I have a standard of Asiaticoside 5mg/10ml methanol.
I need to check it on TLC. I have used mobile phase chloroform/glacial acetic acid/ methanol/ water 60/32/12/8 and 100/40/16/8 and also I used chloroform/methanol/water 10/3/0.2, but nothing showed up.
Only with the 2nd mobile phase did 2 spots show up close to the base line but the resolution was not good.
Any suggestions?
One would need to know the chemical structure of the asiaticoside in order to make any really useful suggestions. Do you know it?
Also, check with Camag for any application notes that they may have - they have a lot of info on TLC of botanicals and the like, and they may have a system worked out already.
Camag is a Swiss Company that specializes in TLC supplies and equipment. They maintain an extensive database of TLC applications that is worth checking. Perhaps it is available through their website, or give them a call. Thay are interested in promoting TLC for obvious reasons, and are often extremely helpful.
How are you trying to visualize the compound? What spray reagents are you using? Does the compound absorb UV light?
I took a loot at the compound. It doesn't really have any good chromophores so seeing it with UV lamp at 254 nm is going to be difficult. You need a reagent that will react with the compound to give it color. Try vanillin/sulfuric acid. It usually gives blue / purple spots for terpenoids.
I assume you are using normal phase silica plates? If so and the spots are so low to baseline you need to increase the polarity of the mobile phase. Try CHCl3 : Ethanol or methanol 1: 1. I don't see need to using acids yet unless the spots tail.
Look for details into the following paper:
"Elite genotypes/chemotypes, with high contents of madecassoside and asiaticoside, from sixty accessions of Centella asiatica of south India and the Andaman Islands: For cultivation and utility in cosmetic and herbal drug applications"
by Munduvelil Thomas Thomasa et al.: Industrial Crops and Products. 2010 Vol 32(3) pp 545–550.
They have used butanol:ethyl acetate:water(4:1:5, v/v).
Dry the TLC for 10 min at 100 C just before using it to reduce tailing.
To my opinion, there is a useful text in the 7.8 Edition of the European Pharmacopoeia. It treats of the full monograph about Centella asiatica in which there is a good analysis tool described to assessing the quality and quantity of asiaticoside by TLC (please see the PDF file attached for your convenience).
Good luck
Jo
thnaks all for your valued answers and suggestions, I am trying to check the ability of each.
Thanks Joseph for the attached Pdf
Thanks Malgorzata for your suggested paper
1. Chloroform: Glacial acetic acid: Methanol: Water (16:8:3:2) is found to be useful with one of my colleagues who worked with asiaticosides. Spraying reagent: Anisaldehyde- Sulphuric acid followed by heating.
2. n-hexane: EtOAc : EtOH (5:3:1). Observe the spots under UV light before revelation by spraying with vanillin (1%) and sulfuric acid (3%) solutions followed by heating.
3. CHCl3-MeOH (9:1) has also been found useful while analysing triperpenes of Centella asiatica.
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