Chapter 7 - Carboxylic Acids

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INTRODUCTION

Under the term “carboxylic acids” we mostly mean the higher members of this vast family, which are the principal components of lipids. On the other hand, there is also often a need to analyse the parent acids. Gas chromatography, liquid column chromatography and, more recently, the electrokinetic approach, can be applied to these frequently very complex mixtures. For chromatographic separation, no matter whether gas or liquid chromatography is used, carboxylic (fatty) acids must be derivatized,

GAS CHROMATOGRAPHY

The vast majority of fatty acid separations are done by gas chromatographic methods. Most of the fatty acids in lipids are bound either through ester or amide bonds. The proportion of non-esterified (free) fatty acids in tissues is rather small. Depending on the information required, these acids can be assayed together with the lipid-bound acids or separately after either extraction or preliminary fractionation of the lipids into their classes. The lipid-bound fatty acids are quite often

DERIVATIZATION FOR GC

As mentioned, chromatographic methods require derivatization. For gas chromatography, the transesterification of lipid-bound fatty acids, and esterification of free fatty acids to fatty-acid methyl-esters is the most commonly applied procedure. A variety of techniques is in use: acid- and base-catalyzed reactions, on-column pyrolytic reactions, and derivatization with other reagents: for reviews see [7, 8, 9, 10, 11, 12, 13].

TYPES OF GAS CHROMATOGRAPHY USED

The sheer complexity of naturally occurring samples of fatty acids, whether free or lipid-bound, makes it clear that a large separation window should be available with any system used for separation. The need of separating positional and conformational isomers, and the overlapping of different critical pairs — depending on the nature of the stationary phase used — made packed columns inadequate for general applications, although they may be used for particular types of analysis or in routine

DETECTION AND IDENTIFICATION IN GC

In general (as, for example, in drug analysis) two approaches are in use, namely retention parameters and GC—MS hyphenation. Identification of the most abundant fatty acids should be easy by comparison with the corresponding fatty acid methyl ester standards. The commercially available products cover the C4–C24 range and contain both the standard and unsaturated species. Additional comparison with natural samples, such as cod- liver- and canola-oil can be exploited because these naturally

LIQUID CHROMATOGRAPHY

Although it is often second in importance, HPLC retains a strong position for free-fatty-acid separations (and for carboxyl-possessing compounds). Although the GC separations described above mostly work with a single type of derivative (methyl esters), for liquid separations they are occasionally used [118, 119] but are usually replaced by a number of other derivatization reactions. However, the main goal in fatty-acid derivatization is to make these entities more easily detectable with the

DETECTION, AND DETECTION LIMITS IN LC

As indicated already, the main purpose of fatty-acid derivatization is to introduce into the molecule a functionality with a high molar absorptivity at longer UV wavelengths where double bonds do not absorb, and where the signa strength is proportional to the mass of the analyte. For phenacyl esters the optimum wavelength detection is at 242 nm. However, not all detection systems are capable of working at this wavelength — frequently the generally provided wavelength of 254 nm is used: however,

EMERGING SEPARATION TECHNIQUES AND THEIR APPLICATION

In addition to the established techniques of fatty-acid separations there are two other approaches that merit some attention. The first, supercritical fluid chromatography, is known to combine some of the advantages of both gas- and liquid chromatography. With this technique, either capillary (open tubular) columns or packed columns can be used in most cases with carbon dioxide as the supercritical fluid (for a recent review see [222]. Fatty-acid methyl esters and free fatty acids have been

CONCLUSIONS

While GC has become a routine method in fatty-acid separations and HPLC has offered results which are comparable but still at a slightly lower level, the future seems to be in techniques that have penetrated this area only recently, i.e., in supercritical fluid chromatography and capillary electromigration procedures.

Although methyl esters are used almost exclusively in separating fatty acids, for liquid chromatography a plethora of suitable derivatives has emerged over the years. At the

ABBREVIATIONS

    ADAM

    9-anthryldiazomethane

    BAN

    α-bromo-2′ -acetonaphthone

    BPB

    p-bromophenacyl bromide

    BrAMC

    3-bromoacetyl-7-methoxycoumarin

    BrAMDC

    3-bromoacetyl-6,7-methylenedioxycoumarin

    BrDMEQ

    3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone

    BrMAC

    4-bromomethyl-7-acetoxycoumarin

    BrMMC

    4-bromomethyl-7-methoxycoumarin

    BrMMEQ

    3-bromomethyl-6,7-methyIenedioxy-1-methyl-2(1H)-quinoxalinone

    DCC

    dicyclohexylcarbodiimide

    DEPC

    diethyl phosphorocyanidate

    DIPEA

    N,N-diisopropylethylamine

    DMEQ-Hz

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