Applications of Thin Layer Chromatography (TLC)
- Analyzing ceramides and fatty acids
- Detection of pesticides or insecticides in food and water
- Analyzing the dye composition of fibers in forensics
- Assaying the radiochemical purity of radiopharmaceuticals
- Identification of medicinal plants and their constituents
What are the principles involved in thin layer chromatography?
Thin Layer Chromatography Principle. Thin-layer chromatography is a method of separation or identification of a mixture of components by using finely divided adsorbent Solid/ liquid over a glass plate and liquid as a mobile phase. Separation of adsorbed substances by the mobile phase. Qualitative and quantitative analysis of eluted substances. 1.
What is the principle of TLC?
Thin Layer Chromatography Principle: TLC is a separation technique where the molecules of mixtures separate using differential migration through a stationary phase, the solvent mixture flowing through the virtue of capillary forces. After chromatography is complete, solutes are detected on the surface of a TLC plate by visualizing the reagents ...
What is chromatography and what is it used for?
Chromatography is an analytical technique used to separate mixture of chemical substances into its individual compounds. Different types of chromatography are used in lab. e.g. column chromatography, thin-layer chromatography, gas chromatography etc. Chromatography consists of two phases: one mobile phase and one contiguous stationery phase.
How to analyze TLC?
always between zero and one. A TLC analysis might be summarized something like, "Using a silica gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots having Rf's of 0.12, 0.25, and 0.87". Comparing these Rf's with the Rf's of known compounds might enable a tentative identification to be made.
Where do we use thin layer chromatography?
TLC is widely used by many industries and research fields, including pharmaceuticals, clinical testing, environmental toxicology, food, water and pesticide analysis, and cosmetics. Typical applications of TLC include: Analysis of drug residues and antibiotics in food and environmental samples.
What is the clinical uses for thin layer and column chromatography?
TLC has been used to quantitatively monitor the purity of several drugs, including sedatives, antihistamines, analgesics, tranquilizers, and steroids.
What was the purpose of using thin layer chromatography in this experiment?
Thin-layer chromatography (TLC) is an extremely valuable analytical technique in the organic lab. It provides a rapid separation of compounds, and thereby gives an indication of the number and nature of the components of a mixture.
What are the most popular TLC applications?
The three largest industrial applications for thin layer chromatography are in clinical, pharmaceutical, and food testing. One of the most common specific types of clinical tests performed with TLC is for the presence of drugs of abuse.
How is TLC used in food analysis?
The TLC assays can detect compounds related to the stability of foods (antioxidant, antimicrobial, antibrowning, etc.), contaminants (antibiotics, pesticides, estrogenic compounds, etc.), and compounds that affect the absorption, metabolism or excretion of nutrients and metabolites or could improve the consumers health ...
Is TLC quantitative or qualitative?
qualitative analysisThin-layer chromatography (TLC) is a widely used method for qualitative analysis to determine the number of components in a mixture, to determine the identity of two substances, or to monitor the progress of a reaction.
How is TLC used in quantitative analysis?
The TLC chromatogram, as an image, is captured by the scanner and the image is then loaded into the Gel Works software....Data processing and treatmentThe series of spots to be manipulated are selected as a lane by lane creation function. ... The background of the TLC chromatogram, if any, is subtracted.More items...
What are the uses of column chromatography?
Column Chromatography is used to isolate active ingredients. It is very helpful in separating compound mixtures. It is used to determine drug estimation from drug formulations. It is used to remove impurities.
How is chromatography used in medical field?
Chromatography has been preferred in the medical field as one of the most important analytical methods used for the identification and quantification of a drug and its metabolites in recent years.
Why is thin layer chromatography preferred over paper chromatography?
TLC tends to produce more useful chromatograms than paper chromatography, which show greater separation of the components in the mixture - and are therefore easier to analyse. The distance a sample travels can depend on the size or the polarity of the molecules involved.
How is thin layer chromatography used in forensics?
Separation of complex mixtures (known as chromatography) is an essential tool in forensic science. It is routinely used to identify and compare samples of drugs, explosives, inks and biological samples such as saliva, urine, blood and other.
Why is TLC used in chemistry?
TLC is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation. TLC functions on the same principle as all chromatography: a compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates.
What is TLC in chemistry?
Thin layer chromatography (T LC) is a chromatographic technique used to se parate the components of a mixture using a thin stationary phase supported by an inert backing. It may be performed on the analytical scale as a means of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation. TLC functions on the same principle as all chromatography: a compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well defined, well separated spots.
What is the backing of TLC plates?
Many plates incorporate a compound which fluoresces under short-wave UV (254 nm). The backing of TLC plates is often composed of glass, aluminum, or plastic. Glass plates are chemically inert and best withstand reactive stains and heat, but are brittle and can be difficult to cut.
How to write on a TLC plate?
Cut the plate to the correct size and using a pencil (never ever use a pen), gently draw a straight line across the plate approximately 1 cm from the bottom. Do not use excessive forces when writing on a TLC plate as this will remove the stationary phase. It is important to use a pencil rather than a pen because inks commonly travel up the plate with the solvent. An example of how black ink separates is shown in the section labeled "examples".
How well does a compound like the stationary phase?
How well the compound likes the stationary phase. If the compound likes the stationary phase, it will stick to it , which will cause it to not move very far on the chromatogram.
Which solvent can be used to separate mixtures of strongly polar compounds?
A solvent that can be used for separating mixtures of strongly polar compounds is ethyl acetate : butanol : acetic acid : water, 80:10:5:5.
How to apply spot on plate?
Spots are applied to the plate using very thin glass pipettes. The capillary should be thin enough to apply a neat spot, but not so thin as to prevent the uptake of an adequate quantity of analyte. Here is a popular method of producing TLC pipettes.
What is thin layer chromatography?
Thin layer chromatography is a kind of chromatography used to separate and isolate mixtures that are non-volatile in nature. Just like other chromatography processes, this one consists of a mobile phase and a stationary phase.
Why do cosmetics use TLC?
The cosmetic industry also uses TLC for checking the presence of preservatives in the products.
What is a TLC plate?
TLC Plates: These are used for applying the thin layer of stationary phase. They are inert or stable in nature. The layer of stationary phase is kept even throughout these plates for better analysis. Usually, ready-to-use plates are preferred by the people conducting experiments.
What is the mobile phase of TLC?
This layer is applied to plastic, glass, or aluminium foil sheets called an inert substrate. The mobile phase in the TLC procedure is a solvent or a mixture of it.
What is filter paper?
Filter Paper: This gets placed inside the chamber after being moistened with the mobile phase solution. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length.
How does TLC work?
The separation principle of the TLC procedure is based on the given compound’s relative affinity towards the mobile and the stationary phase. The process begins here by moving the mobile phase over the stationary phase’s surface. During this movement, the higher affinity compounds gain less speed as compared to the lower affinity compounds. This results in their separation.
Why is relative Rf used in TLC?
Usually, relative Rf comes into use here because keeping all the TLC factors constant may not be possible. These aspects include adsorbent, temperature, adsorbent thickness, spotted material’s amount, and solvent system. The formula used for Rf value calculation is:
Why do chemists use thin layer chromatography?
Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance.
How to run thin layer chromatography?
To run a thin layer chromatography plate, the following procedure is carried out: Using a capillary tube, a small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge.
What is TLC in chemistry?
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose.
What is HPTLC in quantitative analysis?
A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This method is referred to as HPTLC, or "high-performance TLC". HPTLC typically uses thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of resolution due to diffusion .
What is the mobile phase of a TLC plate?
Because different analytes ascend the TLC plate at different rates, separation is achieved. The mobile phase has different properties from the stationary phase. For example, with silica gel, a very polar substance, non-polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase.
How are TLC plates made?
They are prepared 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. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the absorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 0.5–2.0 mm for preparative TLC.
Why is TLC used in chemical reactions?
Because of its simplicity and speed, TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.
Why is thin layer chromatography important?
In many experiments, it is important to be able to separate a mixture into its chemical components in order to isolate one compound or to assess the purity of the mixture. Thin layer chromatography (TLC) is one of the easiest and most versatile methods of doing this because of its low cost, simplici …. ...
Why is it important to separate a mixture into its chemical components?
In many experiments, it is important to be able to separate a mixture into its chemical components in order to isolate one compound or to assess the purity of the mixture. Thin layer chromatography (TLC) is one of the easiest and most versatile methods of doing this because of its low cost, simplicity, quick development time, high sensitivity, ...
How high is a thin layer chromatography plate?
Usually, a thin layer chromatography plate is around 5–7 cm high, and a line is drawn around 0.5–1.0 cm from the bottom. That is the line in which you will spot your mixtures to separate. It is important that you spot the mixtures above the solvent level on your elution chamber!
What is the purpose of chromatography?
It was a technique to separate substances that had different colors. Basically, a chromatography is any lab technique in which we separate different chemical components of a mixture by their affinity to a stationary phase (usually silica gel in TLC) and to a mobile phase (the solvent or mixture of solvents).
How to spot TLC?
Spot the TLC mixtures at the corresponding mark in the line above the bottom of the plate. Then elute the plate and see how many compounds there is in your mixture, and how polar are they, just by checking out the different spots. Try to spot your mixtures as tightly as possible. Make very small spots of sample.
What is TLC in chemistry?
If there is one technique or experiment that every chemist, or student learning chemistry should know, it is Thin Layer Chromatography (or TLC for short). If you want to become a synthetic chemist, or you are planning to ace an experimental course on organic chemistry, TLC is something you really need to master. TLC of black ink.
What happens after you run column chromatography?
But after running the column chromatography, you usually end up with dozens of tubes filled with eluent with the different compounds dissolved. Now we have to use TLC again!
What is preparative TLC?
Well, preparative TLC is just a regular thin layer chromatography separation, but with a bigger plate!
What is the stationary phase of TLC?
In TLC, we use a stationary phase (most frequently silica gel ) which is deposited over a glass or aluminum support. We then can spot mixtures of compounds over the same line. Then we elute the TLC with an organic solvent, and the different compounds will move upwards at different rates, allowing the separation of the different components.
When was thin layer chromatography first used?
Thin layer chromatography was first used by two Russian scientists N.A. Izmailov and M.S. Schreiber in 1938 for separation of plant extracts. They used a slurry medium of 2mm thickness. The plant extract was applied at the center of the layer. The separation was in ring-like shapes. This work was further reviewed in 1941 by M.O’L Crowe. The first reported TLC in analytical chemistry was improved by adding binders in sorbents by J.E. Meinhard and N.F. Hall in 1949.
What is the difference between paper chromatography and thin layer chromatography?
The difference between paper chromatography and thin layer chromatography is the type of stationary phase used. In TLC, the stationary phase is a thin layer of solid adsorbent on an inert support. The adsorbent often used is silica gel. While in paper chromatography, the water content present in cellulose or paper will act as a stationary phase.
What is adsorption chromatography?
Adsorption chromatography is a type of chromatography that involves surface adsorption. And TLC is categorized as adsorption chromatography as it uses a thin layer of adsorbent (silica).
What are the phases of TLC?
A thin layer of silica or alumina on inert support acting as a solid stationary phase. The second one is a developing solvent that acts as a mobile phase.
Why is TLC better than paper chromatography?
TLC is preferred over paper chromatography as it is time saving, more sensitive, non-destructive, and temperature resistant technique. Furthermore, TLC is more precise and accurate than paper chromatography.
Why is 2D TLC used?
Thus, a 2D TLC is used in which two mobile phases separate analyte against a stationary phase in two steps.
What temperature to use for activation of TLC plate?
When we intend to remove the water content or moisture from the TLC plate, silica gel is heated at 100 -110°C for activation. Silica gel is preferred as it is stable at high temperatures and does not interact with any mobile phase.
What is the most common mode of chromatography used in TLC?
TLC conforms to the basic principles of liquid chromatography. The most common mode of chromatography used in TLC is adsorption chromatography , although other modes of chromatography can be employed. With silica, celite, keiselghur, and cellulose, the mechanism is by adsorption chromatography if the adsorbent on the plate is completely free of water, and the solvent system is a nonpolar mixture. However, if water is present on the adsorbent or if the mobile phase contains a highly polar solvent, then separation will be by partition chromatography. In addition, reverse-phase TLC plates also separate by partition chromatography. TLC plates are also available precoated with reverse-phase silica gel that has been impregnated with a chiral reagent and copper ions. These can be used to separate optically active isomers, e.g., amino acids, by chiral chromatography on the basis of ligand exchange.
What is the TLC method used for?
However, this method involves radioactive isotope labeling and the analytical procedure is relatively tedious. TLC method is more commonly used for the qualitative study due to its limited accuracy.
What type of TLC is used for gastrointestinal toxicants?
Another type of TLC that has been used for the detection of gastrointestinal toxicants is high-performance thin-layer chromatography ( HPTLC). This technique is similar to TLC; however, with the advancements in stationary phases and other detection methods, this technique offers more advantages over traditional TLC.
Why is TLC important?
Several samples can be analyzed simultaneously and it provides the possibility of a wide variety of detection methods for the separated components either by using UV or chemical reactions resulting in coloured spots. Therefore, TLC procedures are important especially in resource-limited countries or laboratories. A number of further instrumental developments have been made to improve the sample application on the plates and in applying controlled and pressurized mobile-phase flow. The detection and further analysis of the separated components can be carried out using sophisticated instrumentation such as mass spectrometry and infrared spectroscopy as mentioned in this chapter.
How is TLC used?
TLC involves the use of a stationary phase that is coated onto a plate of glass or plastic. The samples are spotted onto the stationary adsorbent on the plate and placed in a chamber to develop in the presence of a mobile phase. The mobile phase is usually a solvent or mixture of solvents. The sample travels across the plate based on the chemical and physical properties of the analytes present in the sample. These analytes are detected and identified based on their relative migration distance through the stationary phase and based on their color reaction with chromogenic derivatization spray reagents or their ability to absorb ultraviolet (UV) light (Touchstone and Dobbins 1978 ).
How to do preparative TLC?
The steps involved in preparative TLC are (1) application of the sample onto the plates, (2) development of the plates, (3) detection and location of the compound of interest, and (4) extraction of the compound of interest. Detection is usually by ultraviolet light. When the separated compound of interest is located on the plate, the band is scraped and the impurity is extracted from the stationary phase (i.e., silica gel) with an appropriate solvent. The extracted material is filtered or centrifuged, and the solvent collected is evaporated to yield the isolated material. It is essential to remove silica gel and other interferent that may inhibit the identification of the compound. The isolated material is submitted for LC–MS analysis.
What is the role of TLC in drug development?
TLC plays a crucial role in the early stage of drug development when information about the impurities and degradation products in drug substance and drug product is inadequate.
What is the principle of thin layer chromatography?
Thin Layer Chromatography is a separation process based on the principle of adsorption in which the components of sample separate based on their affinity towards the stationary and mobile phase where the stationary phase is a thin layer of adsorbent material such as Aluminium oxid e, Cellulose, or Silica gel while the mobile phase is the solvent.
How to visualize a chromatogram?
Then the developed chromatogram is visualized using UV light or visualizing agents such as KMnO4 stain, Dragendorff’s reagent, etc.
How to make a TLC plate?
To prepare a TLC plate Silica gel is mixed with Gypsum and water to make a thick slurry. Then apply the slurry on a clean glass plate uniformly. Uneven thickness of slurry on the plate can alter the results. After evenly placing the slurry on the place air-dry it .
What is TLC in chemistry?
Thin Layer Chromatography (TLC) is a separation technique used to separate the components of non-volatile mixtures. TLC is significantly a better separation process than Paper chromatography.
How far should you draw a baseline from a TLC plate?
Draw a baseline above 4 cm from one end of a TLC plate. Spot the sample on that baseline at a fair distance such that during the development of the sample, the components of one sample don’t mix with the other.
Why is visualizing agents such as corrosive reagents useful?
Visualizing agents such as corrosive reagents can help in better detection of components.
Is it possible to use TLC for a low detection limit?
The detection limit is too high. Therefore, using TLC for a low detection limit is not possible. Separation of only soluble components of mixtures is possible.
Overview
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin layer chromatography (TLC) is a chromatographic technique used to separate the components of a mixture using a thin stationary phase supported by an inert backing. It may be performed on the analytical scale as a means of monitoring the progress of a r…
Plate preparation
TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared 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. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the absor…
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. Plates can be labeled before or after the chromatography process using a pencil or other im…
Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
• Fluorescent analytes, like quinine, may be detected under blacklight (366 nm)
• 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 UV-C light (254 nm). The adsorbent layer will thus fluoresce light-green by itself, but spots of analyte quench this fluorescence.
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 cross-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 the starting material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this mi…
See also
• Radial chromatography
Bibliography
• F. Geiss (1987): Fundamentals of thin layer chromatography planar chromatography, Heidelberg, Hüthig, ISBN 3-7785-0854-7
• 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, ISBN 0-8247-8335-2.