Organic Chemistry Laboratory
Thin Layer Chromatography
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.
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. |
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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 |
Figure 2.3: TLC Developing Chambers |
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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. |
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).
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.
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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 |
Figure 2.6: Developed in 75:25 |
Figure 2.7: Developed in 90:10 |
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 |
Figure 2.9: Developed in 75:25 Ethyl acetate-hexane |
Figure 2.10: Developed in 90:10 Ethyl acetate-hexane |
distance traveled by the solvent front |
Figure 2.11: Calculating retention factors (adapted from Feiser & Williamson, p. 126) |