Thin layer chromatography (TLC) is one of the most useful tools in phytochemistry and biotechnology for monitoring the progress of organic chemical reactions and determining the purity of organic compounds. Like all chromatographic methods, TLC uses the analyte’s different affinity with the mobile and stationary phases to separate complex mixtures of organic molecules. Chromatography refers to a group of laboratory techniques used to separate mixtures into their constituents. All types of chromatography operate on the same basic principle.
What is Thin Layer Chromatography?
TLC (thin layer chromatography) is an affinity-based method for separating compounds in a mixture. Thin-layer chromatography, like paper chromatography, is a type of planar chromatography in which the stationary phase is a finely divided sorbent spread as a thin layer on a supporting flat plastic, aluminum, or glass plate.
TLC is a versatile separation technique that is widely used for both qualitative and quantitative sample analysis. TLC can be used to analyze almost any class of substance, including lipids, nucleotides, glycosides, carbohydrates, fatty acids, alkaloids, phenols, pesticides, steroids, and glycosides. It is an analytical tool widely used because of its simplicity, relatively low cost, high sensitivity, and speed of separation.
Principle of Thin Layer Chromatography
Thin-layer chromatography is a technique that separates mixture components by differential migration through a planar bed of a stationary phase, with the mobile phase flowing by capillary forces. After the chromatography is completed, the solutes are detected in situ on the surface of the thin-layer plate using visualizing reagents. As the solid phase in thin-layer chromatography, a thin glass plate coated with either aluminum oxide or silica gel is used.
A solvent is selected for the mobile phase based on the characteristics of the mixture’s constituent parts. The principle of TLC is the distribution of a compound between a solid fixed phase applied to a glass or plastic plate and a liquid mobile phase moving over the solid phase.
Whether a compound moves up the plate or stays behind is determined by its physical properties and, as a result, by its molecular structure, particularly functional groups. The mobile phase will transport the most soluble compounds up the TLC plate. Compounds with a lower solubility in the mobile phase and a higher affinity for the particles on the TLC plate will be left behind.
Retention Factor (Rƒ)
The behavior of an individual compound in TLC is characterized by a quantity Known as Rƒ which is expressed as a decimal fraction. The Rƒ is calculated by dividing the compound’s distance from the starting point by the solvent’s distance from the starting point (the solvent front).
The following factors affect the retardation factor (Rf):
- solvent system
- absorbent (grain size, water content, thickness)
- amount of material spotted
Components of Thin Layer Chromatography
The following are some of the components involved in the TLC procedure.
TLC plates: They are used to apply a thin layer of stationary phase. They have an inert or stable nature. For better analysis, the layer of stationary phase is kept even throughout these plates. People conducting experiments usually prefer ready-to-use plates.
Mobile Phase: A solvent is used in the mobile phase (or solvent mixture). The solvent used must be chemically inert, of the highest purity, and particulate-free. The TLC spots won’t be able to develop until then.
TLC chamber: The thin layer chromatography procedure is carried out in the TLC chamber. It keeps dust particles out of the process and prevents the solvent from evaporating. A uniform environment is maintained inside this chamber in order to properly develop the spots.
Filter paper: After being moistened with the mobile phase solution, this is placed inside the chamber. It ensures that the mobile phase rises uniformly along the length of the TLC plate.
The mobile phases used in TLC are extremely diverse, and they are frequently chosen empirically. Combinations of two solvents are frequently used because the eluting power, or strength, of the solvents can be easily changed to optimize a separation by changing the solute distribution ratios. Some general guidelines for selecting and optimizing the composition of a mobile phase are as follows:
- Given that TLC is a highly sensitive analytical technique, solvents should be of the highest purity;
- In order to increase resolution, mobile phase eluting power should be changed such that solute Rf values fall between 0.2 and 0.8;
- For separations on silica gel and other polar adsorbents, solute migration rates and, consequently, their Rf values, are governed by the overall polarity of the mobile phase; Rf values will be significantly increased by small additions of a slightly polar solvent, such as diethyl ether, to a nonpolar solvent, such as methylbenzene;
- The best way to separate polar and ionic solutes is to mix a polar organic solvent, like n-butanol, with water; by adding small amounts of ethanoic acid or ammonia to the water, you can make basic and acidic solutes more soluble, respectively.
Microparticulate sorbents with particle diameters between 10 and 30 mm are the stationary phases used in TLC. In terms of band spreading (efficiency) and resolution, chromatographic performance is improved by a smaller mean particle size and a narrower size range. To create thin-layer chromatography plates, rectangular plastic, aluminum, or glass sheets are coated with adherent, uniform layers of sorbents that are about 250 mm thick. An insoluble fluorescent reagent may be included in commercially produced plates to help with the detection of solute spots. These plates are available in several sizes ranging from 5 cm to 20 cm square. Silica and powdered cellulose are the two most frequently used sorbents, and adsorption and partition are the corresponding sorption mechanisms. Thin layers can also be created using chemically altered silicas, ion-exchange resins, exclusion gels, and chiral-selective cyclodextrins.
Procedure of Thin Layer Chromatography
TLC plates are typically available commercially, with standard particle size ranges to improve reproducibility. They are made by combining an adsorbent, such as silica gel, with an inert binder, such as calcium sulfate (gypsum), and water. This mixture is applied as a thick slurry to a nonreactive carrier sheet, which is typically glass, thick aluminum foil, or plastic. The finished plate is dried and activated in an oven for thirty minutes at 110 °C. The adsorbent layer thickness is typically around 0.1- 0.25 mm for analytical purposes and around 0.5- 2.0 mm for preparative TLC.
Spotting the plate
The spotter’s thin end is immersed in the dilute solution, and the solution rises in the capillary (capillary forces). At the beginning, lightly touch the plate. Allow the solvent to evaporate before re-spotting in the same location.
You will get a concentrated and small spot this way. Avoid spotting too much material, as this will significantly reduce the quality of the separation. The spots should be sufficiently separated from the edges and from one another. If possible, mark the compound or mixture on the plate alongside the starting materials and potential intermediates.
Location of spots
Various methods can be used to determine the position of various solutes separated by TLC. Colored substances can be seen directly when viewed against a stationary phase, whereas colorless substances can only be detected by using a spraying agent that produces colored areas in the region that they occupy.
For spraying the invisible spots in TLC, the following can be used:
- Because corrosive agents are purely inorganic in nature, they can also be used to spray on the invisible spots.
- Potassium dichromate solution in concentrated sulfuric acid. Most organic compounds, particularly those used for sugars, reduce potassium dichromate (yellow) to chromic sulfate (green) during the process.
- Sulfur trioxide vapors are produced by warming fuming sulfuric acid char organic compounds,and thus making them visible as dark spots.
- A potassium permanganate solution.
- Vapors of iodine.
Saturated hydrogen sulphide solution, 0.2N aqueous ammonium sulphide, 0.1% alcoholic quercetin, 0.2% methanolic 1-(2- pyridylazo)- 2- napthol, 1% methanolic oxine, and 0.5% aqueous sodium rhodizonate are other common reagents. If the TLC plate adsorbent contains a fluorescing material, the solutes can be viewed under ultraviolet light.
The nature of the substance and the adsorbent used on the plate influence the choice of a suitable solvent. Thus a development solvent should not react chemically with the substances in the mixture under investigation. Carcinogenic solvents (benzene, for example) and environmentally hazardous solvents (dichloromethane, for example) should be avoided at all costs. Non-polar to polar solvents are used in solvent systems. Because highly polar solvents cause adsorption of any component of the solvent mixture, non-polar solvents are typically used.
Petroleum ether, carbon tetrachloride, pyridine, glycol, glycerol, diethyl ether, formamide, methanol, ethanol, acetone, and n-propanol are also other common development solvents.
Developing a Plate
A TLC plate can be developed in a beaker or closed jar.
Firstly, fill the container halfway with solvent (mobile phase). A small spot of the sample-containing solution is applied to a plate about one centimeter from the bottom.
The plate is then placed in a sealed container after being dipped in a suitable solvent such as hexane or ethyl acetate.
By capillary action, the solvent moves up the plate and meets the sample mixture, which is dissolved and thus carried up the plate by the solvent.
Because of differences in their attraction to the stationary phase and solubility in the solvent, different compounds in the sample mixture travel at different rates.
The separation of components (measured by the Rf value) can also be adjusted by changing the solvent or possibly using a mixture. The solvent level must be lower than the TLC’s starting line, or the spots will dissolve. The plate’s lower edge is then dipped in a solvent. Because of their different degrees of interaction with the matrix (stationary phase) and solubility in the developing solvent, the solvent (eluent) moves the components of the samples at different rates.
Because non-polar solvents dissolve well and do not interact with the polar stationary phase, they will force non-polar compounds to the top of the plate. Let the solvent rise up the plate until it is about 1 cm from the top. Remove the plate and immediately mark the solvent front. Allow no solvent to run off the edge of the plate. After that, allow the solvent to completely evaporate.
If the sample’s constituent parts are colored, it is possible to see them. If not, they can occasionally be seen by shining ultraviolet light on the plate or by letting the plate stand for a short period in a closed container with an iodine-vapor-rich atmosphere. Spraying the plate with a reagent that will react with one or more of the sample’s components can occasionally make the spots visible.
Applications of Thin Layer Chromatography
- Purity of any sample: TLC can be used to determine the sample’s purity. The sample and the standard or authentic sample are directly compared; if any impurities are found, they thus show up as extra spots and are easily detectable.
- Identification of compounds: Natural products like volatile oil or essential oil, fixed oil, waxes, alkaloids, glycosides, steroids, etc. can also be purified, isolated, and identified using thin layer chromatography.
- Analyzing reactions: Thin layer chromatography can be used to determine whether a reaction is complete or not. Other separation and purification processes, such as distillation and molecular distillation, are also checked using this technique.
- Biochemical analysis: TLC is incredibly useful in isolating or separating biochemical metabolites or constituents from its body fluids, such as blood plasma, serum, urine, etc.
- In chemistry: TLC methodology is increasingly used in chemistry for the separation and identification of compounds that are closely related to each other. In inorganic chemistry, it is also used to identify cations and anions.
- In the pharmaceutical industry: TLC technique has also been adopted by a number of pharmacopeia to identify impurities in pharmacopoeial chemicals.
- The TLC method has been used to qualitatively test several medications, including hypnotics, sedatives, anticonvulsant tranquilizers, antihistamines, analgesics, local anesthetics, and steroids.
- Separating multicomponent pharmaceutical formulations is therefore one of the most significant uses of TLC.
- Colors, preservatives, sweeteners, and different cosmetic products are separated and identified using the TLC method in the food as well as cosmetic industries.
Advantages of Thin Layer Chromatography
- This is a very simple method for separating the components.
- Very few types of equipment are used in comparison to other separation methods.
- Because the components elute quickly, they can be separated in a short amount of time.
- All of UV light’s constituents are easily observable.
- Some TLC plates lack lengthy stationary phases. In these circumstances, it puts a cap on how long the mixture can be separated. The separation of the mixture into individual components would be finer the longer the plate was.
- The TCL method is used to separate the non-volatile compounds.
- In TLC, only a small sample size is necessary, and it can be measured in microliters.
- A comparison with standard material allows for a preliminary identification.
- The components in the complex mixture of samples can also be easily separated and recovered by scratching the plate.
Disadvantages of Thin Layer Chromatography
- TLC results are difficult to reproduce.
- Despite its simplicity and convenience, one of TLC’s drawbacks is that it cannot distinguish between a compound’s isomeric and enantiomeric forms. This is because a molecule’s chiral pairs share the same physical characteristics.
- It is only possible to add soluble components to the mixtures.
- Only qualitative analysis is feasible; and hence quantitative analysis is not possible.
- Typically, TLC doesn’t happen automatically.
- Because TCL operates in an open system, changes in temperature and humidity can have an impact on the outcomes.
- F. Geiss (1987): Fundamentals of thin layer chromatography planar chromatography, Heidelberg, Hüthig, ISBN 3-7785-0854-7
- Elke Hahn-Deinstorp: Applied Thin-Layer Chromatography. Best Practice and Avoidance of Mistakes. Wiley-VCH, Weinheim u. a. 2000, ISBN 3-527-29839-8
- Thin Layer Chromatography: A Tool of Biotechnology for Isolation of Bioactive Compounds from Medicinal Plants. International Journal of Pharmaceutical Sciences Review and Research/ISSN 0976 – 044X
- Harry W. Lewis & Christopher J. Moody (13 Jun 1989). Experimental Organic Chemistry: Principles and Practice (Illustrated ed.). Wiley Blackwell. ISBN 9780632020171.
- A.I. Vogel; A.R. Tatchell; B.S. Furnis; A.J. Hannaford & P.W.G. Smith (1989). Vogel’s Textbook of Practical Organic Chemistry (5th ed.). ISBN 978-0-582-46236-6.