Can you please suggest specific spray reagents for TLC visualization of,
1. Falvonoids?
2. Terpenoids?
3. Phenolics?
Thank you
For Flavonoid you can use NP PEG: Natural product reagent [Intense orange fluorescence at UV-365 nm]: Sprayed with 10 ml of 1% methanolic diphenyl boryloxy ethylamine followed by 8 ml of 5% ethanolic poly ethylene glycol-4000 (PEG), See: http://pelagiaresearchlibrary.com/der-pharmacia-sinica/vol2-iss3/DPS-2011-2-3-74-80.pdf or] Wagner H., Bladt S. and Zgainski E.M, Plant drug analysis, A thin layer chromatography atlas, Springer Verlag Berlin Hidelberg, New York, Tokyo, pp.117 – 222.
PHENOLS TEST: https://chem.colorado.edu/genchemdemo/index.php/organic/o638-identification-of-phenols-ferric-chloride-test OR http://academics.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/ClassificationTests/phenol_amine_nitro.html
For various compounds classes see http://www.nitte.edu.in/journal/Dec2012/PPSOVEOPGP.pdf
I have searched several spray reagents. some papers say vaniline sulfuric acid reagents for terpenes. but some says it is a universal reagents. IS there any specific reagent for terpenes?
For terpenoids you can use
Vanillin spray reagent: Methanol (9 mL), Vanillin (0.5 g), glacial acetic acid (0.5 mL), concentrated sulfuric acid (3 drops).
Methanol: sulfuric acid spray reagent: 95 mL methanol and 5 mL concentrated sulfuric acid.
after spraying the tlc plates put then in oven for 5 min at 110 degree celcious for cherring....
Anisaldehyde Sulfuric acid reagent is the best for most of the phytochemical categories. It would be suitable for all you have mentioned. Method of Preparation is as follows:
0.5 ml anisaldehyde + 15 ml glacial acetic acid + 85 ml methanol + 5 ml conc. sulfuric acid (drop by drop from the sidewalls of the beaker)
After spraying, heat at 110 C for 10 minutes. Evaluate in visible light or UV 366 nm for best results.
You can use a cobalt chloride solution: Dissolve 10 g of CoCl2 in 250 ml of distilled water, then add 50 ml of sulphuric acid and gage to 500ml. Spray this solution over your TLC and heat at 100 C for 5-10 minutes. The compounds should appear as violet or pink spots.
For terpenes you can use oleum a mixture made with 160 mL water, 80 mL glacial acetic acid and 10 mL sulfuric acid.
Spray over the TLC and put it in the ofen for less than a minute at 150ºC.
If you cant see it at visible range you can use some UV lamp to see it.
You can use FeCl3 3% for Phenolics compounds and to flavonoids spray plate with a 1% ethanolic solution of aluminum chloride and watch yellow fluorescence in long wavelength UV light (360nm).
For Phenolics 5 - 10 % Ferric Cholride, For Terpenoids and Steriods Ansiladehyde and Vanillin Sulphuric Acid, For Flavanoids go for NP-PEG and 10 % MeOH KOH. Please also go for UV in both wavelength as all compunds gives fluroscence in UV, but keep in mind terpenoids and steroids produced fluroscence even after derivitisation with AS and VS. Where as flavanoids produced intense band after derivitisation with NP-PEG and KOH.
Dear Mr Galeano and Mr Sethiya :
What is the solvent for Ferric Chloride solution? Is it water, methanol or ethanol?
Thanks
A 5%w/v solution of ferric chloride in 90% ethanol is used for the detection of phenols.
for 0.5 M ferric chloride solution you can use water and 20 ML of concentrated HCL and make up the volume to one liter. for tannin detection.
you also can use neutral 5% ferric chloride solution. for Tannin detection.
for phenolic compounds u use 3 ml of 10% lead acetate solution.
I this wil help you.
Additionally, also the 2% aluminium chloride in methanol is used for flavonoids visualisation - they all react with yellow fluorescence. However, I would advice you to perform also 2D TLC on cellulose plates - without derivatization you may observe different fluorescence colour, which may give you some clue about the type of flavonoids present in your sample
What is the Pharmacopoeia standard preparation of Ferric chloride spray reagent for the identification of Phenolics on TLC plates?.
1. Flavonoids : Aluminum chloride : 1 g aluminum chloride is dissolved in 100 mL ethanol (The plate is sprayed and dried UV 366 nm)
2. Terpenoids : Anisaldehyde– sulfuric acid : 10 mL sulfuric acid is carefully added to an ice-cooled mixture of 170 mL methanol and 20 mL acetic acid. To this solution, 1 mL anisaldehyde is added. (The plate is immersed in the reagent for 1 s then heated at 100°C for 2–5 min. White light, UV 366 nm
3. Phenolic Compounds : Fast blue salt B : 0.5 g Fast blue salt B is dissolved in 100 mL water. (The plate is sprayed and dried . White light, UV 366 nm
Ref. Eike Reich & Anne Schibli. High-Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants
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