Dear Sir. Concerning your issue about isolation and purification of indicane glycoside from Indigofera tinctoria leaves . Plant materials were cut to small pieces, fermented in water at different periods and then added twice in volume of Ca(OH)2 solution (pH~11), blew the air for 15 mins to precipitate indigo. The precipitated indigo was washed twice with Ca(OH)2 solution and centrifuged at 9820xg for 10 mins. The dyes were extracted by chloroform and evaporated under reduced pressure to dryness. The red pigment was separated by extracting with methanol after which the blue pigment was extracted with chloroform. Both pigments were evaporated to dryness and separated by column chromatography. A glass column 45x3 containing 60 g of silica gel 60F254 saturated with hexane was used to separate 2 g of dye pigment and eluted with chloroform-hexane (4:1 v/v) and chloroform-hexane-methanol (7:4:1 v/v/v), respectively. The eluates were concentrated. The purity of the pigments was then tested by TLC in several solvent systems. The pure pigments were analysed for their chemical structure. If the pigment was not pure, the subcolumn chromatography was done again. I think the following below links may help you in your analysis:

https://pdfs.semanticscholar.org/c00a/2b18308efb5cbfbffc2cbb6ad6077e4a78a4.pdf

http://krishikosh.egranth.ac.in/bitstream/1/5810015023/1/KERL-172366.pdf

http://cmuj.cmu.ac.th/sites/default/files/styles/thumbnail/may2002-2f.pdf

Thanks

Thanks Dr. Isam Sir for your wonderful explanation about isolation of Indican glycoside. I only have a small confusion that when i will get blue pigment in pure form, So, would it confirmed that the compound is Indican glycoside not a indigo dye? As shown in literature that after hydrolysis Indican converted to indoxyl. So, are you sure after fermenting with water indican would not convert to indoxyl? Are both (hydrolysis or fermentation) totally different or have interrelation with each other? Please let me know your opinion?

Thanking you

Abhay

I agree with the suggestions of Isam. Goodluck.