Fatty acid phenacyl derivatives

Table 1 Elution Conditions Used to Separate Fatty Acid Phenacyl Derivatives...

With samples containing a wide range of components, such as hydrogenated fats, the column was eluted with solvent A for 13 min, then changed in one step to A-B (75 25), with a gradient to 100% B over 20 min. The free fatty acids were converted into the phenacyl derivatives and, prior to HPLC analysis, were purified by elution from a BOND ELUT NH2 column with hexane-diethyl ether (9 1). 

A commercially available column containing silver ions has recently been developed by Chrompack (ChromSpher Lipids HPLC column). A solvent system (43) composed of dichloro-methane, dichloroethane, and small amounts (0.01-0.025%) of acetonitrile (ACN) is used with both the Nucleosil and Chrompack HPLC columns. Because chlorinated solvents are opaque at the wavelengths (200-210 nm) used for FAME analyses, the use of UV detectors is precluded unless the phenacyl derivatives of the fatty acids are first prepared. 

The phenacyl esters are more readily prepared than the other derivatives. The fluorescent tag 4-bromomethyl-7-methoxycoumarin may however offer increased sensitivity. Due to the bulky nature of the fluorescent products, separation factors may be poorer and allow only positive identification of a limited number of fatty acids. 

In the quantitative analysis of fatty acids by HPLC a plethora of reagents have been used to increase the sensitivity of detection. The most popular derivatives formed include benzyl derivatives (Polizer et al., 1973) 2-naphthacyl derivatives (Cooper and Anders, 1974) o-and p-nitrobenzyl derivatives (Knapp and Krueger, 1975) phenacyl derivatives (Borch, 1975) p-bromophenacyl derivatives (Durst et al, 1975) methoxyphenacyl derivatives (Miller et al., 1978), and 1-naph-thylamide derivatives (Ikeda et al., 1983). The benzyl, nitrobenzyl, phenacyl, p-bromophenacyl, methoxyphenacyl, 1-naphthylamide and... 

Fig. 3. Silver-ion high-performance liquid chromatogram of phenacyl esters of cyclic fatty acids derived from heated linseed oil. A column of Nucleosil 5SA (250 x 4.6 mm i.d. 5 pm particle size) was used in the silver ion form. The mobile phase was composed of dichloromethane-dichloroethane (50 50 vol/vol Solvent A) and dichloromethane-dichloroethane-acetonitrile (49 49 2, by vol Solvent B) the flow rate was 1 mL/min. There was a linear gradient from 100% A to 75% A/25% B over 50 min, then to 100% B over a further 5 min. An evaporative light-scattering detector was employed. Source Ref. 10.

Fatty acids can be derivatized with p-bromophenacyl (73, 85, 86], phenacyl [87-89], naphthacyl [90, 91] or p-nitrophenacyl [74] bromide. To prepare p-bromophenacyl esters [73], fatty acids (0.001—0.5 mM) were dissolved in methanol or water and neutralized to a phenolphthalein end-point by methanolic KOH. The solvent was removed by either a rotary evaporator or lyophilization. A 3—10-fold molar excess of alkylating agent, p-bromophenacyl bromide/18-crown-6 (20 1) in acetonitrile, was then added. The mixture was stirred continuously in a sealed Reacti-Vial at 80 °C for 15 min. After cooling, the solution containing the derivatives was directly subjected to RP-HPLC with UV detection at 260 nm. The mobile phase was usually acetonitrile/ water [87, 92, 93], methanol/water [73, 88, 94, 95] or acetonitrile/methanol/water (86, 89], The separation of phenacyl derivatives of palmitoleic (Ci, ) and arachi-donic ( 20 4) acids was not achieved with the acetonitrile/ water system [87, 89], and elution with methanol/water could not resolve linolenic (Cig.3) and myristic (C]4.q) acids [88, 89]. A ternary mobile phase [89] containing a mixture of acetonitrile, methanol and water seemed to be best for the separation of phenacyl derivatives of fatty acids. 

A wide choice of stationary phases is available for separation, such as reversed-phase or silver-ion impregnated phases. The separation of phenacyl derivatives of saturated, monoenoic, polyenoic, and monohydroxy fatty acids, and of the geometric isomers of fatty acids is possible. 

Sep-Pak C18 coated with 300 pg of sodium hydroxide is used for sampling C2-C4 fatty acids. A 1-1001 volume of air is sampled at 0.5-1.5 Imin through the cartridge. The adsorbed substances are eluted with 2 ml of acetonitrile and derivatized by mixing with 0.4 mg of 18 p-bromophenacylbromide and 0.04 mg of 18-crown-6-ether. The phenacyl derivatives produced under catalytic effect of the crown ether are assessed for the fatty acids using LC with UV absorbance detection. 

Figure 5.7 Reversed-phase high-pressure liquid chromatogram of phenacyl ester derivatives of the fatty acids from cottonseed cotyledons and embryos. CIS (50 nun x 4.5 nun, 3 pm particle size) and C8 (150 nun x 4.5 nun, 5 pm) were used in tandem. The mobile phase was composed of acetonitrile and water, and the flow rate was 1.5 ml min . An initial mixture of acetonitrile/ water (80 20 vol./vol.) was maintained for 25 min, followed by a gradient to acetonitrile/water (85 15) for a further 10 min and then to 100% acetonitrile for a final 10 min. A variable-wavelength detector was employed. Redrawn from Wood, R., Comparison of the cyclopropene fatty acid content of cottonseed varieties, glanded and glandless seeds, and various seed structures, Biochem. Arch., 2, 73-80, 1986.

Although most Ag-HPLC analyses have traditionally been done using CLA methyl esters, improved separation and quantitation of CLA isomers has been noted when phenacyl or p-methoxyphenacyl ester rather than methyl ester derivatives were analyzed (58). Nikolova-Damyanova et al. (59) demonstrated improved separation of the p-methoxyphenacyl derivatives of the cis/trans 8,10- through 11,13-18 2 isomers in a commercial sample of CLA when compared with CLAME. Only a single Ag-HPLC column was required. Resolution of the cis/ trans isomers was similar to that obtained for CLAME using Ag-HPLC systems with two or three Ag-HPLC columns connected in series (60). A stepwise solvent gradient of 100% hexane/dichloromethane/ACN (40 60 0.2 vol/vol/vol 30 min) to 100% dichloromethane/ACN (100 1) over 10 min with UV detection at 270 nm (for phenacyl esters) yielded a semiquantitative estimation of fatty acid composition comparable to, but more detailed than that obtainable by GC analysis as FAME. This method was also applied to detamine the CLA isomer composition of a sample of beef/pig fat, but prefractionation by RP-HPLC was required before Ag-HPLC analysis. The prefractionation step was required to concentrate the CLA isomers and to remove octadecenoate and other fatty acids that might interfere with Ag-HPLC analysis. 

Nikolova-Damyanova, B., Christie, W. W., and Herslof, B. (1994). Improved separation of some positional isomers of monounsaturated fatty acids, as their phenacyl derivatives, by silver-ion thin layer chromatography. J. Planar Chromatogr.—Mod. TLC 7 382-385. 

C10-C24 fatty acids as phenacyl derivatives separation from each other... 

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