Keto acid chromatography

A good example of the simplicity and power of the chemistry to rapidly construct complex systems is provided by the Kolbe dimerization of (55) as the key step of a total synthesis of the triterpene (+)-Q -onocerin (57 Scheme 14) [33], Thus, oxidation of (+)-hydroxy keto acid (55) in methanol containing a trace of sodium methoxide and at a temperature of 50 C, followed by acylation and chromatography, provided (+)-diacetoxydione (56) in a 40% yield. 

Quinoxalinones have been suggested for the chromatography of keto acids [184,185]. They are produced by reaction with aromatic o-diamines, as shown in Scheme 4.21 (p. 77). A solution of 60 mmol of sublimed o-phenylenediamine in 100 ml of 10% acetic acid was mixed with a solution of 30 mmol of the keto acid in 30 ml of water. The precipitated quinoxalinone was filtered after 15 min, washed with water, dried and crystallized from methanol. Prior to the GC analysis proper, it had to be converted into a silyl derivative 5—50 /ul of about a 1% solution of quinoxalinone in dry pyridine was mixed with 20 jul of a pyridine solution of the internal standard (6-methyl-2-naphthol, p-nitrophenyl phenyl ether). A 200-/lz1 volume of BSA and 50 of pyridine were added. As the silylation proceeded very quickly, the reaction mixture could be injected immediately. 

A good example of the simplicity of the operation is provided by Stork s total synthesis of the complex triterpene (+)-o -onocerin (13) [5]. In tliis instance, the (+)-hydroxy keto acid 11 was oxidized in a methanol solution containing a trace of sodium methoxide at 0.1 A and 50 V, at a temperature of 50" C. Following acetylation with acetic anhydride/pyridine and chromatography, the (+)-diacetoxydione 12 was isolated in a 40% yield. 

As important losses of a-keto acids occur during paper chromatography of organic acids (as free acids) of blood and urine, special techniques are used for these acids they include the preliminary transformation of the keto acids into more stable derivatives. 

Paper chromatography of a-keto acids has also been achieved after conversion to other derivatives, especially nitroquinoxalinols through 1,2-diamino-4-nitrobenzene (H25, S27, T6). 

Other workers separate by paper chromatography the amino acids obtained by catalytic hydrogenolysis of the 2,4-dinitrophenylhydrazones of the a-keto acids (H19, K19, T24). 

Column chromatography has rarely been used in the special field of a-keto acids in blood and urine (D3, LIO, S3), and the techniques described do not appear to be preferable to the paper chromatographic ones. 

Oxalacetic Acid. Chromatography of the 2,4-dinitrophenyl-hydrazones of the keto acids shows no oxalacetic acid in blood and urine (B18, D15, E2, Kl, Slla) or only traces of it (H19, VI) this fact can be explained by the low concentration and by lack of stability of this acid. 

C6. Cavallini, D., and Frontali, N., Quantitative determination of keto-acids by paper partition chromatography. Biochim. et Biophys. Acta 81, 439 (1954). 

C7. Cavallini, D., Frontali, N., and Toschi, G., Determination of keto-acids by partition chromatography on filter-paper. Nature 163, 568 (1949). 

K19. Kulonen, E., Experiences with paper chromatography in the study of a-keto acids. 11. Reduction of hydrazones to amino acids. Scand. J. CUn. 6- Lab. Invest. B, 72 (1953). 

Slla. Seligson, D., and Shapiro, B., Alpha-keto acids in blood and urine studied by paper chromatography. Anal. Chem. 24, 754 (1952). 

T26. Tumock, D, Paper chromatography of the keto acids. Nature 172, 355 (1953). 

The quinoxalinol derivatives of a-keto acids are stable and sufficiently volatile to allow their separation by gas chromatography [423-427]. Their fluorescence characteristics depend somewhat on the a-keto acid excitation maxima of the quinoxalinol derivatives of most keto acids (pynivic, a-ketobutyric, a-ketoisovaleric, a-ketoisocaproic and a-keto-[ -methylvaleric acid) in methanol are at 375-377 nm, and emission maxima at 407-407.5 nm. The derivative of a-ketoglutaric acid has an emission maximum at 412 nm. Only for the derivative of glyoxylic acid do the values differ considerably its excitation maximum is at 381 nm and its emission maximum is at 390 nm [419]. 

Fatty Acids. Ordinary fatty acids, epoxy-, hydroxy- and keto-acids can be separated into compound classes by adsorption chromatography. This apphes to the corresponding methyl esters also [78, 82, 134, 135, 205]. Even certain esters which differ only in the position of the functional group in the chain, can be separated [140]. An example of this, a series of hydroxystearate positional isomers, is shown in Fig. 138. 

Reversed phase partition TLC may be applied to fractionate alcohols [78, 86], aldehydes and ketones [10, 125], fatty acids [4, 80, 84, 124] and their methyl esters [114, 213], keto-acids, hydroxy-acids and lactones [82]. More complete separations of these compounds or of suitable derivatives, are, however, obtained with gas chromatography and it is appreciably more sensitive than TLC it is moreover more suitable for quantitative determinations. Reversed phase partition TLC offers substantial advantages only in the analysis of lipids of relatively high molecular weight, like wax esters [86], steryl esters [79] (see also Chapter L), carotenoid esters (see Chapter K) and triglycerides [4, 81, 89, 124] some derivatives of naturally occurring lipids are also best fractionated on hydrophobic layers [21, 130, 131, 165]. 

Hutterer, F., and Gaul, G. Determination of a-Keto Acids as Silylated Oximes in Urine and Serum by Combined Gas Chromatography-Mass Spectrometry Clin. Chim. Acta 47(3) 371-379 (1973) CA 80 12013w... 

Paust, 1977). Irradiation of retinoic acid (3) in heptane, in the presence of iodine, with a fluorescent lamp gave the two isomeric 4-keto acids (188) and (193), which can be separated by chromatography (McKenzie et aL, 1978b),... 

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