Use of Reference Substances in Identity Tests— In paper and thin-layer chromatography,the ratio of the distance (this distance being measured to the point of maximum intensity of the spot)traveled on the medium by a given compound to the distance traveled by the front of the mobile phase,from the point of application of the test substance,is designated as the RFvalue of the compound.The ratio between the distances traveled by a given compound and a reference substance is the RRvalue.RFvalues vary with the experimental conditions,and thus identification is best accomplished where an authentic specimen of the compound in question is used as a reference substance on the same chromatogram.
For this purpose,chromatograms are prepared by applying on the thin-layer adsorbent or on the paper in a straight line,parallel to the edge of the chromatographic plate or paper,solutions of the substance to be identified,the authentic specimen,and a mixture of nearly equal amounts of the substance to be identified and the authentic specimen.Each sample application contains approximately the same quantity by weight of material to be chromatographed.If the substance to be identified and the authentic specimen are identical,all chromatograms agree in color and RFvalue and the mixed chromatogram yields a single spot;i.e.,RRis 1.0.