Fluorescence labelling of acidic functions

Although carboxylic acids can be readily determined by GLC methods, fluorescence labelling with Br-Mmc has nevertheless gained some importance for the sensitive derivatization of fatty acids [461-463] dicarboxylic acids, a-keto acids and other organic acids [460,464], prostaglandins and thromboxanes [465], glucuronides [466], giberellins [467], imides [461,468,469] and phenols [460]. 

Derivatives are prepared by refluxing with an excess of Br-Mmc in the presence of a crown ether and anhydrous K2CO3 in an aprotic solvent (e.g. acetone or acetonitrile). 

Suitable instrumentation for derivative formation in microlitre volumes has been described [470], The crown ether accelerates the alkylation reaction by acting as a phase-transfer catalyst [471]. Instead of K COj, other bases may be used (KOH, U2CO3). Yields are quantitative. A modified derivatization procedure suitable for automated samples preparation has been reported [472]. 

Br-Mmc and its derivatives are light-sensitive the derivatives are therefore preferably formed in the dark. Br-Mmc itself shows little fluorescence. 

Fluorescence characteristics of Mmc-carboxylic acid esters have been reported by Lloyd [473]. Excitation maxima in methanol and water methanol (9 1) were at 323 and 325 nm, respectively, and the corresponding fluorescence maxima were at 395 and 402 nm (corrected spectra). Quantum yields were 0.08—0.095 (methanol) and 0.054—0.44 (water/methanol, 9 1). TLC separation is possible, but owing to the sensitivity of the Mmc esters to light column chromatographic methods, especially on reversed phases, are much more suitable. Mmc esters were detected in column eluates (excitation at 340 nm emission at 420 nm) in pmol quantities [461]. 

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