Thin Layer Chromatography and its uses in Pharmacognosy

The word pharmacognosy is derived from the greek words “pharmakon” which means drug and “gnosis” or knowledge.  This is the study of drugs from natural products.  These natural products are typically derived from plant sources, like aspirin, or methanphedamines or caffeine.  However some compounds come from animal sources, such as the novel drug Conotropin, which is derived from conotoxin ( a potent neurotoxin found in geographic cone snails).  Think of the pharmacognosist as the modern day alchemist.

TLC of a plant extract

As their assays typically require identifying the base constituents of a larger compound, these scientists rely on an array of separation techniques to help them sort out compounds into their base components.  One such technique is thin layer chromatography.  Thin layer chromatography or TLC as its more commonly known, is based on the general concept that objects will move through a medium of evenly spaced particles based on their molecular weight.  Think of this analogy.  You have two small children running from two fat men.  If the children run to a play ground and move through the playground equipment they will get out of the climbing structure much faster than the two fat men will.  These children have passed through quickly because of their smaller size.  This is true also of molecules in an aqueous solution.  The larger particles will move slower through a matrix than smaller particles will.  In this case it become possible with a consistent media usually of powered glass (silica) to separate things by molecular weight.  This is the basic idea for chromatography.  Now thin layer chromatography relies on several key factors.  First it relies on the fact that a solute system has been designed that due to its polarity helps to separate out the compounds of interest ( think of this as a liquid to help move the particles through the matrix).  In high school chem lab, many of us performed the simple experiment where we took a sharpie or black inked pen and drew a dot on it.  We then placed one end in water and let the water wick upward.  This separated the ink out by color.  The reason this worked is because the water is an extremely polar compound.  It thus pulls the ink apart into its basic components which happen to be multiple colors of ink.  These components then travel along the plate until the solvent front stops at the top.    This can be seen in the example below.  The ink has separated out based on its components molecular weights.  To help more with this explanation, the yellow ink is the lightest in weight and thus has traveled farthest on the plate.

Black ink on a TLC Plate

So how is this useful you might ask?  Does this assay tell us anything about the constituents themselves?  Can I identify what they are?  Do I know the molecular make up of that yellow banding?  The answer is of course no.  However, TLC can be used as a basic step to help us later answer these questions.  Variations to our TLC assay can help us to actually identify what the banding patterns are.  Special sprays that interact with the separated compounds can produce a color change for example that help to identify the compounds within.  For example, potassium dichromate spray can help to identify the presence of any organic compounds within the original sample.   Also by varying the solvent system, (mixing in water with an alcohol or organic solvent) we can change not only the rate at which the solvent travels but also the clarity and resolution of separation.  We can also use a fluorescent plate that will cause particular samples within the matrix to change color under a UV light.

So how do we setup a basic TLC assay.  First your compound of interest must be obtained in a liquid delivery system.    This allows the liquid to transport through the media. You would first place blot your sample onto the TLC plate making sure to measure the starting position.  Then place your plate upright into a container with a seal able top .  Then place a piece of filter paper to help wick the solvent upward.  Then pour in just enough solvent to cover the base of your plate.  Cover and wait.  Remove your plate before the solvent front runs off the glass slide.  Take a look at the below figure for perhaps a better explanation as to how TLC assays can be setup.  The  final interesting point about TLC assays is that the particular bandings, once separated can be literally cut out and reconstituted in alcohol or an organic solvent for further analysis with other equipment.

Identified bandings

Steps for a TCL assay


Posted on November 18, 2009, in Uncategorized and tagged , , , , , , , , , . Bookmark the permalink. Leave a comment.

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