ACP Home | Organic Chemistry I Organic Chemistry II  | Lecture | Laboratory 

Organic Chemistry Laboratory
Thin Layer Chromatography


What is TLC and why is it useful?
Thin layer chromatography (TLC) is a qualitative,  experimental method that is used primarily for separating organic compounds.  There are two types of  TLC:  preparative and analytical.  Preparative TLC is used to purify organic compounds.  Analytical TLC has multiple purposes, some of which are given in Table 2.2.
 
to determine the number and/or identity of components in a mixture
to determine purity of a compound 
to determine the effectiveness of a purification or separation step
to monitor the progress of a reaction

Table 2.2:  Some Uses of Analytical TLC

Analytical TLC will be used in this experiment to determine the number and identity of components in a mixture.  In future experiments, TLC analysis will be used to determine purity, evaluate separation of mixtures, and to monitor reaction progress. While TLC is very useful for analyzing the number and identity of components in a mixture, it is a qualitative technique, not a quantitative technique, meaning the ratio of components generally cannot be determined by simple TLC analysis.



Experimental Aspects of TLC Analysis
There are five experimental features of conducting a TLC analysis; 1) the mixture to be analyzed, 2) the TLC plates, 3) the developing solvent, 4) the developing chamber, and 5) the detection method.
 
The Mixture to Be Analyzed
Regardless of the number of components in the mixture, or the states of the individual components (solid or liquid), the mixture to be analyzed must be dissolved in an organic solvent that will solubilize all components of the mixture. Suitable organic solvents are those that evaporate rapidly (low bp) such as ether, methylene chloride, chloroform, or hexane.  The specific concentration of the solution is not crucial, however it should not be too dilute.  An approximate concentration range for solutions to be analyzed by TLC is ~10mg/200ml.
Figure 2.1:  TLC Plates
The TLC Plates
TLC plates (Figure 2.1) are the surface upon which the actual analysis takes place.  There are two parts of a TLC plate:  1) the coating and 2) the backing.  The backing is either glass or plastic and serves as a support for the coating material.  The coating material covers one side of the plate and may be any one of a variety of materials used for TLC analysis.  The most common material used for TLC analysis is silica gel.  Silica gel is a highly polar, chalky material that is often embedded with a fluorescent indicator that makes viewing the result of the TLC analysis easier. (See "The Detection Methods" below).  Alumina and hydrocarbon coatings are also used.  The solution of the mixture (~2-8ml) to be analyzed is introduced to the coated side of plate using a very fine capillary tube (Figure 2.2). The coated side is chalky, while the backing side of the plate is smooth and shiny. 

Figure 2.2:  Spotting the TLC Plate

The Developing Solvent
The developing solvent in a TLC analysis is used to move the analytes introduced onto the TLC plate.  The developing solvent is generally an organic solvent or mixture of solvents.  Aqueous solvents are rarely or never used in simple TLC analysis.
 
The Developing Chamber
The developing chamber is the vessel used to carry out the analysis.  Different types of chambers are available to run the analysis (Figure 2.3).  In this experiment, a large beaker (generally 300ml or larger) and watchglass are used  as a developing chamber.  The beaker must be large enough so that the entire plate, bottom to top, fits completely inside the beaker.  The top end of the plate cannot extend over the top lip of the beaker.  The watchglass must cover the entire opening of the beaker.

The Detection Method
The detection method is used to view the components on the TLC plate after the analysis has taken place.  Most organic compounds (with the exception of dyes) are colorless and cannot be seen on the plate with the naked eye.  Therefore, special techniques have been developed to help view these colorless compounds.


Figure 2.3:  TLC Developing Chambers
Figure 2.4:  Ultraviolet Lamp
One of the most common methods used to view organic compounds on a TLC plate whose coating has been embedded with a fluorescent detector is to irradiate the plate under ultraviolet (UV) light.  A UV lamp used for TLC analysis is depicted in Figure 2.4.  Organic compounds that absorb UV radiation will appear purple, while the rest of the plate will fluoresce (yellow).  Organic compounds with conjugated pi bonds absorb UV radiation, and can be detected using this method.  It is necessary to circle the spot generated by the compound while it is under the UV light for future reference (i.e., to calculate Rf)   However not all organic compounds have conjugated pi bonds.  Therefore additional detection methods are available.

Iodine vapors are also commonly used to reversibly or irreversibly "stain" organic compounds on a TLC plate.  The iodine must be contained in a closed vessel and the plate introduced to the closed chamber.  The iodine vaporizes within the chamber and stains susceptible compounds on the TLC plate.  Compounds that contain pi systems (alkenes, carbonyls) and those that contain amine and hydroxyl functionality stain in the presence of iodine vapors.  The spots left by the compounds must also be circle for future reference because when the plate is removed from the chamber, the iodine will frequently evaporate.

Other detection methods that are specific to the compound class or functional group have also been developed.  For example, bromocresol green is a detection reagent used to visualize carboxylic acids, while ferric chloride is used to detect phenols.



Theoretical Aspects of TLC Analysis
Separation of organic components of a mixture using TLC is based on simple principles of polarity.  Generally, organic components of a mixture must have sufficiently different polarities in order to be separated efficiently by TLC.

There are two distinct components of the TLC analysis:  the stationary phase and the mobile phase.  In experimental terms, the stationary phase is the TLC plate, or more specifically, the coating on the TLC plate.  The term "stationary phase", used to describe the plate, refers to the fact that the plate does not move during the analysis, or it remains "stationary".  Silica gel is most commonly used as a stationary phase for simple TLC analysis, but numerous other stationary phases can be employed for more sophisticated experiments. The mobile phase, or the component of the analysis that moves, is the developing solvent.  The developing solvent may be a single organic solvent or a mixture of two or more organic solvents.  When binary (two solvents) or tertiary (three solvents) mixtures are used, they must be completely miscible in each other.  Usually the solvents are of different polarities.  Aqueous solvents are rarely used for simple TLC analyses.

The TLC analysis begins by applying a solution of the component mixture to the stationary phase using a capillary pipet.  Different components of the mixture will adhere to the stationary phase to different degrees depending on the relative polarity between the stationary phase and the specific component of the mixture. Polar components adhere strongly to a polar stationary phase; non-polar components adhere weakly to a polar stationary phase.  For example, silica gel, the stationary phase used in the TLC Analysis of Analgesics, is very polar.  Very polar components of the mixture adhere strongly to the silica gel, while less polar constituents have a weaker attraction.  When the plate is developed, the polar components will tend to stay at the bottom of the plate (bound to the silica gel) and the non-polar components will tend to move with the relatively less polar mobile phase (developing solvent).



How to Choose the Mobile Phase (Developing Solvent)
The purpose of the mobile phase in the TLC analysis is to move components of the mixture up the TLC plate and away from each other.  The degree to which a component of the mixture moves with the mobile phase as opposed to staying adhered to the stationary phase depends on the component's polarity relative to each of these two phases.  If  the component has a polarity more like the mobile phase, then it will dissolve in the mobile phase and move up the plate.  If the component has a polarity more like the stationary phase, it will remain adhered to the stationary phase at the bottom of the plate.  This means for TLC analyses run with silica gel as stationary phase, polar compounds will tend to stay at the bottom of the plate while non-polar compounds tend to move up on the plate.

The polarity of the stationary phase is fixed.  For example, silica gel is polar while C18 stationary phases are non-polar.  However, the polarity of the mobile phase can be adjusted if more than one solvent is used.  Binary (two solvents) or tertiary (three solvents) mixtures are usually used as a developing solvent for simple TLC analyses.  Typically the polarities of the solvents used in binary or tertiary mixtures are different.  The overall polarity of the mobile phase can then be adjusted by changing the ratio of the polar solvent relative to the non-polar solvent of the mobile phase.  Some typical solvent mixtures used as mobile phases in TLC analyses are given in Table 2.3.  The more polar solvent of each mixture is given first.
 

Ethyl Acetate-Hexane
Ether-Pentane
Acetone-Petroleum Ether
Ethanol-Chloroform
Ethanol-Chloroform-Hexane
Acetic acid-Methanol-Benzene

Table 2.3:  Solvent Combinations for Use as Mobile Phase in TLC Analysis

Determining an appropriate mobile phase to achieve maximal separation of components in a mixture is a trial and error process.  Ideally, all components of the mixture should be cleanly resolved (separated) from each other with no overlapping.  All the components should also be located in the bottom/middle two thirds of the plate after it has been developed.  The only way to find a mobile phase that will result in meeting these criteria is to try a solvent mixture of a specific ratio and see what happens.  If the desired results are not achieved, then adjust the solvent ratios.  Consider some simple scenarios for guidance in how to adjust the ratios of solvent of binary or tertiary mobile phases to get the results you want.

For example, let's say you have a mixture of three compounds, A, B, and C.  You decide to use silica gel as the stationary phase and a binary mobile phase of ethyl acetate-hexane in a 50:50 ratio.  The result of the TLC analysis looks like the illustration in Figure 2.5.  None of the components of the mixture moved, suggesting A, B and C are all very polar and adhere strongly to the polar silica gel.  A more polar solvent system is needed to move at least some of the components up the plate.  You decide to increase the ratio of ethyl acetate to hexane to 75:25 resulting in a plate that looks like the one depicted in Figure 2.6.  Two of the three components of the mixture have been resolved, but not the third. You then decide to increase the polarity of the mobile phase even more (90:10 ethyl acetate-hexane) to move the components further away from each other.  The desired result is achieved as shown in Figure 2.7.
 

Figure 2.5:  Developed in 50:50
Ethyl acetate-hexane

Figure 2.6:  Developed in 75:25
Ethyl acetate-hexane

Figure 2.7:  Developed in 90:10
Ethyl acetate-hexane

In an alternative scenario, using 50:50 ethyl acetate-hexane with silica gel, TLC analysis of a mixture of compounds X, Y and Z gave a developed TLC plate shown in Figure 2.8.   All of the compounds moved very high on the plate suggesting they are all non-polar.  It is necessary to make the components less soluble in the mobile phase.  Increasing the polarity of the mobile phase will make the components less soluble and force them to remain lower on the plate.   TLC analysis of the mixture with 75:25 ethyl acetate-hexane, then 90:10 ethyl acetate-hexane, gave the results shown in Figures 2.9 and 2.10.
 

Figure 2.8:  Developed in 50:50
Ethyl acetate-hexane


Figure 2.9:  Developed in 75:25
Ethyl acetate-hexane


Figure 2.10:  Developed in 90:10
Ethyl acetate-hexane



Calculating the Retention Factor (Rf)
The retention factor is a unitless value that is used to indicate how far a compound moves on a TLC plate.  Rf values are calculated for each spot that
appears on a  plate using the formula given below .and illustrated in Figure 2.11.  Distances are typically measured in cm, from the starting line at the bottom of the plate to the center of the spot.
 
Rf     =  distance traveled by the spot
                      distance traveled by the solvent front

Figure 2.11:  Calculating retention factors
    (adapted from Feiser & Williamson, p. 126)

References
Feiser, L.F.; Williamson, K.L. Organic Experiments, 8th Edition, Houghton Mifflin Co.: New York, 1998.
Zubrick, J.W. The organic Chem Lab Survival Manual, 4th Edition, John Wiley & Sons: New York, 1997.
Landgrebe, J.A. Theory and ractice in the Organic Chemistry Laboratory, 4th edition, Brooks/Cole Publishing Co.: Pacific Grove, CA, 1993.