In the Laboratory
What Factors Affect the Separation of Substances Using Thin-Layer Chromatography?
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An Undergraduate Experiment John J. Nash,* Jeanne A. Meyer, and Barbara Everson Department of Chemistry, Purdue University, West Lafayette, IN 47907; *
[email protected] It is well known that Rf values in thin-layer chromatography (TLC) depend strongly on solvent saturation of the atmosphere above the liquid in the TLC developing chamber. Although a multitude of experiments in the literature describe the use of TLC for the analysis of a mixture, we were unable to find one in which the student discovers the importance of maintaining the solvent atmosphere during development of a TLC plate. Here, we summarize an experiment that illustrates the potentially dramatic effects on TLC Rf values that can occur if the solvent atmosphere is not equilibrated during development. The experiment can be performed in a single laboratory period and is applicable to any course in which TLC is introduced (e.g., general chemistry, organic chemistry). Equipment The following equipment is needed for this experiment: silica-gel TLC plates, 250-mL beakers, wide-mouth jars with screw-cap lids, filter paper, glass capillary tubes, heat gun, plastic wrap, and watch glasses. Chemicals The known compounds that are analyzed in the experiment are cyclohexanone, diethylmalonate, 1-hexadecene, and 1-octanol. Various mixtures of pentane and diethylether are used as eluents. The reagent (stain) that is used to visualize the TLC plates requires para-anisaldehyde, glacial acetic acid, concentrated sulfuric acid, and ethyl alcohol. Table 1. TLC R f Values Determined Using Different Eluents Eluent Composition
R f (Av ± SD, N = 10) Cyclohexanone
Diethyl Malonate
1-Hexadecene
1-Octanol
100% ether
.64 ± .02
.70 ± .02
.83 ± .02
.59 ± .03
90% ether 10% pentane
.63 ± .02
.69 ± .01
.82 ± .01
.58 ± .02
70% ether 30% pentane
.55 ± .02
.63 ± .01
.81 ± .01
.48 ± .01
50% ether 50% pentane
.48 ± .03
.55 ± .03
.78 ± .02
.34 ± .02
30% ether 70% pentane
.35 ± .02
.38 ± .02
.79 ± .02
.23 ± .02
10% ether 90% pentane
.15 ± .01
.15 ± .01
.77 ± .02
.07 ± .01
100% pentane
.01 ± .01
.01 ± .01
.68 ± .02
.00 a
NOTE: The developing chamber was a wide-mouth jar with a filterpaper wick and screw-cap lid. a Compound remained on the baseline.
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Hazards All of the chemicals and reagents used in the experiment are flammable. The TLC visualization stain is corrosive. Results and Discussion In general, the “best” eluent to use for any particular TLC analysis is not known in advance and must be determined. The first part of the experiment involves preparing seven mixtures of ether and pentane (ranging from 100% pentane to 100% ether) and then analyzing the four known compounds (cyclohexanone, diethylmalonate, 1-hexadecene and 1-octanol) by TLC using each eluent (identical developing chambers are used for these seven TLC analyses, see below). The Rf values for the four known compounds are determined from the seven TLC analyses, compared, and then used to determine the best eluent for separating these compounds. The results for the different eluents are shown in Table 1. The results show that the eluent composition has a striking effect on the R f values for the four compounds. The students are told that the best eluent is one that 1. does not allow any of the compounds to remain on the baseline, 2. does not allow any of the compounds to travel with the solvent front, and 3. provides the greatest differences in the R f values for the compounds being separated.
Based on these considerations, the students generally choose either 50% ether/50% pentane or 70% ether/30% pentane as the best eluent (either of these two is satisfactory). The students are also told that ether is a more polar solvent than pentane, and they are asked to describe how their R f values change as the relative polarity of the eluent increases or decreases. Ultimately, this leads them to a better understanding of the partitioning of a substance between the mobile phase and the stationary phase in a chromatographic separation. After the students have identified the best eluent, they study how the separation of the four compounds is affected by the type of developing chamber employed. They evaluate how the separation of the compounds is affected by 1. the type of container used (e.g., 250-mL beaker, widemouth jar), 2. the presence or absence of a filter-paper “wick” inside the container, and 3. how well the container is sealed (open, plastic wrap, watch glass, screw cap).
Journal of Chemical Education • Vol. 78 No. 3 March 2001 • JChemEd.chem.wisc.edu
In the Laboratory
Eight TLC analyses (each utilizing a different set of the variables listed above) are performed and the results are used to identify the best type of TLC developing chamber. The results for the different types of developing chambers are shown in Table 2. Clearly, the R f values are very sensitive to the type of developing chamber used. Using a filter-paper wick and sealing the developing chamber (with either a screw cap or plastic wrap) are essential for preventing 1-hexadecene from traveling with the solvent front during the development of the TLC plates. The type of container, beaker vs wide-mouth jar, does not seem to matter. Interestingly, when the developing chamber consists of a 250mL beaker, filter-paper wick, and watch-glass lid (last chamber listed in Table 2), the R f values are significantly different from those obtained from chambers with tightly sealed lids. Many undergraduate laboratory textbooks depict this very type of developing chamber when TLC is discussed. Although the experimental results indicate that using a filter paper wick and sealing the developing chamber tightly are important for the development of TLC plates, the reasons for this behavior are not immediately obvious to the students. Experience has shown that students generally conclude that “containing” the (eluent) vapor is important because this prevents the eluent from evaporating. However, they have difficulty explaining why the filter paper wick is important. At this point, a discussion of the reasons for equilibrating the eluent atmosphere is typically needed. It is noteworthy that the sensitivity to the type of developing chamber is due primarily to the volatility of ether and pentane. These solvents were chosen for this experiment not only because they nicely illustrate the importance of sealing the developing chamber, but also because they are quite effective at separating the compounds used in this experiment. Although the sensitivity to the type of developing chamber used is expected to diminish as the volatility of the eluent decreases, the experiment does serve to illustrate some of the problems that can potentially occur in a TLC analysis. Finally, the students explore the “reproducibility” of TLC R f values. Three separate TLC analyses of the known compounds are performed using the same eluent and the same type of developing chamber (the “best” eluent and developing chamber determined previously). The R f values for the four compounds for each analysis are then compared. This part of the experiment appears to be especially important because many students believe that R f values obtained from TLC are
Table 2. TLC R f Values Determined Using Different Types of Developing Chambers Developing Chamber
R f (Av ± SD, N = 10) Cyclohexanone
Diethyl Malonate
1-Hexadecene
1-Octanol
wide-mouth jar no wick no lid
1.00 ± .00
1.00 ± .00
1.00 ± .00
1.00 ± .00
wide-mouth jar no wick plastic-wrap lid
0.75 ± .02
0.82 ± .03
1.00 ± .01
0.63 ± .02
wide-mouth jar filter-paper wick plastic-wrap lid
0.49 ± .04
0.56 ± .04
0.83 ± .04
0.42 ± .04
wide-mouth jar filter-paper wick screw-cap lid
0.48 ± .03
0.55 ± .03
0.78 ± .02
0.34 ± .02
250-mL beaker no wick no lid
1.00 ± .00
1.00 ± .00
1.00 ± .00
1.00 ± .00
250-mL beaker filter-paper wick no lid
1.00 ± .00
1.00 ± .00
1.00 ± .00
1.00 ± .00
250-mL beaker filter-paper wick plastic-wrap lid
0.51 ± .04
0.58 ± .04
0.82 ± .05
0.42 ± .03
250-mL beaker filter-paper wick watch-glass lid
0.69 ± .08
0.78 ± .06
0.99 ± .03
0.53 ± .06
NOTE: The eluent was 50% ether/50% pentane.
not generally reproducible. However, as long as the eluent atmosphere in the developing chamber is equilibrated during the development of the TLC plate, the students find that R f values are quite reproducible. Acknowledgments We thank the students in Chemistry 126, Jason Milligan, and Steve Peters, for their help in the development of this experiment. W
Supplemental Material
The detailed instructions for students are available in this issue of JCE Online.
JChemEd.chem.wisc.edu • Vol. 78 No. 3 March 2001 • Journal of Chemical Education
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