Abstract-One of the recent uses of fluorescence has been in the assay of substances directly on solid surfaces such as paper chromatograms, thin-layer chromatography (TLC) plates, potassium bromide disks, electrophoresis strips, and silicone-rubber pads. Chromatography and electrophoresis are widely used analytical techniques and it is not surprising that fluorometric methods are used in conjunction with them. Advantages are increased sensitivity and rapidity of analysis.In this paper the use of fluorescence in the assay of substances after separation on paper and TLC chromatograms, as well as the direct assay of substances on a solid surface placed directly in a commercial fluorometer, are discussed. Particular emphasis is placed on the direct analysis of enzymatic reactions.
ANALYSIS OF PAPER CHROMATOCRAMSDifferent grades of filter paper vary in their content of fluorescent materials, which may interfere with attempts to measure fluorescence emission from the paper. Also problems with the purity of filter paper arise; the seriousness of interference depends on the compound under analysis. Van Horst et al. (1959) 4, and S and S No. 597 were best suited for the fluorometric assay of reducing oligosaccharides on paper chromatograms.Any one of the commercially available fluorometers or fluorescence spectrometers can be used (with appropriate modification) to measure fluorescence emitted from paper chromatograms. Fluorescence can be excited by passing the exciting light through the paper, as was done by Wadman et al. (1954). Semm and Fried (1952) used a similar arrangement, although complete details were not given. The paper 403 404 GEORGE G. GUILBAULT strip can also be placed at a 45-degree angle across the cell compartment, and the fluorescence emitted from the surface can be measured.If the fluorescence from paper is measured in a spectrophotometer (reflectance) or in a fluorescence spectrometer, it should be possible to determine the entire fluorescence spectrum of a given compound. Korte and Weitkamp (1958) report procedures for measuring the fluorescence spectra of berberine, fluorescein, xanthopterin, and thiochrome on paper chromatograms. Van Horst et al. (1959) carried out a quantitative study of amino acids on paper with a Photovolt Model 525 densitometer with a phototube detector connected to a recorder. A mercury lamp was used for excitation.
ANALYSIS OF TLC PLATES
A. instrumentationEarly applications of fluorescence in quantitative measurements of solid surface analysis consisted principally of scraping off the substance from the solid TLC plate, or cutting the substance out of the paper chromatogram, dissolving the substance in an appropriate organic solvent, and then measuring the fluorescence in solution with a commercial fluorometer. This technique was time consuming and had limited accuracy. Hence much attention was paid to the development of instrumentation capable of direct measurement of the solid surface. As a result several attachments to commercial instruments, capable of direct measurem...