Fatty acid methyl ester,stationary phases

Normal alkanes or fatty acid methyl esters are generally used as the standard homologous compounds. The column separation number is dependent on the nature of the stationary phase, the column length, column temperature, and carrier gas flow rate [42-44]. Referring to Figure 1.2, at a sufficiently high capacity factor value either n, N, or SN provides a reasonable value for comparing... 

Table 12.13 Polar stationary phases for GLC of fatty acid (methyl esters)...

Figure 12.19 GLC of fatty acid methyl esters. Schematic representation of isothermal separation on a PEGA stationary phase.

Haken has considered the applicability of "Rohrschneider/ McReynolds constants" for the classification of stationary phases for the separation of fatty esters (13). He concluded that the approach was limited since the measurements used to determine the aforementioned "constants" are made at 100°C and most fatty acid methyl ester separations are carried out at about 200°C. He had previously shown significant variation in the, what will now be called, Rohrschneider/McReynolds coefficients, with temperature (14). Polar polysiloxanes such as XF-1150 demonstrated greatest variability in the coefficients and nonpolar types such as SE-30 demonstrated least variation. Supina pointed out that the X factor in the McReynolds coefficients should be indicative of extent of interaction with olefinic substituents (15). Figure 9.5 demonstrates the utility of this approach the 18 3 and 20 0 methyl esters are used as markers for the consideration of... 

Fig. 18 RP-HPLC of methylesters derived from three different fish oil sources sardine (A), menhaden (B), and cod liver (C). Identified fatty acid methyl esters in order of their elution 1. C20 5o 3 2. C14 0 3. C16 lw9 4. C22 6o>3 5. C18 2a>6 6. C16 0 7. C18 1 >9. BHT is eluted right after the void volume remaining peaks have not been positively identified. Mobile phase acetonitrile/THF/water (9 5 11) at 2.0 ml/min stationary phase WHATMAN ODS-3 RAC II (100 X 4.6-mm ID) detection, refractive index at ambient temperature.

In the characterization of volatiles, the most commonly applied reference series is -alkanes. However, the latter commonly present fluctuant behavior on polar stationary phases. In consideration of the fact that retention index values are correlated to retention mechanisms, alternative standard series of intermediate polarity have been introduced, such as 2-alkanones, alkyl ethers, alkyl halides, alkyl acetates, and alkanoic acid methyl esters [22]. Shibamoto [36] suggested the use of polar compounds series, such as ethyl esters, as an alternative. The most feasible choice, when analyzing volatiles, is to apply reference series as n-alkanes, fatty acid ethyl esters (FAEEs), or fatty acid methyl esters (FAMEs), employed according to the stationary phase to be used. 

Figure 9. Isothermal analysis (200 °C) of fatty acid methyl esters on a packed column with stationary phase ethylene glycol succinate (EGS-X)...

As stationary phase for the resolution of fatty acid mixtures polar (polyesters of short chain dicarbonic acids and low molecular diols) and unpolar (hydrocarbons, silicones) substances can be used. Gas-chromatography under standardized conditions permits the tentative identification of separated fatty acids from the time elapsing between application of the sample and the emergence of the acid in question. Figure 6 shows the separation of serum fatty acid methyl esters using a polar stationary phase. 

As fatty alcohols are comparable in structure and molecular weight to fatty acid methyl esters, they are usually subjected to GC on the same stationary phases and under near-identical conditions. It is certainly possible to separate alcohols in the free form by GC, especially on modem WCOT columns of fused silica, but sharper peaks are obtained if less polar derivatives such as the acetates, trifluoroacetates or TMS ethers are prepared. Suitable preparation procedures are described in detail in Chapter 4. Jamieson and Reid [438] studied the relative retention times of many different saturated and unsaturated fatty alcohols in the free form and as the acetates on packed GC columns containing polar polyester phases, and concluded that very similar separation factors applied as with the equivalent fatty acid methyl esters. The order of elution was - methyl ester < alcohol acetate < free alcohol. A TMS ether derivative would be expected to have a lower retention time than an acetate, but the separation factors for double bonds in the alkyl chain in this instance were found to be lower than with the acetates and resolution in general was poorer some changes in retention sequence for specific isomers was noted, depending on the type of derivative [439]. In contrast, the free alcohol eluted before derivatized forms on non-polar phases [944]. It is therefore possible to use equivalent chain-length data for the provisional identification of fatty alcohols in the same way as with methyl ester derivatives of fatty acids (see Chapter 5). 

Figure 3.2 Separation of fatty acid methyl esters by gas-liquid chromatography on a 2mx4mm column. Stationary phase 10% polyethylene glycol adipate (stabilized with orthophosphoric acid) on Diatomite C-AAW. Carrier gas argon. Flow rate 50ml/1 min. The oven temperature was programmed from 65°C to 185°C at 8°C per min. The sample consists of fatty acids derived from a milk diet for calves. Heptanoic and heptadecanoic acids were added as internal standards in concentrations of 1.55 mg 7 0 and 4.94 mg 17 0 in 10 ml sample solution and the computer was programmed to print out the concentration in mg/10 ml of each fatty acid In the mixture. IS = internal standard BHT = butylated hydroxytoluene (antioxidant). Reproduced with kind permission of Mr J.D. Edwards-Webb.

Fatty acids are GC analysed after their conversion to non-polar derivatives such as methyl or silyl esters (Evershed, 1992a). Often, esters are prepared as derivatives of the fatty acids liberated from wax esters or sterol esters by hydrolysis (Summchen et al., 1995 Szafranek Synak, 2006). The details and other important considerations of fatty add analysis have been well summarized by Christie (1994). Generally speaking, fatty add methyl esters (FAME) can be GC analysed employing non-polar stationary phases like other cuticular wax components. Unsaturated compoimds should be analysed on polar stationary phases (e.g. Carbowax). The determination of FAME often requires high resolution for the separation of positional and configurational isomers. The elution order is as follows (Evershed, 1992a) ... 

It is usual to convert fatty acids to their methyl ester derivatives before separation by GLC, although it may be possible to analyse those with short chain lengths (two to eight carbon atoms) as the free fatty acids. Polar or non-polar stationary phases can be used and capillary (open-tubular) or SCOT columns will separate positional and geometric isomers. The cis isomers have shorter retention times than the corresponding trans isomers on a non-polar phase and visa versa on a polar phase. 

Capillary and packed (GC) columns are of value in the analysis of complex mixtures of lipids. The best capillary column length will depend on the complexity of the material injected, however, 30-m columns are often employed. In packed columns, many types of stationary phases are available for lipid separation, and these include silicone and alkylated or cyanogenated derivatives, polyesters, polyglycol, and carboranes. It is also common to derivatize the fatty-acid side chains to the corresponding methyl esters by reaction in BF3/ methanol prior to chromatographic analysis to achieve more distinct and uniform separations. 

Figure 22-4 illustrates excellent separation of 22 fatty acids for the measurement of trans fat for food labels (Figure 5-3). Peaks 1, 2, 3, 4, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21 are all trans fats. Fatty acids were converted (derivatized) to methyl esters to make them volatile enough for gas chromatography. The stationary phase is an ionic liquid that melts below room temperature and has a wide liquid...

FIGURE 3.13 Effect of concentration of stationary phase and column temperature on sample resolution (methyl esters of fatty acids). (Reproduced from Reference 20 W. A. Supina, in Modern Practice of Gas Chromatography, 2nd ed., R. L. Grob, ed., copyright 1985, John Wiley Sons, Inc. Reprinted by permission of John Wiley Sons, Inc.)... 

The acyl residues of an acylglycerol are released as methyl esters (cf. 3.3.1.3) and are analyzed as such by gas chromatography. However, free fatty acid analysis is also possible by using specially selected stationary solid phases. Capillary-column gas chromatography should be used to... 

Figure 5.1. GC analysis of a complex mixture of natural fatty acids (as the methyl ester derivatives) on packed columns with EGSS-X and EGSS-Y as stationary phases (see footnote to Table 5.1 for further chromatographic details).

Deuterated fatty acids can be separated from non-deuterated in part at least on WCOT columns coated with polar stationary phases [693,695], t-Butyldimethylsilyl ester derivatives (see Chapters 4 and 7) are useful for GC-mass spectrometric estimation of these compounds [692,709,1004], Long-chain dicarboxylic acids have been separated by GC and identified by GC-MS in the semm of patients suffering from Reye s syndrome [663] and in the lipids of royal Jelly [542,543], Brominated vegetable oils are added to soft drinks to disperse flavouring agents after acid-catalysed methanolysis, the methyl ester derivatives of fatty acids containing two, four and six bromine atoms have been separated on packed [538] or fused silica [151] columns with non-polar silicone stationary phases. 

Fatty acids are usually analyzed by gas-liquid chromatography (GLC). This method is applicable to oils containing fatty acids with chain length in the range C14 to C24. GLC analysis of fatty acids is performed following their conversion to methyl ester derivatives. Columns with polar phases are used, for example polyethylene glycol stationary phase (Carbowax) (Jennings 1987). 

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