Photochemical Reaction Detection in HPLC

Abstract

Post-column photochemical reactions may be used to enhance the sensitivity or selectivity of detection in high-performance liquid chromatography (HPLC). Several new photochemical reaction detection schemes are discussed. Anthraquinones may be detected with extremely high selectivity based on a photocatalytic reaction in the presence of oxygen to produce hydrogen peroxide. The hydrogen peroxide is subsequently detected by the highly sensitive peroxyoxalate chemiluminescence reaction. Catalytic factors ranging from 40 to 70 are achieved for many quinone analytes. Use of an immobilized fluorophore and solid-bed reagent addition makes this detection scheme particularly easy to implement. In the absence of oxygen, quinones are photoreduced in the presence of certain hydrogen-atom-donating substrates to produce highly fluorescent dihydroquinones. Thus, quinone derivatives may be used as photochemical reagents for the detection of analytes such as aliphatic alcohols, ethers, aldehydes, and amines. This post-column photochemical reaction is particularly useful for the detection of compounds having no chromophore such as saccharides and steroids. The fast reaction of singlet oxygen with either 2,5-diphenylfuran or 2,5-dimethylfuran forms the basis of a photochemical amplifier for UV-absorbing compounds. This method is photocatalytic and leads to improved detection limits in UV absorption by factors of one to two orders of magnitude. This photochemical reaction allows non-fluorescent compounds to be detected in HPLC using a UV detector with sensitivities rivaling those of fluorescence detection (typically 1 to 10 pg of compound).