Information about Thin Layer Chromatography
| 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 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
- determination of the pigments a plant contains
- detection of pesticides or insecticides in food
- analysing the dye composition of fibers in forensics, or
- identifying compounds present in a given substance
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
)
Chromatogram of 10 essential oils coloured with vanillin reagent.
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.
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
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.
References
1. ^ 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
2. ^ 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
2. ^ 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
- Hand book of Thin Layer Chromatography,Sherman, J.and Fried, B. (authors) 3rd ed. Marcel Dekker, New York.
See also
Chromatography (from Greek χρώμα:chroma, colour and γραφειν:"grafein" to write) is the collective term for a family of laboratory techniques for the separation of mixtures.
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Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
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SDS-PAGE, officially sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight
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Chromatography (from Greek χρώμα:chroma, colour and γραφειν:"grafein" to write) is the collective term for a family of laboratory techniques for the separation of mixtures.
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Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or, more rarely, a liquid (adsorbent), forming a molecular or atomic film (the adsorbate).
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Silica gel is a granular, porous form of silica made synthetically from sodium silicate. Despite the name, silica gel is a solid.
Silica gel is most commonly encountered in everyday life as beads packed in a semi-permeable plastic.
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Silica gel is most commonly encountered in everyday life as beads packed in a semi-permeable plastic.
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Aluminium oxide is an amphoteric oxide of aluminium with the chemical formula Al2O3. It is also commonly referred to as alumina in the mining, ceramic and materials science communities.
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Cellulose is an organic compound with the formula (C6H10O5)n. It is a structural polysaccharide derived from beta-glucose.[1][2] Cellulose is the primary structural component of green plants.
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Capillary action, capillarity, capillary motion, or wicking is the ability of a substance to draw another substance into it. The standard reference is to a tube in plants but can be seen readily with porous paper.
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Forensic science (often shortened to forensics) is the application of a broad spectrum of sciences to answer questions of interest to the legal system. This may be in relation to a crime or to a civil action.
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Silica gel is a granular, porous form of silica made synthetically from sodium silicate. Despite the name, silica gel is a solid.
Silica gel is most commonly encountered in everyday life as beads packed in a semi-permeable plastic.
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Silica gel is most commonly encountered in everyday life as beads packed in a semi-permeable plastic.
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In English, to be inert is to be in a state of doing little or nothing.
In chemistry, the term inert is used to describe something that is not chemically active. The noble gases were described as being inert because they did not react with the other elements or themselves.
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In chemistry, the term inert is used to describe something that is not chemically active. The noble gases were described as being inert because they did not react with the other elements or themselves.
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Calcium sulfate is a common laboratory and industrial chemical. In the form of γ-anhydrite (the nearly anhydrous form), it is used as a desiccant. While natural, unrefined calcium sulfate is a translucent, crystalline white rock, a form sold under the name Drierite®
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Glass is a noncrystalline material that can maintain indefinitely, if left undisturbed, its overall form and amorphous microstructure at a temperature below its glass transition temperature.
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Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary schools in ink experiments.
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chemical reaction is a process that results in the interconversion of chemical substances.[1] The substance or substances initially involved in a chemical reaction are called reactants.
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A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. The most common solvent in everyday life is water. Most other commonly-used solvents are organic (carbon-containing) chemicals. These are called organic solvents.
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Ethanol, also known as ethyl alcohol, drinking alcohol or grain alcohol, is a flammable, colorless, slightly toxic chemical compound, and is best known as the alcohol found in alcoholic beverages.
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Water is a common chemical substance that is essential to all known forms of life.[1] In typical usage, water refers only to its liquid form or state, but the substance also has a solid state, ice, and a gaseous state, water vapor.
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A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. The most common solvent in everyday life is water. Most other commonly-used solvents are organic (carbon-containing) chemicals. These are called organic solvents.
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Capillary action, capillarity, capillary motion, or wicking is the ability of a substance to draw another substance into it. The standard reference is to a tube in plants but can be seen readily with porous paper.
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Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.
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Black light (also Wood's light) is the common name for a lamp emitting electromagnetic radiation that is almost exclusively in the soft near ultraviolet range, and very little visible light.
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Iodine (IPA: /ˈaɪədaɪn, ˈaɪədɪn/, or /ˈaɪədiːn/; from Greek: iodes
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A reactant or reagent is a substance consumed during a chemical reaction.[1] Solvents and catalysts, although they are involved in the reaction, are usually not referred to as reactants.
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In chromatography, a retardation factor (Rf) (also known as retention factor) is a ratio defined as follows:
For example, if particular substance in an unknown mixture travels 2.5cm and the solvent front travels 5.0cm, the retention factor would be 0.5.
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For example, if particular substance in an unknown mixture travels 2.5cm and the solvent front travels 5.0cm, the retention factor would be 0.5.
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Organic chemistry is a specific discipline within chemistry which involves the scientific study of the structure, properties, composition, reactions, and preparation (by synthesis or by other means) of chemical compounds consisting primarily of carbon and hydrogen, which may
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capillary is used to describe any very narrow tube or channel through which a fluid can pass. See capillary action for details.
Capillaries are the smallest of a body's blood vessels, measuring 5-10 μm, which connect arterioles and venules, and are
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S. oleracea
Binomial name
Spinacia oleracea
L.
Spinach (Spinacia oleracea) is a flowering plant in the family of Amaranthaceae, native to central and southwestern Asia.
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Binomial name
Spinacia oleracea
L.
Spinach (Spinacia oleracea) is a flowering plant in the family of Amaranthaceae, native to central and southwestern Asia.
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