Abstract: Investigations have shown that the peroxyoxalate chemiluminescence reaction can be effectively used for a postcolumn detector in high-performance liquid chromatography (HPLC). The reaction generates the fluorescent state of certain suitable acceptors, such as 2-cyanobenz[f]isoindole (CBI) derivatives of amino acids, peptides, amines, and related primary amines. The derivatization method makes use of the reaction of 2,3-naphthalenedicarboxaldehyde and cyanide (NDA/CN) as a new fluorogenic and chemilumigenic reagent for analysis of primary amine functions. While the method has proven to be superior to standard fluorescence assays for low-level detection of amino acids and peptides, optimizing the method's sensitivity remains the current goal of this research. For this reason, the contributing parameters for efficient generation of chemiluminescence for the assay of CBI derivatives based on oxalate-hydrogen peroxide activation have been identified and investigated. Among the important factors are: (i) the nature of the leaving group on the oxalate ester, (ii) the fluorescence properties of the NDA/CN analyte, (iii) the fluorescence-quenching capabilities of the oxalate ester and its reaction products, (iv) solvent effects, and (v) the effects of added catalysts. In order to optimize the chemiluminescence response, we have investigated the mechanism of the complex reactions leading to chemical generation of chemiluminescence. A new peroxyoxalate-hydrogen peroxide reaction mechanism has emerged from our preliminary studies on the five contributing factors listed above. Two kinetic models are discussed, one for the mechanism of the reaction in organic solvents and the second for mixed aqueous-organic solvents. The latter model is particularly adaptable to quantitative rate studies and has become the focus of efforts to develop the peroxyoxalate chemiluminescence reaction as a postcolumn detector for HPLC. Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. The potential for improved chemiluminescent detection is large, since the efficiency for activation of the acceptor is less than 0.01%. A thousand-fold increase in signal could be anticipated from this reaction.
Publication Year: 1989
Publication Date: 1989-12-30
Language: en
Type: book-chapter
Indexed In: ['crossref']
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Cited By Count: 5
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