Source: Laboratory of Dr. Yuri Bolshan — University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method used to separate mixtures of non-volatile compounds. A TLC plate consists of a thin layer of adsorbent material (the stationary phase) fixed to an appropriate solid support such as plastic, aluminum, or glass1. The sample(s) and reference compound(s) are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The TLC plate is developed by immersing the bottom edge in the developing solvent consisting of an appropriate mobile phase. Capillary action allows the mobile phase to move up the adsorbent layer. As the solvent moves up the TLC plate, it carries with it the components of each spot and separates them based on their physical interactions with the mobile and stationary phases.
Chromatography involves the separation of a mixture of compounds by distributing the components between two phases2. The stationary phase is fixed in place while the mobile phase is allowed to flow through the stationary phase, carrying the components of the mixture with it. The properties of compounds such as solubility in the mobile phase and the strength of interaction with the stationary phase affect the rate at which they are carried through the medium. Since different types of compounds have different physical properties, they are carried at different rates allowing for separation of the components of a mixture.
This video will demonstrate the separation and visualization of mixtures of compounds using the technique of one-dimensional thin layer chromatography (TLC).
1. TLC Plates
- Common adsorbents for TLC are silica gel, alumina, and cellulose. TLC plates are commercially available with a variety of properties. Choose a TLC plate and cut it down to an appropriate size (approximately 5 cm x 5 cm is sufficient for most applications). For glass-backed TLC plates, score the glass using a ruler and a glass cutter, then carefully break along the line.
- Dissolve the sample in a suitable solvent to make an approximately 1% solution. If possible the solvent should be nonpolar. Column chromatography fractions and other dilute solutions may be used without dilution if the solute is present at a concentration between 0.2% and 2.0%.
- Mark the baseline with a pencil about 1.0 cm from the bottom of the plate. Position the spots at least 1.0 cm from the edge of the plate and label them appropriately.
- Spots may be applied using a glass capillary. To spot the TLC plate, dip the capillary into the solution to draw in a small quantity of liquid. Gently touch the tip to the desired location on the TLC plate and remove it immediately.
- Alternatively, apply spots with a microliter syringe by delivering approximately 1 μL of solution for each application.
- Spots may be applied successively at each location, taking care not to disturb the surface of the adsorbent with the spotter. Allow solvent to dry in between applications.
3. Choosing a Developing Solvent
- It is preferable to use the least polar solvent possible for good separation. Common solvents for TLC include hexane, ethyl acetate, dichloromethane, and methanol (Table 1).
- A convenient way to find an appropriate mobile phase is to spot the TLC plate with a sample. Apply enough solvent directly to the spot to form a circle of solvent 1–2 cm in diameter. Mark the circumference of the circle. Upon visualization an appropriate solvent will show well-separated rings, with the outermost ring about 50% of the distance from the center to the solvent front.
- It may be necessary to adjust the polarity of the mobile phase by choosing two miscible solvents and testing them in varying proportions. Examples of common mixtures are hexanes with ethyl acetate and dichloromethane with methanol.
- Place the spotted TLC plate in a developing chamber containing the appropriate developing solvent. The solvent line should be below the baseline marked on the TLC plate.
- The developing chamber can be a jar with a lid, or a beaker covered with aluminum foil or plastic film. Use the smallest container available that will accommodate the TLC plate.
- Do not allow the solvent to reach the top edge of the plate. When the solvent front (the boundary where the wet part of the adsorbent ends) is 5–10 mm from the top the plate, remove the TLC plate from the developing chamber and mark the solvent front with a pencil before the solvent dries.
- Colored spots may be visualized immediately and marked with a pencil. Often, spots are not visible and therefore must be visualized by some other method.
- Often, the TLC adsorbent contains a fluorescent indicator. Spots may be visualized using a hand-held ultraviolet (UV) lamp. Compounds that quench fluorescence will appear as dark spots when the plate is irradiated with short-wave (254 nm) UV light. Fluorescent compounds will produce bright spots when irradiated with UV light of an appropriate wavelength. Mark the center of each spot with a pencil.
- Spots may also be visualized by applying a visualizing reagent or stain to the TLC plate. The visualizing reagent may be applied by dipping the plate into the reagent, or by wiping the plate with a cotton ball saturated with a noncorrosive reagent.
- A 20% solution of phosphomolybdic acid in ethanol is useful for the visualization of most organic compounds. The spots appear upon heating of the plate with a heat gun or in an oven.
- Other reagents may be used for visualization of specific classes of compounds. For example, ninhydrin reagent is used for amino acid visualization, and 2,4-dinitrophenylhydrazine for aldehydes and ketones.
- The retardation factor (Rf) is the ratio of the distance a compound travels up a TLC plate to the distance the solvent travels. This may be determined by measuring the distance from the baseline to the center of the spot and dividing the value by the distance from the baseline to the solvent front.
- The Rf of a compound is characteristic of its physical properties and dependent on factors such as temperature, sample size, and thickness and activity of the adsorbent, temperature, and sample size.
- To confirm that an unknown is identical to a known compound, a standard should be spotted on the same TLC plate. Identical substances will have the same characteristic Rf.
Thin layer chromatography, or TLC, is a chromatographic method used to separate mixtures of non-volatile compounds, commonly used in organic chemistry.
TLC is performed on a glass- or plastic-backed plate. A baseline is marked on the plate, along with labels. The mixture being examined and reference compounds are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The plate is placed in a jar, and a solvent (the mobile phase) separates the mixture based on physical properties of each component.
Despite the fact that more instrument-heavy separation techniques have more resolving power than TLC, it is the speed and low cost that makes TLC an attractive technique for on-the-fly qualitative analysis. This video will demonstrate the preparation, operation, and analysis of thin layer chromatography.
Chromatographic techniques include a stationary and mobile phase. In TLC, the stationary phase consists of a thin layer of material fixed to the plate. The material is a polar substance, such as silica gel. The mobile phase is a non-polar liquid that moves up the adsorbent layer by capillary action. As the mobile phase moves up the plate, it drags along the components of each spot, which are subsequently separated based on polarity.
Compounds that are less polar will spend more time in the mobile phase as it is pulled up the plate. Compounds that are more polar are more attracted to the stationary phase, and will therefore not move up the plate as far.
The separation takes place in a developing container. These can be jars with lids or beakers covered with aluminum foil. Use the smallest container available that will accommodate the TLC plate to speed up the separation.
The mobile phase, or developing solvent, should be as nonpolar as possible for a good separation. Shown here is an eluotropic series for silica gel, a list of common mobile phases in order of increasing extracting power.
A number of mobile phases can be tested simultaneously. On a clean plate, spot the dissolved sample multiple times, at least 2 cm apart. Apply enough mobile phase to each spot to form a circle 1-2 cm in diameter.
Mark the distance the mobile phase travels. If the mobile phase is not polar enough, the sample will remain close to the initial spot. If the mobile phase is too polar, all of the sample will migrate with the solvent front. An appropriate mobile phase will show well-separated rings, with the outermost ring about 50% of the distance to the solvent front.
If necessary, two miscible mobile phases can be mixed in varying proportions to obtain the desired properties. Here, a 1:1 mixture of ethyl acetate and hexane was too polar, but a 1:20 mixture was appropriately separated.
With the mobile phase chosen, you’re ready to start developing the plate.
To begin the procedure, cut a commercially available TLC plate to the desired size. If the plate has a glass backing, score it with a glass cutter and carefully break along the line.
With a pencil, mark a baseline about 1 cm from the bottom of the plate. Mark the location where the samples will be spotted along the line. Be sure the spots are at least 1 cm from the edge and 3 mm apart. Label them appropriately.
Solid samples must be dissolved in a suitable solvent. Common solvents include hexanes, ethyl acetate, or dichloromethane. Use the least polar solvent that will dissolve the sample.
Draw up the sample/solvent mixture with a glass capillary. Gently touch the tip to the desired location on the TLC plate and remove it immediately. It is important to not disturb the stationary phase.
Keep the spot as small as possible, as this leads to a better separation. If more sample is needed, spots may be applied successively at each location. Allow the solvent to dry between applications. A stream of air can be used to dry less volatile solvents.
The TLC plate is now ready to be developed. Place a piece of filter paper in the bottom of the jar to increase the vapor pressure. Add the mobile phase to a depth that does not reach the baseline. Cap the jar when not in use so the solvent vapors do not escape.
Carefully place the spotted TLC plate in the developing jar. Make sure the mobile phase is below the baseline. Watch the progress of the solvent front— the leading edge of the mobile phase— as it will move quickly up the plate.
Do not allow the mobile phase to reach the top edge of the plate, as sample bands will begin to expand via diffusion. Once the solvent front nears the top, remove the plate from the developing chamber and mark the solvent front with a pencil before the solvent dries.
If the compounds are not colored, a UV lamp can be used to visualize the spots. The compound will block the background fluorescence of the plate. Set the lamp to the short wave setting, and illuminate the dry plate. Using a pencil, outline any spots visible under the lamp. Using a pencil, outline any spots visible under the lamp.
Another possible visualization technique is to use potassium permanganate, an oxidizing agent. Using tweezers, dip the plate into the permanganate stain.
Remove and dab away excess solution with a paper towel. In a fume hood, carefully heat the plate with a heat gun to visualize the spots. Use a pencil to mark any spots that appear.
Once the spots have been visualized, the substance of interest can be compared to standards, as shown here. In this example, the unknown is 1,3-diphenylpropynone, a building block in organic synthesis. By comparing the band to a known standard and benzoyl chloride, one of the starting materials, the product can be identified.
The retardation factor, or Rf, is used to identify the unknown compound. The Rf is the ratio of the distance a compound travels up a TLC plate to the distance the mobile phase travels. The factor is determined by measuring the distance from the baseline to the spot and dividing by the distance from the baseline to the solvent front.
The Rf of a given compound is dependent on the conditions used in the experiment, including solvent choice, thickness and activity of the adsorbent, temperature, and sample size. Care should be taken to keep these factors consistent between experiments.
There are several applications of thin layer chromatography.
In this example, the triacylglyceride content of bat sebaceous glands was studied. The lipid surface fraction was first separated by polarity on a TLC plate. The triacylglyceride band was then removed from the plate with a spatula. The silica powder was transferred to a microcentrifuge tube with solvent. After centrifugation, the stationary phase was left at the bottom of the tube, while the compounds remained dissolved in the solvent. The triacylglycerides were then further separated by another physical property. In this case, the second dimension of separation was molecular size.
TLC can also be used to monitor the progression of a chemical reaction. In this example, the starting material of the reaction was used as a standard, and ran alongside the reaction solution on a TLC plate. This process was repeated at specific intervals over the course of the reaction. As the reaction progressed, the starting material band diminished and the product band enlarged. When there was no change in bands, or all of the starting material was consumed, the reaction was complete.
Finally, TLC plates can be used in bioassays. In this example, compounds were separated from red clover with TLC. Each band was then placed on bacteria growing on agar plates. Molecules that showed inhibited bacterial growth were further analyzed for their antimicrobial properties.
You’ve just watched JoVE’s introduction to thin layer chromatography. You should now understand the underlying theory of the separation, how to choose an appropriate mobile phase for your experiment, and how to set up and operate a TLC plate. Thanks for watching!
An example of a typical TLC plate is shown in Figure 1. An unknown compound 'A' may be compared to known standards 'B' through 'E'. Determination of the Rf value for each component is achieved by spotting of each respective compound, developing the TLC plate, and visualization. The Rf of unknown compound 'A' is calculated by measuring the spot height (y) and dividing by the solvent height (z). Comparing this value to the Rf determined for each of the standards allows for the identification of the unknown compound.
Figure 1. Schematic of a TLC plate. The retardation factor (Rf) should be consistent between experiments as long as the conditions are held constant.
Table 1. Eluotropic Series for Silica Gel. A list of common mobile phases in order of increasing eluting power.
Applications and Summary
TLC has a number of practical applications in the laboratory. TLC may be used to identify unknown compounds and unknown components of mixtures via comparison with standards. TLC is commonly used to monitor the course of a chemical reaction, and to assess the purity of the product through the comparison of relative amounts of reactants, products, and by-products on successive chromatograms over time. TLC can also be used to determine if a substance purified by other methods (such as recrystallization or distillation) still contains a significant amount of impurity.
When similar compounds cannot be resolved with TLC, they can be further separated based on another physical property, known as a two-dimensional separation. In one example, TLC was used to separate broad lipid classes (triglycerides, sterols, fatty acids, etc.) secreted by mammalian sebaceous glands. The different classes were then further separated by mass spectrometry3.
In microbiology, TLC is used in bioautography screenings for novel plant-based antimicrobial compounds4. Once compounds have been separated from an extract of the plant in question, the bands can be applied directly to microbial cultures. Bands that are observed to inhibit growth are then further studied as likely candidates.
TLC is a straightforward method to determine an appropriate mobile phase for separation of a mixture by column chromatography. Additionally, it is used to determine the composition of the various fractions collected during a column chromatography separation, so that fractions containing the desired compound can be identified and collected.
No conflicts of interest declared.
- Lehman, J. W. The student's lab companion: laboratory techniques for organic chemistry: standard scale and microscale. Pearson College Div, (2008).
- Pavia, D. L., Lampman, G. M., Kriz, G. S., & Engel, R. G. Microscale and Macroscale Techniques. Thomson Wadsworth, (2006).
- Pannkuk, E. L., Risch, T. S., Savary, B. J. Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry. J. Vis. Exp. (79), e50757, (2013).
- Kagan, I. A., Flythe, M. D. Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds. J. Vis. Exp. (85), e51411, (2014).