ANALYSIS OF FATTY ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

ANALYSIS OF FATTY ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Abstract: Although gas chromatography is the dominant technique for fatty acid analysis, high-performance liquid chromatography has an important role to play in applications such as the handling of less usual samples, avoidance of degradation of heat-sensitive functional groups, and for micro-preparative purposes. Several approaches for development of improved methods are suggested, especially for reversed-phase applications. There can be little doubt that gas chromatography (GC) is the only technique that need be considered for routine analysis of most fatty acid samples. The flame-ionization detector is robust and has an enormous dynamic range, so accurate quantification is rarely a problem. Therefore, is there any place for high-performance liquid chromatography (HPLC) for the analysis of fatty acids? The answer is an undoubted yes, perhaps not for mainstream samples but certainly for the less usual. A major advantage can be that HPLC operates at ambient temperature so there is relatively little risk to sensitive functional groups. It should also be remembered that HPLC is not merely an analytical technique, but can be used equally easily for micro-preparative purposes. For example, it is easy to collect fractions for analysis by other techniques such as by chemical degradation or by mass spectrometry (MS) [1] or nuclear magnetic resonance spectroscopy [2]. Indeed, direct analysis by HPLC-MS with electrospray ionization may offer an advantage in terms of sensitivityTHE EVAPORATIVE LIGHT-SCATTERING DETECTOR FOR HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF LIPIDS Abstract: Evaporative light-scattering detectors have many benefits for lipid analysis and a number of commercial instruments are now available. Analysts should not let the need for careful calibration deter them. Lipid analysts were initially slow to come to terms with the potential of high-performance liquid chromatography (HPLC), largely because of the non-availability of a sensitive universal detector. In contrast the flame-ionisation detector, commonly used in gas chromatography (GC), is highly sensitive and exhibits a linear response over a wide range of sample sizes. Transport-flame ionisation detectors for HPLC have always looked promising, but have never been a commercial success. We may wait for ever if we want an HPLC detector with the simplicity, ease of operation and linearity of GC, and it may cause us to overlook an extremely useful, proven and versatile HPLC detector - the evaporative light-scattering or "mass" detector (ELSD). Principles With this instrument, the solvent emerging from the end of the column is evaporated in a stream of air in a heated chamber (see Figure 1); the solute does not evaporate, but is nebulized and passes in the form of minute droplets through a light beam, which is reflected and refracted. The amount of scattered light is measured and bears a relationship to the concentration of material that is eluting. The commercial detector based on this principle with which the author was first familiar was available at a cost comparable to that of other optical detectors from Applied Chromatography Systems (A.C.S.) Ltd, later taken over by Polymer Labs. Now, several different manufacturers produce excellent instruments, including Alltech and Sedere. There are no special wavelength requirements for the light source, and in some commercial instruments, it is simply a projector lamp. Such a detector can be considered to be universal in its applicability, in that it will respond to any solute that does not evaporate before passing through the light beam. The instrument gives excellent results under gradient elution conditions, and it is simple and rugged in use. The sensitivity is comparable to that of a refractive index detector, but the evaporative light-scattering detector is not affected by changes in the mobile phase or small variations in the room temperature or in the flow-rate of the mobile phase, as is the former. Figure 1. Schematic diagram of the ACS evaporative-light-scattering detector The latest generation of such instruments have much greater sensitivity and improved linearity. Some of these detectors have distinctive design features, and for example in the Cunow and Sedere detectors, the larger droplets in the spray from the nebulizer are condensed out before they reach the heater chamber. A consequence is reported to be a more uniform particle size and improved linearity. The Alltech detector has a laser light source and a photodiode detector instead of a photomultiplier tube. Thus, there is now a good choice of commercial instruments. Once the instrument has warmed up and is running, there is little base-line drift during continuous operation even with abrupt changes in solvent composition. Most organic solvents, including acetone and chloroform, for example, can be used, and these can contain up to 20% water and small amounts of ionic species even. The minimum detection limit is dependent to a certain extent on the nature of the mobile phase, the nature of the sample, and the specific instrument, but it is certainly less than one microgram. As with all detectors, there are disadvantages. A source of dry, filtered compressed air, that is capable of delivering 5 litres/min, is required and in practice, this means that an air compressor must be used; a standard cylinder of air or nitrogen is emptied in about 4 to 8 hours. The stream of air containing the evaporated solvent must be conducted to the outside of the laboratory or into a fume cupboard. Although the detector is destructive in that the sample is lost, it is possible to insert a stream splitter between the end of the column and the detector to divert much of the sample to a collection device. To my knowledge only one company offers a stream-splitter commercially, and then at a very high price. My own is home-made and consists merely of a low-dead-volume T-piece with narrow-bore (0.25 mm) HPLC tubing to the detector and wider-bore (1 mm) tubing to the collection vessel. I can add a tap to the outlet end if required. This is simple and apparently effective, but it would be helpful if instrument manufacturers would offer a more professional splitter as a standard item with their equipment. I used to receive more correspondence on this than on any other single topic in relation to the detector. When used in this way, the evaporative light-scattering detector is a splendid research tool, since samples can easily be collected for analysis by other means, e.g. for determination of fatty acid compositions by GC. A further advantage in research applications is that it is easy to make rapid adjustments, as is often required during method development, since changing the solvent has virtually no effect on the base line. Detector Response Many analysts appear to be deterred from using ELSDs because of the well-established fact that the response is not rectilinear for purely analytical applications. I find this attitude to be short sighted. The first requisite for any analysis is that the required compounds are adequately resolved. With an ELSD, we can use the whole range of solvent groups described by Snyder and colleagues [1] to optimise the selectivity of the mobile phase, and complex gradients can be used, if need be, to achieve the desired separation. With most other types of HPLC detector, we are restricted to a limited number of solvents and isocratic elution. Secondly, it is essential that we can monitor the separation. With UV detection at low wavelengths, for example, it is not at all easy to detect saturated lipids; there is no such problem with an ELSD. Having obtained and proved that we have a suitable separation, only then do we need to consider quantification. It is known that the detector response follows the equation: - where A is the peak area, and a and b are numerical constants, i.e. a plot of peak area against the log of sample concentration is linear and has the slope b. The value of b lies between 1 and 2, and appears to depend on the design of the specific detector, especially the nebulizer system. When the value of b is close to 1, there can be a useful linear range of 1 to 2 decades. Righezza and Guiochon [2] showed that the size distribution of solute droplets formed in the aerosol is variable and depends on the nature of the solvent (probably on the rate of vaporisation). This obviously has an effect on response. In addition, they found that the amount of scattered light depended strongly on the molar absorbtivity of the solute. Calibration In practice, this means that it is essential to calibrate the detector carefully for each analytical system. Once the conditions have been optimised for a particular separation, these must be kept constant while the calibration is carried out with lipid standards as similar as possible in nature to the samples for analysis. This is of course particularly important when absolute amounts of particular components must be determined. On the other hand, when relative proportions only of different lipids are required, as in molecular species analysis for example, small changes in the chromatographic conditions or in the nature of the fatty acid constituents of the lipids have little effect on calibration. I would not recommend an ELSD for quantitative analysis of simple fatty acid derivatives, such as methyl esters, because of their relatively high volatility, though this may be less of a problem with some commercial instruments than others. Lipid analysts have perhaps been spoiled by the ease of using the flame ionization detector in gas chromatography, and expect a perfect linear response in all circumstances. Most workers in other fields realise that careful calibration is an essential part of any analytical procedure, and that non-linear calibrations are not unusual. What the lipid analyst must consider is which detector is any better than an ELSD for his purpose. All alternatives are dependent on the nature of the lipid, some greatly so, and few can be used with gradients. Applications The first important applications to the analysis of lipids were published in the early 1980s, so this is now a mature technology. Two review articles on the use of the detector have appeared [3,4] and a comprehensive bibliography of applications is available on this website. To consider a few examples, one of the more important tasks for HPLC and lipids is the separation and quantification of the different lipid classes in tissues. Ideally, this should be accomplished on a small scale, e.g. 0.2 to 0.4 mg, and in as short a time as can conveniently be managed. I made use of the A.C.S. evaporative light-scattering detector with a ternary solvent delivery system and a short (5 x 100 mm) column, packed with SpherisorbTM silica gel (3 micron particles) for the purpose [5]. In selecting a mobile phase, the choice of solvents was constrained at first by the need for sufficient volatility for evaporation in the detector under conditions that do 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 mobile phase of intermediate polarity was then needed to effect the transfer from one extreme to the other, and mixtures based on isopropanol gave satisfactory results. At the end of the analysis, 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) assisted the separation greatly. In later work [6], 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 was used in place of 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 liver is shown in Figure 2. 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, such as phosphatidic acid and to a lesser extent phosphatidyl-serine, 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. While this work has been superseded by that of others in recent years, the principle has not changed. Figure 2. Separation of rat lipids by HPLC with evaporative light-scattering detection (CE = cholesterol esters, TG = triacylglycerols, C = cholesterol, PG, PE, PI, PC and SPH are various phospholipids). Another important application of HPLC in lipid analysis is to the separation of molecular species of lipids, especially triacylglycerols. For example, silver ion HPLC separates solely on the basis of the degree of unsaturation of the molecules has been used with the ELSD, and there is further information here. Much more use has been made of HPLC in the reversed phase mode in which separation depends on the combined chain-lengths and the number of double bonds in the fatty acid constituents (reviewed in some detail elsewhere [7] and briefly here). Very many different stationary phases of the octadecylsilyl type have been utilised, with acetonitrile and a modifier solvent such as acetone, dichloromethane or tetrahydrofuran as the mobile phase. With the evaporative light-scattering detector, the choice of the mobile phase has little effect on the response and gradients can be used, a feature that is especially important with such complex natural fats as fish oils or milk fat. Indeed with the latter, there may even be virtues in hydrogenating the sample prior to analysis so that resolution is based solely on chain-length and is not complicated by double bond effects. With such separations, there has also been some debate about the efficacy of the evaporative light-scattering detector in quantification. However, Herslof and Kindmark [8] obtained good reproducibility for the relative proportions of different molecular species in analyses of the triacylglycerols of soybean oil. When the technique is used in research with triacylglycerols differing widely in composition, the best approach to quantification consists in collecting fractions and adding an odd-chain fatty acid as an internal standard prior to transesterification and GC analysis, i.e. the technique long used with thin-layer chromatography. The fatty acid composition and the amount of each fraction are thereby obtained simultaneously. In summary then, if you expect the evaporative light-scattering detector in HPLC to be the equivalent of the flame-ionisation detector in GC, you will be disappointed. If you look for its virtues in terms of ease of use, universality and its capacity to handle any combination of solvents and gradients, you will find much to commend. For example, it can be used with all classes of lipid separations and it is highly flexible in that it can be rapidly changed from one mode of analysis to another (adsorption, reversed-phase, silver ion, etc.). It is my opinion that, when combined with a stream splitter, it is the most useful HPLC detector in its price range currently available to lipid analysts. The charged aerosol detector, which is new to the market, has the potential to change this view, but we must await further work [9]. Similarly, mass spectrometry is becoming more affordable as a detection/identification system, but it is still expensive and requires a high degree of technical skill to operate the instrument, not to mention interpretation of the data. References Rutan, S.C., Carr, P.W., Cheong, W.J., Park, J.H. and Snyder, L.R. Re-evaluation of the solvent triangle and comparison to solvatochromic scales of solvent strength and selectivity. J. Chromatogr. A, 463, 21-37 (1989).Righezza, M. and Guiochon, G. Effects of the nature of the solvent and solutes on the response of a light-scattering detector. J. Liqu. Chromatogr., 11, 1967-2004 (1988).Christie, W.W. Detectors for high-performance liquid chromatography of lipids with special reference to evaporative light-scattering detection. In: Advances in Lipid Methodology - One, pp. 239-271 (edited by W.W. Christie, Oily Press, Ayr) (1992).Moreau, R.A. and Christie, W.W. The impact of evaporative light-scattering detectors on lipid research. INFORM, 10, 471-478 (1999).Christie, W.W. Rapid separation and quantification of lipid classes by high performance liquid chromatography and mass (light-scattering) detection. J. Lipid Res., 26, 507-512 (1985).Christie, W.W. Separation of lipid classes by high-performance liquid chromatography with the 'mass detector'. J. Chromatogr. A, 361, 396-399 (1986).Nikolova-Damyanova, B. Reversed-phase high-performance liquid chromatography: general principles and application to the analysis of fatty acids and triacylglycerols. In: Advances in Lipid Methodology - Four, pp. 193-251 (edited by W.W. Christie, Oily Press, Dundee) (1997).Herslof, B. and Kindmark, G. HPLC of triacylglycerols with gradient elution and mass detection.Lipids, 20, 783-790 (1985). Moreau, R.A. The analysis of lipids via HPLC with a charged aerosol detector. Lipids, 41, 727-734 (2006). This article is based on two previous publications (Lipid Technology, 1, 23-25 (1989) and Lipid Technology, 5, 68-70 (1993)) (by kind permission of P.J. Barnes & Associates (The Oily Press Ltd)). When amalgamating the two, they were substantially updated. HPLC separation of lipid classes With the previously described techniques, the quantification of the separated lipid classes represent a serious drawback since each fraction may need a separate treatment. Considerable progress has been made to, simultaneously, separate by HPLC and quantify with an efficient and near universal detector, the evaporative light-scattering detector (LSD). The separation of the various simple and complex lipids present in natural extracts is easily managed through an isocratic or better with a gradient elution procedure. The non-specific LSD enables the quantification of non-polar and polar lipids in the same run. It is recommended to first separate the crude lipid extract in two or three fractions by low pressure chromatography and analyze the fractions by HPLC . Thus, lower complex gradients and analysis time will be required. Among several published procedures devoted to the separation of all lipid classes in one run, we have selected a simple and efficient procedure initially applied to the separation of lipid classes from plant lipids (Christie WW et al, J High Resol Chromatogr 1995, 18, 97). Apparatus: column: YMC PVA-Sil (250 x 4.6 mm, 3 µm from Hichrom), the phase is prepared by bonding a layer of polymerized vinyl alcohol to silica. Ternary HPLC pump Evaporative light-scattering detector Reagents: solvent A: isooctane/methyl tert-butyl ether (98/2, v/v) solvent B: isopropanol/acetonitrile/chloroform/acetic acid (84/8/8/0.025, v/v) solvent C: isopropanol/water/triethylamine (50/50/0.2, v/v) The author has replaced isooctane by isohexane for safety reasons but with similar results. Procedure: A ternary gradient was generated during 40 min with a flow rate of 1 ml/min followed by a 10 min reequilibration time. The optimum gradient is described below. Time (min)ABCFlow rate (ml/min)0100001580200115445241403452141.440.1307001.44510000250100002 SE: sterol esters, S: sterols, DAG: diacylglycerols, MGDG: monogalactosyldiglycerides, SG: sterol glycosides, CERE: cerebrosides, DGDG: digalactosyldiglycerides, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PC: phosphatidylcholine The quantification of lipid compounds is made using appropriate standards, the relationships between sample size and response being dependent of the instrument used and the concentration ranges. An HPLC method with LSD detection was optimized and validated for the simultaneous quantitation of cholesteryl esters, triglycerides, cholesterol and phosphatidylcholine in human plasma. A silica Spherisorb column was used with a multistep gradient system. The calibrations were made at levels of 0.14-14 mg lipid/injection (Seppanen-Laakso T et al., J chromatogr B 2001, 754, 437). A reliable method was established to evaluate the lipid composition of plants. The procedure focused on the polar lipid distribution of rapeseed oil but was also applied to the estimation of waxes, triacylglycerols, and sterols (Beermann C et al., JAOCS 2003, 80, 747). The eluent system was modified from the method described above and the water eluent was supplemented with 1 mM ammonium sulfate to improve reproducibility. The gradient system was adapted to be suitable for the separation of major lipid classes of plant materials. A precise quantification was made on about 30 mg of total lipids using an evaporative light scattering detector.