Physical methods of detection

When a coloured spot of the authentic reference substances lies in an identically coloured band of the naturally occurring mixture, the identity of the substances is suspected. If this is repeated in various solvents of widely differing separation properties, the identity is probable. In any case, reference substances and mixture of naturally occurring substances should be chromatographed together (co-chromatography) as shown, since colour and i /-value can be altered considerably by contaminants. 

The procedure described is satisfactory for one-dimensional separations, also with multiple development or when stepwise technique is used (see p. 87). In two-dimensional separation, suitable bands may be applied outside the separation square itself. Further, reference substances which come into consideration after the first 

If there is enough substance available, the eluates of the scraped-off zones can be qualitatively and quantitatively examined by the usual spectrophotometric, colorimetric and fluorometric methods. 

SEHiER et al. [110] describe a direct quantitative determination of anthranilic acid and some indole derivatives, without elution. Layers on 5 X 20 cm plates were chromatographed using solvent XIV, Table 89, sprayed with the Prochazka reagent (No. 123, Table 88), illuminated with UV light of wave-length 365 nm and the fluorescence measured with a slightly modifled Zeiss-Spektralphotometer PMQ II (Firm 155). The surface areas under the lines registered with a recorder were found to be directly proportional to the amount of material between 10 and 2000 ng. A TLC-attachment for the Zeiss Spektralphotometer has been developed with which direct routine qualitative and quantitative measurements on normal TLC-plates have become possible [41, 42]. 

Wakhloo [133] has obtained information about tryptophan, separated by TLC from Solarium nigrum. he chromatographed the plant extract under investigation and 3—4 known amounts of tryptophan on the same layer and made densito-metric measurements on a contact diapositive of the TLC-layer. 

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B. Physical Methods of Detection and Analysis Toward Gallane Preparations for the Hunt... 

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