Thin layer chromatography
From Wikipedia, the free encyclopedia
| Thin layer chromatography | |
|---|---|
Separation of black ink on a TLC plate | |
| Acronym | TLC |
| Classification | Chromatography |
| Other Techniques | |
| Related | Agarose gel electrophoresis SDS-PAGE |
Thin Layer Chromatography (TLC) is a chromatography technique used to separate chemical compounds [1]. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution.
Its wide range of uses include
- determination of the pigments a plant contains
- detection of pesticides or insecticides in food
- analysis the dye composition of fibers in forensics, or
- identification compounds present in a given substance
It is a quick, generic method for organic reaction monitoring.
Contents
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[edit] Plate preparation
TLC plates are made by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC. Every type of chromatography contains a mobile phase and a stationary phase.
[edit] Technique
The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.
A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as ethanol or water, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase. Results also vary depending on the solvent used. For example, if the solvent were a 90:10 mixture of hexane to ethyl acetate, then the solvent would be mostly nonpolar. This means that when analyzing the TLC, the nonpolar parts will have moved further up the plate. The polar compounds, in contrast, will not have moved as much. The reverse is true when using a solvent that is more polar than non-polar (10:90 hexane to ethyl acetate). With these solvents, the polar compounds will move higher up the plate, while the non-polar compounds will not move as much.
The appropriate solvent in context of Thin layer chromatography will be one which differs from the stationary phase material in polarity. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other. Hence, to restrict the radial growth of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice-versa.
[edit] Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
- Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight(UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
- Iodine vapors are a general unspecific color reagent
- Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate
Once visible, the Rf value of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.
[edit] Applications
In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis is qualitative, and it will show if starting material has disappeared, product has appeared, and how many products are generated. Unfortunately, TLC's from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary. One such reaction is DIBALH reduction of ester to aldehyde.
As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.
| Step 1 | Step 2 | Step 3 | Step 4 |
| Step 5 | Step 6 | Step 7 |
In one study TLC has been applied in the screening of organic reactions [2] for example in the fine-tuning of BINAP synthesis from 2-naphtol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are place separately on the base line, then reacted and then instantly analyzed.