Aniline phthalate, sugar detection

Solvent systems used for thin layer chromatography were 1) n-butanol acetic acidiwater (4 1 5 upper phase), 2) acetic acid water (15 85), 3) ethyl acetate pyridine water (12 5 4), and 4) chloroform acetic acid water (50 45 5). Silica gel plates were used for chromatography of flavonoid aglycones and cellulose plates for all other components. Aluminum chloride was used for detection (under long UV light) of flavonoids, aniline phthalate for sugars, ninhydrin for amino acids and iodine for other components. Cellulose thick layer plates were developed with solvents 1 or 2. 

The protein polysaccharide is hydrolyzed at 100-110° for 3 hours with N sulfuric acid, neutralized with baryta solution, filtered, and the filtrate concentrated to small volume. Zone electrophoresis in borate buffer (pH 8.6) is then carried out for a suitable time, and the paper strip is dried and sprayed with ninhydrin (to locate the amino acids) and with aniline hydrogen phthalate (to detect the reducing sugars). 

The reducing methylated sugars are detected by the same reagents used for paper chromatograms such as aniline phthalate (No. 10), p-anisidine phthalate (No. 12), 2,3,5-triphenyltetrazolium chloride (No. 255), alkaline silver nitrate (No. 234) and, in addition, the sulfuric acid sprays and iodine vapor. Brief exposure to iodine vapor does not cause a detectable destruction of the methyl ethers of the sugars [83] and is therefore a convenient method for visualizing the components on preparative plates [73] or for quantitative analysis. 

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