Ultra-violet Spectroscopy in the Analysis of Chromatograms

Journal/Book: Reprinted from Nature Vol. 208 No. 5012 pp. 784-785 November 20 1965. 1965;

Abstract: Department of Medicine The Graduate Hospital University of Pennsylvania. The sensitivity of the normal human eye to light does not extend far below 400 mµ. Nevertheless ultra-violet-absorbing compounds appear as dark spots an paper chromatograms illuminated with ultra-violet light. This is due to absorption of the light before it can excite the fluorescence of the paper and constitutes one variety of quenching. Compounds which fluoresce in ultra-violet light will be detected an paper chromatograms if their fluorescence exceeds that of the surrounding paper. The light sources commonly employed emit the mercury lines at 254 mµ or 366 mµ. In chromatograms of biological extracts the short-wave lamp detecta principally the absorbing compounds and the long-wave lamp fluorescing compounds. Beyond this the method possesses little inherent specificity. Thus compounds such as cortisone nicotinic acid caffeine and uric acid (max 240 260 273 and 292 mµ respectively) all appear indistinguishable as dark spots under the short-wave lamp. Furthermore for substances such as oestrogens the absorption maximum (280 mµ) of which is very different from the 254 mµ emission of the handlamp the method is relatively insensitive. A need therefore exists for an Instrument with which absorption and activation maxima can be rapidly determined an the paper. I have accordingly designed an ultra-violet spectroscope in which the ultra-violet light is shifted to the visible region by the fluorescence of either the paper itself or a superimposed screen. The light source an ´Osram' 450-W xenon arc provides an ultra-violet continuum. After passing through a quartz prism monochromator (Schoeffel Instrument Co. Westwood N.J.) with variable exit slit the light emerges through a 1 3/16 x 3/4 in. rectangular aperture as a slightly divergent beam which illuminates the chromatogram. The lattor is attached by spring clips to a frame which can be moved manually in three planes or by a motor in the direction of solvent flow only. A screen coated with a suitable phosphor can be placed in position over the illuminated portion of the chromatogram if desired. Among several phosphors examined ZnSiO4 : Mn and (Sr Mg)3(PO4)2 : Sn were found to be the most generally useful. The former is slightly phosphorescent but not excessively so. It provides a bright illumination over the range 230-295 mµ and 330-400 mµ. The latter exhibits a continuous though lass intense fluorescence over the range 230-370 mµ and is therefore suitable for rapid routine searching. ___MH

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