV detection in the reversed-phase separation of triacylglycerols. Shukla et al.  showed that by using a wavelength of 220 nm, i.e. away from the region in which isolated double bonds absorb, sufficiently sensitive detection and good quantification could be obtained. They employed tetrahydrofuran-acetonitrile (73:27, v/v) as the mobile phase with two columns of a 3 μm ODS phase in series, and obtained the chromatogram of cocoa butter shown in Figure 9.7. Each of the main groups with the same ECN value were well resolved from the others, as were molecular species within each group. Indeed the resolution obtained is at least as good as any published to date. It may, however, be necessary to purify the tetrahydrofuran component of the mobile phase immediately prior to use by distillation from lithium aluminium hydride to remove UV-absorbing oxidized material.
Figure 9.7. Separation of triacylglycerols from cocoa butter by reversed-phase HPLC using two columns of a 3 micron ODS phase in series . The mobile phase was acetonitrile-tetrahydrofuran (73:27, v/v) at a flow-rate of 1 mL/min, with spectrophotometric detection at 220 nm. See Figure 9.6 for a list of abbreviations. (Reproduced by kind permission of the authors and of Fette Seifen Anstrichmittel, and redrawn from the original paper.)
Several research groups have found propionitrile on its own to be an excellent mobile phase for the elution of triacylglycerol molecular species from reversed-phase columns, and indeed some would claim that it may be the best available, although it is costly and highly toxic. Schulte  first made use of it for the fractionation of cocoa butter, but it has been since been employed for many different samples and with most detection systems. In Kuksis' laboratory, a gradient of 30 to 90% propionitrile in acetonitrile has been used to effect separations of triacylglycerols from vegetable oils and animal fats [514,516,522-524]. Chemical ionisation MS, with the solvent as the ionizing agent, was utilized to detect and identify components in this research (and incidentally to correct some misidentifications made in earlier work from other laboratories).
3. Phospholipids and diacylglycerol derivatives
Methods for the separation of intact glycerophospholipids by means of HPLC in the reversed-phase mode have evolved rapidly in recent years. Although it has always been considered that nonpolar derivatives of phospholipids are capable of being resolved more cleanly than are the intact compounds, the difference is now much less marked than it was formerly. With reversed-phase HPLC of molecular species of phospholipids, the nature of the separation is similar to that discussed above for triacylglycerols, in that it is dependent on the combined chain lengths and degree of unsaturation of the fatty acyl or alkyl chains. Similarly, the detector limitations are the same as for triacylglycerol analyses. It has become apparent that some ODS phases are much better than others for phospholipid separations, although the reason for this is not known, and a number of analysts have obtained excellent resolution of phospholipids with Ultrasphere™ ODS.
A particularly important paper on the subject of separations of phospholipid molecular species was published by Patton, Fasulo and Robins in 1982 . They fractionated molecular species from different phospholipid classes on a column (4.6 × 250 mm) of Ultrasphere™ ODS, with methanol-water-acetonitrile (90.5:7:2.5, by volume) containing 20 mM choline chloride as the mobile phase. UV spectrophotometric detection at 205 nm was used to locate the fractions, and these were collected for phosphorus assay and fatty acid analysis. In Figure 9.8, the separation obtained for phosphatidylethanolamine from rat liver is illustrated. (Note that with UV detection at 205 nm, the response is highly dependent on the degree of unsaturation, and peak heights do not immediately reflect the relative abundances of the components.) The separation can be considered as bimodal, with in essence those molecular species containing a 16:0 fatty acyl group eluting before those containing 18:0. As with triacylglycerols, the position of the acyl group within the molecule has no effect on separation in the reversed-phase mode, although the saturated components are known to be located predominantly in position sn-1 in this particular sample. All of the major components are clearly resolved, and a few only of the minor fractions contain two distinct species. The problems of identification of molecular species are somewhat less than with triacylglycerols, since only two fatty acids are present.
Figure 9.8. HPLC separation (reversed-phase) of molecular species of phosphatidylethanolamine from rat liver . An ODS column was used with methanol-acetonitrile-water (90.5:7:2.5, by volume) containing 20 mM choline chloride as the mobile phase at a flow-rate of 2 mL/min, and with UV detection at 205 nm. Only a few of the peaks are identified here for illustrative purposes. (Reproduced by kind permission of the authors and of the Journal of Lipid Research, and redrawn from the original paper.)
Phosphatidylcholine and phosphatidylinositol from rat liver were separated into molecular species under exactly the same conditions. Indeed, fractions identical in composition were obtained, although the relative proportions were rather different because of variations in fatty acid profiles, as expected. After modifying the mobile phase to 30 mM choline chloride in methanol-25 mM K2HPO4-acetonitrile-acetic acid (90.5:7:2.5:0.8, by volume), molecular species of phosphatidylserine were resolved. Closely related elution schemes have been used by many other workers in separating molecular species of phospholipids.
Molecular species of phosphatidylglycerol from plant chloroplasts were fractionated by HPLC on a column (4.6 × 250 mm) containing Rainin Microsorb™ reversed-phase packing material, and with 1-ethylpropylamine-acetic acid-methanol-acetonitrile (0.3:0.5:34.7:64.5, by volume) as the mobile phase at a flow-rate of 0.8 mL/min [674,860]. Of particular interest here was what appears to have been the first published application of the Tracor transport-flame ionization detector to lipid analysis. Excellent base-line stability was recorded in spite of the presence of ionic species in the mobile phase, and minor components present at a level of as little as 1.2 nmole could be determined. It was confirmed that direct quantification of components by integration of peaks from the detector gave results which were comparable to those obtained by alternative methods. A stream splitter ahead of the detector enabled fractions to be collected for determination of radioactivity or for GC analysis of the fatty acid constituents. In the sample studied, species eluted in the order 16:1t-18:3, 16:1t-18:2 and 16:0-18:2.
The alternative approach is to prepare diacylglycerol derivatives by phospholipase C digestion as described in Section C.2 above and in Chapter 8. Some improvement in resolution may be obtained, with no requirement for inorganic ions in the mobile phase, while UV-absorbing or fluorescent derivatives of diacylglycerols may be employed, simplifying detection and quantification. In addition, complementary chromatographic techniques can more easily be brought to bear for the further resolution of fractions.
This is probably the only appropriate procedure for the separation of diradylglycerols. For example, the alkenylacyl-, alkylacyl- and diacylglycerol acetates, isolated by adsorption HPLC as described in Chapter 8, were each separated into molecular species by HPLC in the reversed-phase mode . For the alkenylacyl and alkylacyl derivatives, a column (4.6 × 250 mm) containing Zorbax™ ODS and maintained at 33°C was eluted with acetonitrile-isopropanol-methyl-t-butyl ether-water (63:28:7:2, by volume) at 0.5 mL/min; for the diacyl form, the same solvents were used but in the proportions 72:18:8:2, respectively, as illustrated in Figure 9.9. UV detection at 205 nm was again employed. Each of the diradyl forms was separated into as many as 22 fractions, and the recorder traces resembled those published by others for intact phospholipids, although the resolution was perhaps slightly better here with the acetate derivatives. This sample also contains a higher proportion of polyunsaturated fatty acids than did that in the previous figure (9.8). Fractions were collected once more for identification and determination of the alkyl and acyl moieties by GC methods. The resolution was still far from complete, and the most abundant peak for example contained 18:0-22:6(n-3), 18:1-20:3(n-6) and 18:1-18:2(n-6), three fractions that would readily be resolvable by high-temperature GC.
Figure 9.9. Reversed-phase HPLC separation of diacylglycerol acetates prepared from the phosphatidylethanolamine of bovine brain on an ODS column, with acetonitrile-isopropanol-methyl-t-butyl ether-water (72:18:8:2, by volume) at a flow-rate of 0.5 mL/min . Detection was at 205 nm and the column was maintained at 33°C. Only a few of the peaks are identified here for illustrative purposes. (Reproduced by kind permission of the authors, and of the Journal of Lipid Research, and redrawn from the original paper.)
Because disaturated molecular species of phosphatidylcholine, such as those predominating in lung, could not easily be detected and quantified spectrophotometrically at 205 nm, the use of refractive index detection for the purpose was explored and was found to give satisfactory results . BDMS ether derivatives can be analysed in the same way. Methodology of this kind could be used to obtain fractions for further resolution and identification by GC-MS, although there is little to suggest that the two techniques are being used in concert at the moment.
By converting diacylglycerols prepared from phospholipids to UV-absorbing derivatives, it has proved possible to use the high sensitivity and specificity of spectrophotometric detection in the analysis of molecular species. Dinitrobenzoate derivatives of diacylglycerols were employed to obtain some impressive separations of molecular species of phosphatidylcholine from rat tissues, for example [479,905]. With a column (4.6 × 250 mm) of Ultrasphere™ ODS and elution with acetonitrile-isopropanol (4:1, v/v) as the mobile phase, 29 distinct fractions were detected, identified and quantified. 12:0-12:0 or 18:0-18:0 species could be added as an internal standard if required. When methanol-isopropanol (19:1, v/v) was the mobile phase, only 17 fractions were seen but some not separated by the previous system were in fact resolved. Thus, by collecting fractions containing more than one component from the first eluent, a more comprehensive analysis could be obtained by re-running with the second eluent. In this way, as many as 36 distinct molecular species were obtained from each lipid class.
This method used in sequence with silver ion chromatography might prove to be an even more profitable approach. As methods for the separation of the various diradyl forms of the benzoate derivatives of diacylglycerols involving HPLC in the adsorption mode have recently been described [104,260], a fairly comprehensive HPLC methodology is now potentially available.
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.
Updated July 12, 2011