Pyrolytic methylation assisted enantioseparation of chiral hydroxycarboxylic acids

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Abstract

Quaternary ammonium salts and dialkylformamide acetals have been demonstrated to be effective reactants for the injector port gas phase derivatization of organic compounds which have insufficient chromatographic properties. A derivatization method is described, in which dimethylformamide dimethylacetal is used as a methylating reagent for hydroxycarboxylic acids in the heated insert zone directly before their gas chromatographic enantioseparation. The results show that the chiral lactic, malic, mandelic, and tartaric acids or mixtures thereof were transformed into their methyl esters very quickly; these esters could easily be resolved into their enantiomers by gas chromatography employing chiral stationary phases. The amount of required samples depends on the analyte and could be limited to a few nanomoles; the entire derivatization procedure could be performed in less than 1 min. We examined the potential reagents systematically to select one of them for application in the COSAC experiment within ESA's ROSETTA mission, where solvent chemistry should be avoided because the transformation of polar organic compounds into derivatives suitable for chromatographic separation has to be performed on the surface of a cometary nucleus under near-zero gravity.

Introduction

The need for an efficient esterification procedure for submicrogram amounts of organic acids became apparent in our work on gas chromatographic enantioseparation as preparation for the Cosac experiment [1], [2] to be performed on the Lander of the ESA ROSETTA Mission on the nucleus of comet 46P/Wirtanen. In order to separate optical antipodes of organic matter expected to be abundant on comets, we validated several chiral stationary phases in gas chromatography with respect to their suitability for enantiomeric separation. As a result it was reported that racemic pairs of underivatized alcohols, diols and phenylsubstituted amines could be successfully resolved [3], whereas underivatized carboxylic acids and amino acids [4], [5] resist enantioseparation on the GC column. We have therefore developed and optimized an esterification technique, in which chiral hydroxycarboxylic acids are transformed into their methyl esters by pyrolytic methylation within the injector port of a gas chromatograph (Py-GC).

Because of their polar character, hydroxycarboxylic acids exhibit low vapour pressure. In a GC injector they are slow evaporating and rarely sweep into the analytical column. Apart from this, underivatized hydroxy acids tend to ‘tail’ because of associative effects with the stationary phase. Compared with the parent hydroxycarboxylic acids their alkyl esters are less polar, much more volatile, and well resolved by gas chromatography. Esterification is therefore the first choice for derivatization of hydroxycarboxylic acids, particularly for the intended gas chromatographic analysis during the ROSETTA project.

Conventional methods of esterification include reaction with diazomethane [6] or higher diazoalkanes or transesterification with methanol–acid mixtures. Diazomethane is unstable, toxic, and explosive; the transesterification step requires solvent chemistry, taking several hours for reaction and work-up. These properties exclude the conventional methods from our project. There was thus a need for a reliably working, rapid method for the preparation of alkyl carboxylates.

For 26 years [7] it has been known that acids are transformed into their methyl esters if they are heated in the presence of specific quaternary ammonium salts like tetramethylammonium hydroxide I [8], [9], [10], [11], [12], [13]. These reactions were realized by injecting the reagent together with the acid into the heated injector port of the gas chromatograph, where rapid pyrolysis occurred (Fig. 1), and the products were immediately swept into the analytical column. The elegance of this Py-GC technique is that it is all achieved within the injector system of the gas chromatograph.

According to literature data [14] for the volatile formic acid and for lactic acid, the tetrabutylammonium salts II were synthesized instead and pyrolysed in the same way. Later trimethylanilinium hydroxide III was found to be preferable to the tetraalkyl compounds [15], [16], because dimethylaniline is a better leaving group than trialkylamines, and thus a lower inlet temperature could be used [17]. III has been widely used as a methylating agent for a variety of different classes of compounds, particularly drugs, but also acids [18], [19], [20]. Recently the chemically related reagent tetramethylammonium acetate IV (TMAAc) was reported to be used as a selective reagent for the methylation of free acids [12]. Apart from the described alkylammonium salts, trimethylsulfonium hydroxide V was used for a one-step thermally assisted hydrolysis and methylation of a polyunsaturated fatty acid [21].

N,N-Dimethylformamide dimethylacetal VI (DMF-DMA) belongs to another class of methylating reagents which have been applied in the Py-GC technique [22], [23], [24]. The mechanism of this reaction is believed to involve the carboxylate ion of the parent acid and the alkoxycarbonium ion of the reagent (Fig. 2). In this study we report the pyrolytic methylation of chiral hydroxycarboxylic acids, in the context of gas chromatographic enantioseparation by chiral stationary phases.

Section snippets

Experimental

The pyrolytic methylation reagents DMF-DMA VI (pract., 7.5 molar, bp 102–104°C, sensitive to humidity), tetramethylammonium hydroxide I (purum, 0.9 molar and pract., 2.2 molar in methylalcohol), trimethylanilinium hydroxide III (puriss., 0.1 and 0.5 molar in methylalcohol) and the optically pure hydroxycarboxylic acids d(−)-mandelic, l(+)-mandelic, d(+)-malic, l(−)-malic, d(−)-tartaric, and l(+)-tartaric acid were purchased from Fluka. Racemic d,l-lactic acid, the achiral benzoic acid (cf. Fig.

Pyrolytic methylation of benzoic acid

In order to investigate both the Py-GC/FID equipment and the pyrolysis reagents, the gas phase methylation reaction of the optically inactive benzoic acid was studied. Preliminarily the chromatogram of underivatized benzoic acid on an achiral Ultra 1 stationary phase is depicted (Fig. 4a), which illustrates the insufficient chromatographic properties of this compound resulting in a small and broad peak. On the other hand, the chromatographic elution of the corresponding methyl benzoate

Conclusions

Pyrolytic methylation assisted gas chromatographic enantioseparation is a proven analytical technique for transforming polar chiral organic compounds into their alkyl esters, so that optical antipodes can easily be resolved. These properties make the described Py-GC techniques useful for the intended enantioseparations within the COSAC experiment on board the ROSETTA mission, in which cometary matter will be analysed in situ. The exact chemical composition of the nucleus of the comet is still

Acknowledgements

We thank co-workers of the COSAC team composed of Jean-Francis Brun, David Coscia, Pascale Ehrenfreund, Fred Goesmann, J. Mayo Greenberg, Guy Israel, Laurent Janin, Elmar K. Jessberger, Alexandra J. MacDermott, Takekiyo Matsuo, Guillermo Muñoz-Caro, François Raulin, Reinhard Roll, Robert Sternberg, Cyril Szopa, and Sandine Zubrzycki for their scientific support. For additional suggestions and discussions we were glad for the participation of F.R. Krueger, K. Kobayashi, and T. Saito. One of the

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