Dual Wavelength Excitation Method for the Off-Line Liquid Chromatographic Analysis of Derivatized Amino Acids

Abstract

Naphthalenedialdehyde (NDA) in the presence of cyanide ion reacts with primary amines to produce highly fluorescent cyano[f]benzisoindole (CBI) derivatives. Fluorescent detection with NDA as the reagent, however, has one major limitation. Namely, analytes with more than one derivatizable amine site, such as lysine, show quantum efficiencies that are much lower in value than those of mono-derivatized CBI-amines. The reason for this lowering is that the multi-derivatized CBI amines undergo fluorescent quenching due to the close proximity of the CBI groups. This quenching can be circumvented, however, when these CBI-amines are deposited onto solid matrices, such as TLC plates. This method of depositing the liquid chromatographic (LC) effluent linearly on a solid matrix and then post-detecting the analytes has been referred to as a “chemical diskette” method in analogy to information storage and retrieval with computer diskettes. The implementation of this method was done by connecting a narrow bore flex-tubing from an LC microbore column to the writing head of a computer-controlled x-y recorder so that the LC effluent served as the “ink” in place of the usual writing pen. A thin layer chromatographic (TLC) plate replaced the recorder paper. The pattern of the deposition onto the TLC plate was preprogrammed and precisely controlled by a computer. The excitation light from a Xe arc lamp and the fluorescence emission to the optical detection system were transferred by optical fibers appropriately mounted on the writing head of the recorder. Thus, the deposited LC analytes could be fluorescence detected by precisely retracing the preprogrammed pattern as stored in the memory of the computer. The objective herein is therefore to evaluate the sensitivity and application of a dual wavelength excitation method (DWEM), particularly for multi-derivatized CBI amines. A detection limit of 6 fmol at a signal-to-noise of 2 has been demonstrated for desmosine, as one example of a multi-functional amine compound.