Principles, instrumentation and applications for fluorimetric studies of photochemically unstable compounds utilizing digital integration

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This investigation is concerned with the feasibility of utilizing photochemical reaction in the fluorescence assay of biologically important compounds. Photochemical reaction has been previously treated as an interference in fluorescence. Often steps are taken to inhibit photochemical reaction in fluorescence assay at the expense of sensitivity. In this study the instrumentation is designed so as to tolerate and, in fact, encourage photochemical reaction, with the hope of improving sensitivity and precision of fluorescence assay. Several different ultraviolet radiation sources were investigated for their ability to both excite fluorescence and initiate photochemistry. High intensity polychromatic radiation was used in the assay of: the indoles, tryptophan, 5-hydroxytryptophan, tryptamine, 5-hydroxytryptamine; the catecholamines, dopa and dopamine; and the aminoquinolines quinine and chloroquine. Decay of native fluorescence signal for each of these compounds was used to develop a method of assay over the concentration range of 10 ppb to 1 ppm. Precision of replicate measurements, expressed as relative standard deviation was on the order of 0.5%. Two compounds possessing basic phenol structures, tyrosine and norepinephrine were also studied. Of the two, a practical method of assay, over the same concentration range and at the level of precision mentioned above, could be developed only for norepinephrine. A major instrumental consideration is the application of digital data handling techniques. Through the application of digital integration of the fluorescence signal significant increase in precision and sensitivity of fluorescence assay was accomplished. An additional advantage of digital integration was the ability to distinguish between very small concentration differences at the ppb level. A mathematical treatment of the photochemical kinetics involved and the fluorescence emission is presented. The effects of instrumental parameters, including blank subtract, integration of signal and digital readout are discussed. An evaluation of the sensitivity of the instrumentation used was carried out using quinine.