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 sensitivity.
Adsorption Chromatography
HPLC on columns of silica gel can be used for the analysis or isolation of fatty acids with polar functional groups, especially oxygenated moieties such as hydroperoxides. With care, isomers differing in the position of hydroperoxy or hydroxy groups on an aliphatic chain can be separated [3].
Chiral Chromatography
Fatty acids containing enantiomeric functional groups, and hydroperoxy or hydroxy groups especially, can be resolved on a variety of commercial chiral stationary phases [4]. This is probably still the realm of the specialist, but it is extremely important in say pharmaceutical chemistry. Chiral separations are also possible by GC, but they appear to be less efficient in general.
Silver-Ion Chromatography
This is a topic dealt with elsewhere in this section of the website, and as a major topic in its own right, so I will mention only a few brief applications. In my laboratory, we used the technique frequently to simplify complex mixtures of fatty acids in order to make it easier to confirm the identities of fatty acids by GC-MS. It would be my method of choice for quantitative separation of cis and trans fatty acids for further detailed analysis and quantification by GC. Finally, it is possible to separate positional and geometrical isomers of some unsaturated fatty acids on analytical and micro-preparative scales in ways no other technique can match.
Reversed-Phase Chromatography
An enormous number of papers have appeared on the topic of HPLC analysis of fatty acids by reversed-phase HPLC, most of which have not advanced knowledge to any significant degree. We are indebted to Boryana Nikolova-Damyanova for reviewing the topic and bringing a critical eye and some sense to it [5].
The principles of the separation are well known, and the instrumentation is straightforward. The stationary phases used are almost exclusively of the octadecylsilyl ("ODS") type, with an octyl phase being recommended occasionally as an alternative. The mobile phase is either acetonitrile (mainly) or methanol containing some water. If free fatty acids are analysed, a little acetic acid can be added to ensure sharp peaks. These solvents are transparent to UV light at 205 to 210 nm, so UV detection at such wavelengths can be employed. However, much greater sensitivity is possible if phenacyl or related derivatives of fatty acids are prepared for detection at higher wavelengths. Then, the detector responds only to the ester moiety giving a quantitative molar response. Astonishing sensitivity is obtainable, down to femtomole levels, by using specific derivatives with fluorescence detection although quantification then presents problems. HPLC-MS with electrospray ionization can also be extremely sensitivity, while giving structural information on components [6].
In reversed-phase HPLC, fatty acids are separated both by chain length and by degree of unsaturation. The first double bond reduces the effective chain length by a little less than 2 carbon units, so an 18:1 fatty acid elutes just after 16:0. A second and further double bonds have smaller effects on retention so an 18:3 fatty acid elutes just before 14:0, for example. A separation of a standard mixture of fatty acids, as phenacyl esters, is illustrated in Figure 1 [7].
Fig. 1. Separation of a standard mixture of fatty acids in the form of the phenacyl esters by reversed-phase HPLC with spectrophotometric detection at 254 nm [7]. The column (900 x 6.4 mm) was packed with µBondapak™ C-18, and was eluted with acetonitrile-water in the proportions 76:33 (by volume) initially, changed to 74:26 at "a", to 4:1 at "b", and to 97:3 at "c", at a flow rate of 2 mL/min. (Reproduced by kind permission of the author and of Analytical Chemistry, and redrawn from the original paper).
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