Carboxylic Acids

Both free and bound carboxyl groups are almost exclusively derivatized to volatile esters - predominantly silyl and methyl - by a variety of methods. These employ a number of silylation reagents, acid-and base-catalysed reactions, on-column pyrolysis,

Silyl esters Silylation is now one of the most extensively used techniques for esterifying free acids primarily because of its speed, convenience and the simultaneous derivatization of other polar functional groups containing an active hydrogen (-OH, -SH, -NH2). The trimethylsilyl (TMS) group is the most commonly introduced substituent by the many silylating agents available, of which N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) is the most widely used. It reacts with all the common polar functionalities and yields volatile by-products that are usually eluted with the solvent. Even more volatile by-products are produced by substituted reagents, e.g. N-methyl-N-trimethylsilyltrifluoroacetam-ide (MSTFA), which are also more reactive toward the polar functional groups. Although all silylating reagents and their products are sensitive to moisture, considerably greater hydrolytic stability is exhibited by i-butyldimethylsilyl (TBDMS) derivatives that are best prepared with N-i-butyldimethylsilyl-N-methyl-trifluoroacetamide (MTBSTFA), which can also serve as its own solvent. It yields excellent results with both volatile and nonvolatile carboxylic acids (Figure 1). A limitation of silylation is that bound acids such as lipids (triacylglycerols) are not converted and their derivatization to methyl (or other alkyl) esters is necessary.

Alkyl esters Methyl esters are most frequently prepared by acid-catalysed reactions with methanol. The principal advantage of this method is the concurrent esterification of free acids and the transesterification of bound ones. The most extensively used catalysts are BF3, HCl and H2SO4, usually as 14%, 5% and 2% solutions, respectively. The reaction is fastest with BF3, requiring the mixture to be boiled for 2 min for free acids and 30-60 min for lipids. With HCl and H2SO4 about twice the time is required. The higher concentration of BF3 used compared to the other catalysts may be responsible not only for the faster reaction, but also for partial degradation of un-saturated acids and reported artefact formation. These problems can be reduced by prior saponification with methanolic KOH, followed by re-esterification of the free acids formed under mild conditions. Several official methods are based on this procedure.

Figure 1 Chromatogram of a mixture of carboxylic acids as the t-butyldimethylsilyl derivatives. GC conditions: 30 m x 0.32 mm i.d., DB-1 fused-silica capillary column initially at 60°C for 2 min, then programmed to 280°C at 4°C min-1; 0.8 L sample, injected with split ratio of 15 : 1; both injector and detector temperatures at 300°C; nitrogen as the carrier gas at 0.9 mL min-1. Peaks: 1, Formic; 2, acetic; 3, propionic; 4, isobutyric; 5, butyric; 6, isovaleric; 7, valeric; 8, caproic; 9, enanthic; 10, benzoic; 11, caprylic; 12, lactic; 13, phenylacetic; 14, glycol; 15, oxalic; 16, pelargonic; 17, malonic; 18, capric; 19, succinic; 20, methylsuccinic; 21, undecanoic; 22, fumaric; 23, 5-phenylvaleric; 24, p-aminobenzoic; 25, lauric; 26, mandelic; 27, adipic; 28, 3-methyladipic; 29, tridecanoic; 30, phenyllactic; 31, hippuric; 32, myristic; 33, p-hydroxybenzoic; 34, malic; 35, suberic; 36, pentadecanoic; 37, vanillic; 38, palmitic; 39, syringic; 40, tartaric; 41, margaric; 42, a-resorcylic; 43, p-hydroxymandelic; 44, y-resorcylic; 45, stearic; 46, homogentisic; 47, protocatechuic, 48, nonadecanoic; 49, citric; 50, arachidic acid. (Reproduced with permission from Kim KR, Hahn MK, Zlatkis A etal. (1989) Simultaneous gas chromatography of volatile and nonvolatile carboxylic acids as tert-butyldimethylsilyl derivatives. Journal of Chromatography 468: 289.

Figure 1 Chromatogram of a mixture of carboxylic acids as the t-butyldimethylsilyl derivatives. GC conditions: 30 m x 0.32 mm i.d., DB-1 fused-silica capillary column initially at 60°C for 2 min, then programmed to 280°C at 4°C min-1; 0.8 L sample, injected with split ratio of 15 : 1; both injector and detector temperatures at 300°C; nitrogen as the carrier gas at 0.9 mL min-1. Peaks: 1, Formic; 2, acetic; 3, propionic; 4, isobutyric; 5, butyric; 6, isovaleric; 7, valeric; 8, caproic; 9, enanthic; 10, benzoic; 11, caprylic; 12, lactic; 13, phenylacetic; 14, glycol; 15, oxalic; 16, pelargonic; 17, malonic; 18, capric; 19, succinic; 20, methylsuccinic; 21, undecanoic; 22, fumaric; 23, 5-phenylvaleric; 24, p-aminobenzoic; 25, lauric; 26, mandelic; 27, adipic; 28, 3-methyladipic; 29, tridecanoic; 30, phenyllactic; 31, hippuric; 32, myristic; 33, p-hydroxybenzoic; 34, malic; 35, suberic; 36, pentadecanoic; 37, vanillic; 38, palmitic; 39, syringic; 40, tartaric; 41, margaric; 42, a-resorcylic; 43, p-hydroxymandelic; 44, y-resorcylic; 45, stearic; 46, homogentisic; 47, protocatechuic, 48, nonadecanoic; 49, citric; 50, arachidic acid. (Reproduced with permission from Kim KR, Hahn MK, Zlatkis A etal. (1989) Simultaneous gas chromatography of volatile and nonvolatile carboxylic acids as tert-butyldimethylsilyl derivatives. Journal of Chromatography 468: 289.

Substituting microwave irradiation for conventional heating may substantially reduce reaction times and lipid degradation. Thus, using the BF3-methanol reagent, a reaction time of 30 s sufficed for the transesterification of most lipids to their fatty acid methyl esters (FAMEs) with less oxidation of the unsaturated species.

Base-catalysed reactions are used extensively for the transesterification of lipids because they proceed faster than those in acid media without degradation of the unsaturated fatty acids. However, they do not esterify free fatty acids. The most commonly used reagents are methanolic solutions of NaOCH3 or KOH. Transmethylation of lipids is usually complete in 5 min at room temperature.

Strong organic bases can be used similarly and possess the great advantage of forming salts which, unlike their inorganic analogues, can be pyrolysed to methyl esters at the high temperatures of a GC injection port. This permits simple one-step determination of both free and bound acids. The organic bases that have been recommended for such pyrolytic conversions include (m-trifiuoromethyl-phenyl)-trimethylammonium, trimethylphenylam-monium and trimethylsulfonium hydroxides. The latter reagent requires the lowest pyrolysis temperature and yields innocuous by-products. It is simply added to the sample solution, mixed and injected.

Esterification of free acids with diazomethane proceeds rapidly in high yield under mild conditions, with minimal side reactions. Special microequipment, reagents and procedures have been developed that allow its relatively safe handling despite its toxic and explosive nature. Other reagents of interest include alkyl chloroformates that can esterify free acids even in the presence of a considerable amount of water (40%). Another reagent, dimethylformamide dimethylacetal, can be simply mixed with the sample of acid and injected into the GC; the reaction occurs in the hot injection port. Silver or potassium salts of acids can be converted to esters with methyl iodide or sulfate. Many other reactions have been reported.

Short chain acids are frequently derivatized to higher esters with butanol or isopropanol and acid catalysts in order to mitigate losses due to volatility and substantial water solubility. Higher diazoalkanes may also be used if the methyl esters are too volatile.

Enantiomers of optically active carboxylic acids have been separated following acid-catalysed esterifi-cation with a chiral alcohol such as S( + )-2-butanol, R( — )-2-octanol, or (— )-methanol or transesterifica-tion with sodium menthylate. Diastereometric esters have also been prepared from optically active acids by reaction with O-( — )-menthyl-N,N-diisopropylisourea.

The above silyl and alkyl esters are most commonly detected by a flame ionization detector (FID). Greater sensitivity, however, can be achieved by forming halogenated silyl esters, e.g. chloromethyldimethyl-silyl, and monitoring with an electron-capture detector (ECD). Similarly, very small amounts of volatile acids may be detected via their penta-fluorobenzyl (PFB) esters with an ECD. Special derivatives for this detector include the 2-chloroethyl and trichloroethyl esters.

Other derivatives The silyl and alkyl esters described are generally also suitable for detection by MS. However, special derivatives are necessary for unsaturated fatty acids to prevent double-bond migration during fragmentation. The most widely used derivatives are those of 3-hydroxymethylpyridine (picolinyl) and 4,4-dimethyloxazoline (DMOX). Picolinyl esters must be prepared from the acid but DMOX derivatives can be prepared even from their esters.

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