A thin layer chromatographic method for the separation and identification of the tryptophan metabolites present in whole human urine.

This paper reports a new thin layer chromatographic system which is able to separate a total of thirtyeight (38) tryptophan metabolites from a synthetic mixture in a period of three hours. The system consists of thin layers of silica gel GF254 which are eluted in two dimensions, employing in the first dimension a mixture of propanone, 2-propanol, water, ammonia (0.88) (50 : 40 : 7 : 3 V/V) and in the second dimension a mixture of chloroform, glacial acetic acid, methanol and water (65 : 20 : 10 : 5 V/V). A method whereby the separated tryptophan metabolites may be unambiguously identified in situ following the thin layer chromatography is also presented and discussed. This method utilises the effect of ultraviolet light, at 254 and 350 nm, and the colour reactions between the metabolites and four reagents namely, a modified Ehrlich, o-phthaldehyde, Pauly and Ninhydrin-cadmium acetate. A summary of the unsuccessful attempts to quantitate the modified Ehrlich positive metabolites by in situ reflectance densitometry is also given. Finally, it is demonstrated that the new system may be used to advantage to separate and identify tryptophan metabolites in studies of whole human urine.