Thin layer chromatography
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{{Infobox chemical analysis
| name = Thin layer chromatography
| image = TLC_black_ink.jpg
| caption =Separation of black ink on a TLC plate
| acronym = TLC
| classification = [[Chromatography]]
| analytes =
| manufacturers =
| related = [[Agarose gel electrophoresis]]<br>[[SDS-PAGE]]
| hyphenated =
}}
'''Thin layer chromatography''' (TLC) is a [[chromatography]] technique used to separate mixtures.<ref>Vogel's Textbook of Practical Organic Chemistry (5th Edition) (Hardcover) by A.I. Vogel (Author), A.R. Tatchell (Author), B.S. Furnis (Author), A.J. Hannaford (Author), P.W.G. Smith ISBN 0582462363</ref> It involves a ''stationary phase'' consisting of a thin layer of <!-- note: the following word really does have a "d" not a "b" -->[[adsorbent]]<!-- yes, a "d" not a "b" --> material, usually [[silica gel]], [[aluminium oxide]], or [[cellulose]] immobilized onto a flat, inert carrier sheet. A ''liquid phase'' consisting of the solution to be separated is then dissolved in an appropriate solvent and is drawn up the plate via [[capillary action]], separating the experimental solution based on the polarity of the components of the compound in question.
Its wide range of uses include
*assaying radiochemical purity of radiopharmaceuticals
*determination of the pigments a plant contains
*detection of pesticides or insecticides in food
*analysing the dye composition of fibers in [[forensic]]s, or
*identifying compounds present in a given substance
*monitoring organic reactions
A number of enhancements can be made to the original method to automate some steps, to increase the resolution achieved with TLC and to allow more accurate quantitation. This method is referred to as [[HPTLC]], or "high performance TLC".
==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.
==Technique==
[[Image:TLC-Essential-Oils.jpg|thumb|right|350px|Chromatogram of 10 [[essential oil]]s coloured with [[vanillin]] reagent. ]]
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 reaction]]s 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 [[chemical compound|compounds]] in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent.
Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will not result in reversed order of running of the compounds on the TLC plate. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.
[[Image:Tlc sequence.png|thumb|right|250px| Tlc plate development sequence: a mixture of a red and blue compound is separated as the plate develops, i.e. as the light blue solvent moves up the plate.]] 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''.
==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]] (UV<sub>254</sub>). 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
* In the case of lipids, the chromatogram may be transferred to a [[PVDF]] membrane and then subjected to further analysis, for example [[mass spectrometry]], a technique known as [[Far-Eastern blot]]ting.
Once visible, the ''R''<sub>''f''</sub> value , or [[Retention factor]], 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.
==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.
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image:Chromatography of chlorophyll - Step 1.jpg|Step 1
image:Chromatography of chlorophyll - Step 2.jpg|Step 2
image:Chromatography of chlorophyll - Step 3.jpg|Step 3
image:Chromatography of chlorophyll - Step 4.jpg|Step 4
image:Chromatography of chlorophyll - Step 5.jpg|Step 5
image:Chromatography of chlorophyll - Step 6.jpg|Step 6
image:Chromatography of chlorophyll - Step 7.jpg|Step 7
</gallery>
</center>
In one study TLC has been applied in the screening of [[organic reaction]]s<ref>''TLC plates as a convenient platform for solvent-free reactions'' Jonathan M. Stoddard, Lien Nguyen, Hector Mata-Chavez and Kelly Nguyen [[Chem. Commun.]], '''2007''', 1240 - 1241, {{DOI|10.1039/b616311d}}</ref> 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.
==References==
{{reflist}}
*''Hand book of Thin Layer Chromatography'',Sherma, J.and Fried, B. (authors) 3<sup>rd</sup> ed. Marcel Dekker, New York.
==See also==
*[[Gas chromatography]]
*[[Analytical chemistry]]
*[[Chromatography]]
[[Category:Chromatography]]
[[Category:Forensics]]
[[ar:كروموتغرافيا الطبقة الرقيقة]]
[[bs:Tankoslojna hromatografija]]
[[ca:Cromatografia de capa fina]]
[[cs:Chromatografie na tenké vrstvě]]
[[de:Dünnschichtchromatografie]]
[[es:Cromatografía en capa fina]]
[[fr:Chromatographie sur couche mince]]
[[it:Cromatografia su strato sottile]]
[[nl:Dunnelaagchromatografie]]
[[ja:薄層クロマトグラフィー]]
[[no:Tynnsjiktkromatografi]]
[[pl:TLC (chemia)]]
[[ru:Тонкослойная хроматография]]
[[sk:Chromatografia na tenkej vrstve]]
[[fi:Ohutkerroskromatografia]]
[[sv:Tunnskiktskromatografi]]
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[[zh:薄板层析]]