In the Laboratory
Thin-Layer Chromatography Experiments That Illustrate General Problems in Chromatography M. Lederer* and E. Leipzig-Pagani Université de Lausanne, Institut de Chimie Minérale et Analytique, Bôite Postale 115, Centre Universitaire, CH-1015, Lausanne 15, Switzerland
The first three experiments described here have been used in our institute for a practical examination for laboratory technicians. They were designed so that they can be carried out readily in a three-hour session. At the same time they illustrate a number of general principles such as pattern identification (of unknown dyes), displacement chromatography (of the constituents of mercurochrome), and salting-out adsorption (of halides in ammonium sulphate solutions). To these three we have now added an experiment of great teaching value: namely, that identification by chromatography alone is impossible. For this purpose some azo dyes are chromatographed alone and also mixed with cyclodextrins—which form inclusion compounds with them, and thus modify the Rf value depending on the cyclodextrin present in the sample. All the experiments are carried out with aqueous solvents only. This is preferable from the point of view of risks in the laboratory. It also illustrates that chromatography is still possible with quite simple means, notwithstanding the sophisticated equipment that some workers prefer.
* Corresponding author.
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Felt Tips In this experiment, dyes from 12 felt-tip pens of different colors are chromatographed (12 Visa, Conté, made in France). Small spots (Br{>I{. The Problem of Identification by Chromatography Our recent work has dealt with thin-layer chromatography on cellulose layers using cyclodextrin solutions
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Small spots (≤2 mm in diameter) of the mercurochrome solutions are placed at 1 cm intervals on a Macherey-Nagel (Düren, Germany) Cel 300 thin layer (about 8 × 11 cm), and eluted with a 1M NaCl solution. The spots produced are better visible under UV light at 366 nm (Fig. 2), and show how the different components of mercurochrome are separated by a displacement process. This happens when there is heavy loading and strong adsorption, and one substance is more strongly adsorbed than Figure 2. Thin-layer chromatograms the others, therefore ocof mercurochrome on Machereycupying the active sites Nagel Cel 300 thin layers eluted with and displacing the other 1M NaCl, as seen under UV light components forward. (366 nm).
Figure 4. Thin-layer chromatograms on Merck 5577 microcrystalline cellulose layers. Left: developed with aqueous 1% borax. Right: developed with aqueous 1% borax containing 1% α-cyclodextrin. On both chromatograms (left to right): methyl orange, methyl yellow, methyl red, ethyl orange, and ethyl red. Note that some of the compounds yield two spots.
Vol. 73 No. 10 October 1996 • Journal of Chemical Education
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In the Laboratory
Figure 5. Thin-layer chromatograms on Merck 5577 microcrystalline cellulose layers developed with aqueous 1% borax. Left to right: 2 drops of methyl orange indicator with addition of about 2 mg of αcyclodextrin, the same with addition of β-cyclodextrin, the same with addition of γ-cyclodextrin, and methyl orange without any addition.
as eluents (2). We find remarkable Rf value changes between aqueous solvents with and without addition of αcyclodextrin, as shown for example in Figure 4. Because these interactions revealed themselves as rather strong, we wanted to examine what would happen if the α-cyclodextrin were present not in the eluent, but in the sample to be analyzed. As shown in Figure 5, methyl orange is strongly linked even under these conditions to the respective cyclodextrin and thus travels with an Rf value that depends on the cyclodextrin. It may be argued that cyclodextrins are rather unusual compounds in analytical problems and thus the example is a bit farfetched. Starches, however, also form inclusion compounds with methyl orange (3); and as shown in Figure 6, the presence of a starch alters the Rf value of methyl orange. The experiment with the cyclodextrins has the advantage that the R f shifts are large and illustrate that methyl orange can have almost any Rf value, depending on the other compound(s) present in the solution to be analyzed. One to 2 mg of α-, β-, or γ-cyclodextrin is mixed on a spot plate or in a small sample tube with 2 or 3 drops of methyl orange indicator solution. Spots of about 3 mm diameter are placed on Merck microcrystalline cellulose thin layers no. 5577 and developed with aqueous 1% borax for about 15 min. The chromatogram shown in Figure 5 is obtained. The whole experiment can be performed in less than half an hour and requires no expensive equipment. Figure 6 shows another chromatogram. Here are samples of methyl orange mixed with potato starch and wheat starch, chromatographed side by side with a solution containing only methyl orange. This chromatogram also shows a measurable change, although it is not as spectacular as that shown in Figure 5.
Figure 6. Thin-layer chromatograms on Merck 5577 microcrystalline cellulose developed with aqueous 1% borax. Left to right: methyl orange indicator with addition of potato starch, methyl orange with addition of wheat starch, and pure methyl orange.
Figure 7. The equipment used in all the experiments described above. Shown here is the elution of felt-tip dyes in a glass container (16 × 12 × 6 cm), commercially available, covered with a thin glass plate.
eral chromatographic principles, and show how the experimental conditions can influence the results—in this case Rf values. These experiments can be included in a laboratory exercise framework, or can simply be used as a classroom demonstration to illustrate the most valid points about identification by chromatography, with the added advantage of being simple and inexpensive (Fig. 7). Literature Cited
Conclusion The experiments described here outline the problem of identification by chromatography, as well as some gen976
1. Schuster, G. Ann. Chim. Anal. 1946, 27, 173. 2. Huynh Thi Kieu Xuan; Lederer, M.; Leipzig-Pagani, E. J. Chromatogr. 1995, 695, 160–164. 3. Sensse, K.; Cramer, F. Chem. Ber. 1969, 102, 509.
Journal of Chemical Education • Vol. 73 No. 10 October 1996
