In the Laboratory
Thin-Layer Chromatography: Four Simple Activities for Undergraduate Students Jamil Anwar*, Saeed Ahmad Nagra, and Mehnaz Nagi Institute of Chemistry, University of the Punjab, Lahore-54590, Pakistan Thin-layer chromatography (TLC) is a very simple and relatively rapid technique for the separation and, sometimes, characterization of chemical compounds in mixtures. The factors responsible for its worldwide popularity include high sensitivity, low cost, and wide range of applications. Although a large variety of TLC experiments is described in undergraduate texts and in the chemical literature (1–4), the need for chromatographic tanks, glass plates, and mechanical applicators makes the technique expensive and a little difficult to practice, especially in high schools and colleges of developing countries. A limited period of time, usually 50 minutes, for laboratory work in such institutions is another factor responsible for not introducing TLC experiments at the undergraduate level. This work was started to simplify conventional TLC exercises so that this important analytical technique could be introduced at the undergraduate level in relatively less developed countries. The major object was to develop TLC experiments that could easily be performed with very simple and commonly available apparatus in high schools and colleges. These exercises are more rapid than conventional ascending TLC and can easily be performed within the laboratory hour. Adsorbent, Sample, and Mobile Phase A single set of adsorbent, samples, and mobile phase was used throughout the work. However, the experiments can also be performed with a different set of materials available in one’s laboratory. Silica Gel 60 HF254 from Merck was used as the TLC adsorbent. The slurry was prepared by mixing 30 g of the adsorbent in 100 mL of water. Five percent starch, also from Merck, was added as binder to the aqueous suspension. The mixture was thoroughly stirred before use. A four-component mixture of red, blue, green, and yellow inks of “Pelikan 4001” was used as the sample in each experiment. The solvent mixture, used as the mobile phase, contained n-butanol, ethanol, and water in a 3:1:1 ratio. TLC with a Test Tube This is probably the most simple form of TLC that can be performed in a school or college lab. It requires a glass test tube, petri dish and a large beaker, items which are usually readily available.
Procedure A clean glass test tube was coated (outside only) by dipping it into the adsorbent slurry, drying it in air, and activating it in an oven for 30 minutes. At least four sample spots were applied on the circumference of the tube about one-half inch from the open end, using a fine *Corresponding author.
Figure 1. TLC with a test tube.
glass capillary. Then the tube was inverted and placed vertically in a petri dish containing the solvent mixture to about half of its depth. The petri dish with tube was then covered with a large beaker as shown in Figure 1. After 40 to 50 minutes, the tube was removed, dried in air, and Rf values were calculated. TLC with a Capillary Feeder In circular paper chromatography, a cotton wick is usually used to feed the solvent to the paper. A cotton wick has also been employed as a solvent carrier in some TLC procedures (5, 6). The use of a wick can, however, cause certain problems. First, the fixation of a cotton wick to the paper or to a glass plate is a difficult task for students. Second, the shape and geometry of the wick are never reproducible. Third, an unsymmetrical wick does not provide a uniform circular flow of solvent and the wick may fall from the paper or plate due to becoming heavy with solvent. In this work a thick-walled glass capillary was used instead of a cotton wick. The optimum length of the capillary made the flow more rapid and uniform.
Apparatus and Procedure A small hole (about 1 mm) was made in the center of an ordinary 6 × 6-inch TLC glass plate. A thick-walled capillary tube of one-half inch length was permanently fused (preferably by glass blowing) to the hole in the center of the plate. The clear side of the plate was coated with the adsorbent by pouring and smearing the slurry with a glass rod. It was dried and activated. Four sample
Vol. 73 No. 10 October 1996 • Journal of Chemical Education
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In the Laboratory
spots were applied in a circle of one-inch diameter around the central hole of the plate. The plate was placed on a small petri dish (3- to 4inch diameter). The plate and dish were then covered with a large petri dish. The solvent was allowed to flow uniformly along all sides of the plate. After adequate development, the plate was removed and dried, and R f values were determined. A sketch of the apparatus used and the separation obtained are shown in Figures 2 and 3, respectively.
Figure 2. TLC with a capillary feeder.
TLC with a Burette This exercise requires only a TLC glass plate and a burette. Separation of mixture components is more rapidly achieved than is possible with conventional ascending TLC.
Procedure Sample spots were applied around an imaginary circle of one-inch diameter in the center of a 6 × 6-inch glass plate, previously coated and activated. The solvent was placed in 10-mL burette, which was clamped so that its tip was 1 mm above the plate. The flow of the solvent was controlled so that a continuous thin stream of solvent developed between the burette tip and the plate surface. The solvent spread uniformly over the plate and separated the mixture components. After adequate development the solvent flow was stopped, the plate was air-dried, and Rf values were computed.
Figure 3. Resolution of dyes with a capillary feeder.
TLC with a Rotating Plate To further reduce resolution time, the coated glass plate was rotated using an electric motor as the solvent was applied to the center of the plate from a burette. Because of the double effects of solvent flow under gravity and the fast development due to centrifugal force, mixture components could be resolved even more rapidly.
Apparatus and Procedure A 1/2-hp electric motor of adjustable rpm was fixed in a wooden box with its shaft protruding. A 6 × 6-inch plastic plate 2 mm thick was permanently screwed on the shaft. A 6 × 6-inch TLC glass plate coated with adsorbent was attached to the plastic plate using two metal clips on each side. The solvent was continuously added to the middle of the rotating plate with a burette (as in the previous procedure). The apparatus and the separation obtained are shown in Figures 4 and 5, respectively. The speed of the motor was controlled with a variable resister. The optimum motor speed (around 300 rpm) was determined by repeated experiments. A very high motor speed (>400 rpm) caused smearing of the sample and tunneling of the mobile phase, whereas a low speed (< 200 rpm) caused a significant delay in resolving of the sample.
Figure 4. Apparatus setup for rotating the TLC plate.
Discussion Data from the different experiments performed in this work are summarized in Table 1. For the purpose of comparison, the results obtained from identical experiments by conventional TLC on a glass plate are also reported. Virtually identical results were obtained by conventional TLC and the coated test tube procedure. Both involved ascending development on the same adsorbent with the same solvent mixture; hence very little differ978
Figure 5. Resolution obtained by rotating TLC plate.
Journal of Chemical Education • Vol. 73 No. 10 October 1996
In the Laboratory
Table 1. Comparison of the Data Obtained for Various TLC Experiments. Technique
Separation Time (min)
Rf Values for Different Dyes (Mean ± SD) Yellow
Blue
Green
Red
Common plate TLC
20
0.64 ± 0.06
0.54 ± 0.05
0.36 ± 0.05
0.32 ± 0.05
TLC with test tube
20
0.66 ± 0.05
0.52 ± 0.05
0.38 ± 0.05
0.30 ± 0.05
TLC with glass capillary
16
0.64 ± 0.04
0.48 ± 0.03
0.34 ± 0.03
0.28 ± 0.04
TLC with burette
12
0.62 ± 0.05
0.46 ± 0.04
0.36 ± 0.04
0.30 ± 0.04
5
0.68 ± 0.06
0.54 ± 0.06
0.40 ± 0.06
0.32 ± 0.06
TLC with revolving plate
ence in the R f values or separation time was expected. The major attraction of the test tube procedure is that it uses simple glass test tubes coated merely by dipping into the adsorbent slurry. The procedure that utilized a capillary feeder fused onto a glass plate allowed satisfactory component separation in significantly less time than the ascending methods. The reproducibility was also excellent. As shown in Table 1, in the case of the capillary feeder, minimum deviations were obtained in the Rf values of the dyes. Component resolution time was further reduced when a burette was used to disperse the developing solvent. The deviations found in R f values were slightly less than those for either ascending TLC procedure. The shortest separation time was noted for the procedure in which the plate was rotated by an electric motor. The deviations in Rf values were a little higher than those of the other procedures, but short separation times (3–5 minutes) make this technique worth employing in certain cases. Advantages • Generally all the experiments described in this
• • •
•
work are simple and easy to perform in a high school or college laboratory. The apparatus used in these experiments is relatively inexpensive and usually available. Most of these experiments are significantly faster than the classical TLC. Compared to classical TLC, smaller quantities of developing solvent were required, thus causing few problems with respect to pollution and ventilation. Reproducibility in certain cases, such as TLC with a capillary feeder, was significantly better than in normal ascending TLC.
Literature Cited 1. Moore, J. A.; Dalrymple, D. L. Experimental Methods in Organic Chemistry; W. B. Saunders: Philadelphia, 1971; pp 51–64. 2. Scism, A. J. J. Chem. Educ. 1985, 62, 361. 3. McKone, H. T.; Nelson, G. J. J. Chem. Educ. 1976, 53, 722. 4. Freese, J. M.; Oleson, B.; Pinnick, H. R., Jr.; Useted, J. T. J. Chem. Educ. 1977, 54, 684. 5. Hashmi, M. H.; Shahid, M. A.; Ayaz, A. A. Talanta 1965, 12, 713. 6. Tubaro, E.; Rustici, E. L. Boll. Chim. Farm. 1964, 103, 205.
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