On-column methylation reaction

Figure 10. Examples of on-column methylation reactions of organophosphorus...

Valentine et al. applied, in principle, the method of Midha and Charette for a quantitative assay of quinidines (quinidine and hydroquinidine) in plasma, using methylene chloride instead of benzene for the extraction of a small volume of plasma after basification. To the extract was added the internal standard, cinchonine evaporation to dryness and reconstituting in a methanolic solution of trimethylanilinium hydroxide followed. An aliquot of this solution was analyzed by GLC via on-column methylation reaction. Evaluation of the method over the range 0.5 - 10 ug/ml in human plasma gave a precision and accuracy overall of 4.5 %... 

Another class of esterification reagents are halogenated compounds (alkyl iodides, substituted benzyl, and phena-cyl bromides), which need basic media for their reaction [K2CO3 or DMFA (dimethyl formamide) is normally used for the neutralization of HHal as acid by-product]. For methy-lation of carboxylic acids, some tetra-substituted ammonium hydroxides or halides can be used, e.g., tetramethylammo-nium hydroxide (in aqueous solutions) or trimethylanilinium hydroxide (in methanol solution). The intermediate ammonium carboxylates are thermally unstable and produce methyl alkanoates when the reaction mixtures are heated or when the carboxylates are introduced into the hot injector of the gas chromatograph (flash or on-column methylation) ... 

This reagent, stable in water in slightly alkaline conditions, is ideal for reducing sugars. The aldehyde function is reduced to a primary alcohol and the ketone function to a mixture of secondary alcohol epimers. The simplest treatment after reduction consists in eliminating the sodium on a cation exchanger column, then reaction with boric acid in the form of volatile methyl borate by co-evaporation with methanol. The alditol is recovered by evaporation of the solvent to dryness or lyophilization. 

Methyl 3-acetamido-2,4-di-0-acetyl-3,6-dideoxy-a-D-mannopyranoside on column containing alkaline alumina eluted with benzene and benzene-ethanol methyl 3-acetamido-4-0-acetyl-3,6-dideoxy-a-D-mannopyranoside. Y 90%. K. Capek, J. Steffkova, and J. Jary, Coll. 33, 1750 (1968) reactions on basic alumina, review, s. F. Serratosa, J. Chem. Educ. 46, 302 (1969). 

Baughman et al 7 studied the gas chromatographic behaviour of methylmercury compounds on a glass column (6ft x 0.25in) packed with 5% of DECS on Chromosorb W and operated at 160°C, and of phenylmercury compounds on a similar column packed with 3% of OV-1 on Chromosorb W and operated at 150 C. Flame ionization and Ni electron-capture detectors were used. Dimethyl- and diphenylmercury were stable under these conditions, but combined glc-ms confirmed that methyl- and phenylmercury salts decompose during gas chromatography. Reliable determination of methylmercury salts were achieved only on columns specially treated so as to make the decomposition reaction reproducible. Phenylmercury salts, which decompose extensively, could not be determined by gas chromatography. 

Thrash et al studied the analysis of phenylmethyldichloro-silane by reaction chromatography with sodium fluosilicate involving an on column conversation in a pre-column to the corresponding fluorosilanes which are then separated on the chromatographic column. Due to the similarity of their boiling points, phenyl methyl silicon dichloride and phenyl silicon trichloride cannot be separated by normal chromatographic procedures. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

The purity of the product was determined by the checkers by GLC analysis using the following column and conditions 3-nm by 1.8-m column, 5% free fatty acid phase (FFAP) on acid-washed chromosorb W (60-80 mesh) treated with dimethyldichlorosilane, 90 C (1 min) then 90 to 200 C (15°C per rain). The chromatogram showed a major peak for methyl 2-methyl-l-cyclohexene-l-carboxylate preceded by two minor peaks for methyl 1-cyclohexene-l-carboxylate and l-acetyl-2-methylcyclohexene. The areas of the two impurity peaks were 5-6% and 0.5-2% that of the major peak. The purity of the product seems to depend upon careful temperature control during the reaction. The total amount of the two impurities was 14-21% in runs conducted at about -15 to -20°C or at temperatures below -23°C. 

A mixture of 50 g of betamethasone, 50 cc of dimethylformamide, 50 cc of methyl orthobenzoate and 1.5 g of p-toluenesulfonicacid Is heated for 24 hours on oil bath at 105°C while a slow stream of nitrogen is passed through the mixture and the methanol produced as a byproduct of the reaction is distilled off. After addition of 2 cc of pyridine to neutralize the acid catalyst the solvent and the excess of methyl orthobenzoate are almost completely eliminated under vacuum at moderate temperature. The residue Is chromatographed on a column of 1,500 g of neutral aluminum oxide. By elution with ether-petroleum ether 30 g of a crystalline mixture are obtained consisting of the epimeric mixture of 170 ,21 -methyl orthobenzoates. This mixture is dissolved without further purification, in 600 cc of methanol and 240 cc of methanol and 240 cc of aqueous 2 N oxalic acid are added to the solution. The reaction mixture is heated at 40°-50°C on water bath, then concentrated under vacuum. The residue, crystallized from acetone-ether, gives betamethasone 17-benzoate, MP 225°-231°C. 

The reaction mixture is then warmed on the steam bath for an additional two hours (90°C to 95°C). The excess hydrazine hydrate is removed in vacuo. The residue of viscous 1-hy-drazlno-3-morpholinyl-2-propanol Is not distilled, but is mixed with 10.16 g (0.0B6 mol) diethyl carbonate and a solution of 0.3 g sodium metal in 15 ml methyl alcohol. The mixture is refluxed about 2 hours under a 15 cm Widmer column, the alcohol being removed leaving a thick, green liquid residue, which is cooled and the precipitate which forms is removed by filtration and washed well with ether. Yield B2%, MP114°C to 116°C. Recrystallization from isopropanol gives purified 3-amino-5-(N-morpholinyl)-methyl-2-oxazolidone, MP 120°C as the intermediate. 

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