I made alkaloid extract in chloroform and want to run TLC to detect probable number of compounds in same by staining it with Munier Reagent (to visible alkaloids). However I used 5 of the mobile phase to get proper resolution but couldn't. Anyway how to get clear bands on TLC? What kind of mobile phase should I make to get rid of trailing?
Also please suggest the mobile phase for Column Chromatography, if the extract is in the same solvent (chloroform).
Dear Shiniwas : As you know there are many factors causes this process such as: nature of the your mixture (acid ,base or neutral) ,solubility (poor soluble sample in mobial phase concentration , polarization , mobial phase polarity and the nature of stationary phase.Generally when your sample basic its necessary to add a base about (0.2- 0.9% ) to your eluent. try to use (ethyl acetate; methanol; conc. ammonia) (8.5:0.1 :0.05 %), this mixture has been used for long time and found to be excellent for basic compounds. If It's not work u can use CH2Cl2,MeOH,conc.aqu. ammonia (9,0.9, 0.1%) this ratio for both TLC and column chromatography of alkaloids.
Addition of 2-3 drops of formic acid or acetic acid will reduce tailing. You should also be careful while applying the spot. Spots should be discrete, distant and concentrated properly. Then you should also saturate the chamber properly using filter paper on the sides of the tank which allows an even distribution of the vapors.
Dear Sriniwas,
I want to point out one more thing which is responsible for tailing is concentration of the extract.
If you extract will be more concentrated then you may get tailing in better solvent system too.
So go with 4-5 different diluted solution and see the results.
Good Luck
There are different solvent systems and spray reagents to detect Alkaloids from plant system. Here with i am attaching file for sample preparation and Spray reagents and solvent system.Please find. Please standardize the method for your Plant Material by trial and error ranging the loading volume of the extract form 5 micro liter to 50 micro liter. The 100 micro liter should contain 50-60 micrograms of Total alkaloids. Keep the concentration low to avoid trailing of the Alkaloid Bands. load the spot in Rectangular Shape with proper spacing between to prevent intermixing of Bands
One thing that is worth trying is to make sure that the plate is free of water, i.. they must be very thoroughly dried in oven to remove all the polar water molecules in the silica gel surface (if we are using silica based packing materials. The principle of trailing or tailing is that the molecules of interest has relatively high affinity to the packing materials or literally 'keeps binding' to the packing materials.
Don't expose plate to UV until you make sure that your plate is dry
Leave it on hot plate or air
- Badges
- Science topic