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Author Topic: THIN LAYER CHROMATOGRAPHIC TECHNIQUES- Running the Thin layer chromatography  (Read 5023 times)
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Francis Umeoguaju
Expert in Bioscience Issues
Posts: 657

« on: April 10, 2010, 07:57:18 am »

Running the Thin layer chromatography

Marking the plate: It is necessary for the plate to be well labeled i.e the name and quantity of each material spotted, the points of origin and the intended solvent front. All markings on the plates should by make with pencil since the solvent can early separate the constituents of ink. Direct labeling of the plate is possible when using pre-coated plate, when self coated plates are used, the replica of the plate is drawn on a paper and all labeling are done there.

All spotting should be done at least 1.5 cm from the edges of the plates. Inter spots should be at least 1.5cm away.

Loading samples of the TLC plates: The diameter of each spotted materials should not be more than 4mm, large spot gives a wider and blurred band; sometimes, there is an overlapping of bands with those of the neighbor's to giver a non distinct result. The quantity of materials to be loaded into each spot is of critical importance, this in a way determinate success of the chromatography. Too little sample in a spot usually give no observable band while too much sample per spot leads to the developed plates having background trailing as a result of the solvents inability to left all the spotted materials. If a dilute sample cannot be concentrated, it would be necessary to apply large quantity of it in a spot; this is usually done over an oven.

When spotting samples of unknown concentration, three varied amount of it are spotted, this will give insight on the best quantity of the sample to be spotted in subsequent runs.

The amount of standard to be spotted depends on the type of plates used. It also depends on the compound of interest. Running TLC of amino acids and sugar using self coated plated required a concentration range of 0.08 to 0.14 milligram of the substance per spot to give a clear and distinct result.

Running of the TLC plate; Running the TLC requires a transparent plastic or glass container with a plat bottom and a tight cover. The smaller the container, the better. The choice of mobile phase used to run the TLC depends on the compound to be separated, suitable mobile phase for running the thin layer chromatography of amino acid include;
1.   Ethanol and ethyl acetate in ratio 9:1
2.   Ethanol and acetic acid in the ratio 9:1
3.   Water and n-propanol in the ratio 50:50
4.   Water + glacial acetic acid + butanol 1:1:4
5.   N-propanol + ethyl acetate + water 14:2:7 (this gave nice result with commercial plate but poor separation with self
coated plates)

Suitable mobile phase for running the TLC of reducing sugar include: Butanol: Acetone: water 5:4:1

Suitable mobile phase for running the TLC of Raffinose series sugar is 90% isopropyl alcohol

The tank is usually left to be saturated with the suitable mobile phase 30-60min before dipping the plates.

The mobile phase usually take about 1hr to 3hrs to move up the TLC plate.

Staining the TLC and colour development; A general method of developing the colour of TLC  is by placing it in a tank saturated with iodine vapour, after a while the bands becomes visible. This regions is immediately encircle with a pencil before the colour fades off.

A more specific method of colour development requires the use of specific reagent which can react with the compound of interest to produce a characteristics colour.

Developing the colors of amino acids in TLC plates. The spray or developing reagent is prepared by mixing 30mg  of Ninhydrin, 0.3ml of glacial acetic acid and 10ml of Butanol together. The dried chromatogram  is sprayed with this reagent. The plate is then heated over the oven the until the colour develops.

Developing the colour of sugar on TLC plates: The spray reagent of reducing sugar s 5% H2S04, the colour is developed by heating the sprayed plate over the oven for about 3 minutes.

A more general carbohydrate spray reagent is prepared by dissolving 10 grams of ammonium molybdate (NH4)2MoO4  in 50ml of water. This is followed by the addition of 15ml of concentrated HCL to the resultant solution. The entire mixture is then made up to 100ml by the addition of 35ml of distilled water. 25g of NH4Cl is then dissolved in the solution (Murray 1973). The reagent is used to visualize raffinose, sucrose and stacheous.

Likely problems to be encountered while running a thin layer chromatography work.

The appearance of background trailing: this is as a result of spotting highly concentrated sample, the sample should be diluted down for the next run.
Observation of uneven solvent front: This occurs when the thickness of the plate is uneven, it may also occur when the plate is tilted in the developing tank.
When the sample moves close to the solvent front, try a less polar solvent, if the sample did not move at all, try a more polar solvent.

Chances favours the trained minds
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