Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures.

It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. This is called the stationary phase. This solvent then moves up the plate via capillary action. As with all chromatography, some compounds are more attracted to the mobile phase, while others are more attracted to the stationary phase. Therefore, different compounds move up the TLC plate at different speeds and become separated. To visualize colourless compounds, the plate is viewed under UV light or is stained. Testing different stationary and mobile phases is often necessary to obtain well-defined and separated spots.

TLC is quick, simple, and gives high sensitivity for a relatively low cost.

There are four main stages to running a thin-layer chromatography plate: Different compounds in the sample mixture travel at different rates due to the differences in their partition coefficients. Different solvents, or different solvent mixtures, give different separations.

If the stationary phase is non-polar, like C18-functionalized silica plates, it is called reverse-phase TLC. In this case, non-polar compounds move less and polar compounds move more. The solvent mixture will also be much more polar than in normal-phase TLC. Using solvents with different elution strengths or different selectivity groups can often give very different results.

In normal-phase TLC, the most common solvent mixtures include ethyl acetate/hexanes (EtOAc/Hex) for less-polar compounds and methanol/dichloromethane (MeOH/DCM) for more polar compounds. Different solvent mixtures and solvent ratios can help give better separation. In reverse-phase TLC, solvent mixtures are typically water with a less-polar solvent: Typical choices are water with tetrahydrofuran (THF), acetonitrile (ACN), or methanol.

  • Potassium permanganate (no heating, for oxidisable groups)
  • Ninhydrin (heating, amines and amino-acids)
  • Acidic vanillin (heating, general reagent)
  • Phosphomolybdic acid (no heating, general reagent)
  • In the case of lipids, the chromatogram may be transferred to a polyvinylidene fluoride membrane and then subjected to further analysis, for example, mass spectrometry. This technique is known as far-eastern blot. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. Other adsorbent coatings include aluminium oxide (alumina), or cellulose. The researchers react an alcohol and a catalyst directly in the co-spot of a TLC plate before developing it. This provides quick and easy small-scale testing of different reagents.

thumb|332x332px|TLC for reaction monitoring and choosing a purification solvent mixture (left)TLC from the resulting [[Column chromatography|flash column chromatography (right)]]

Compound characterization with TLC is also possible and is similar to reaction monitoring. However, rather than spotting with starting material and reaction mixture, it is with an unknown and a known compound. They may be the same compound if both spots have the same R<sub>F</sub> and look the same under the chosen visualization method.

Purity and purification

TLC helps show the purity of a sample. A pure sample should only contain one spot by TLC. TLC is also useful for small-scale purification. Because the separated compounds will be on different areas of the plate, a scientist can scrape off the stationary phase particles containing the desired compound and dissolve them into an appropriate solvent. A compound elutes from a column when the amount of solvent collected is equal to 1/R<sub>F</sub>. The eluent from flash column chromatography gets collected across several containers (for example, test tubes) called fractions. TLC helps show which fractions contain impurities and which contain pure compound.

Furthermore, two-dimensional TLC

TLC is also an analytical method for the direct separation of enantiomers and the control of enantiomeric purity, e.g. active pharmaceutical ingredients (APIs) that are chiral.<gallery class="center" caption="Separation of green plant matter in spinach (note that images from steps 1–6 are zoomed into the bottom of the plate)">

File:Chromatography_of_chlorophyll_-_Step_1.jpg|Step 1

File:Chromatography_of_chlorophyll_-_Step_2.jpg|Step 2

File:Chromatography_of_chlorophyll_-_Step_3.jpg|Step 3

File:Chromatography_of_chlorophyll_-_Step_4.jpg|Step 4

File:Chromatography_of_chlorophyll_-_Step_5.jpg|Step 5

File:Chromatography_of_chlorophyll_-_Step_6.jpg|Step 6

File:Chromatography_of_chlorophyll_-_Step_7.jpg|Step 7

</gallery>

See also

  • Column chromatography
  • HPTLC
  • Radial chromatography
  • Chiral thin-layer chromatography

References

Bibliography

  • F. Geiss (1987): Fundamentals of thin layer chromatography planar chromatography, Heidelberg, Hüthig,
  • Justus G. Kirchner (1978): Thin-layer chromatography, 2nd edition, Wiley
  • Joseph Sherma, Bernard Fried (1991): Handbook of Thin-Layer Chromatography (= Chromatographic Science. Bd. 55). Marcel Dekker, New York NY, .
  • Elke Hahn-Deinstorp: Applied Thin-Layer Chromatography. Best Practice and Avoidance of Mistakes. Wiley-VCH, Weinheim u. a. 2000,