The ABC's of Chromatography A Colorful Demonstration Robert C. Reynolds and Robert N. Comber Southern Research Institute, P.O. Box 55305, Birmingham, AL35255
Scientific demonstrations i n local area schools a n d tours of our laboratories are regularly used to introduce the community to our work a t Southern Research Institute. In the tour of our organic chemistry laboratories, a discussion of drug design problems and chemical reactions is coupled with a n explanation of how organic chemists use thin-layer chromatography in the laboratory for separation, characterization, and isolation of organic compounds. A colorful demonstration is used in conjunction with our discussion to demonstrate the technique of thin-layer chromatography (TLC).This demonstration is based on an earlier publication A Thin-Layer a n d Column Chromatography Experiment Adapted for Use in Secondary Schools [J. Chem. Educ. 1992,69,989-9911 and involves the separation of combinations of seven highly colored dyes by TLC to help explain separation science while capturing the attention of the audience by spelling words with these dyes. Figure 1 shows a sample alphabet created by this method. Judging by the number of requests for this particular demonstration, it has been extremely well received. The entire demonstration takes about 45 min and integrates well into our discussion of drug design and synthesis, hut would also serve a s an excellent visual program for a discussion of chromatography a t the high school level. The experiment requires small quantities of common dyes and solvents that are less toxic or flammable relative to solvents routinely used in TLC demonstrations or experiments. Experimental Procedure
Materials and Supplies Dyes: Methylene Blue, New Methylene Blue, Basic Fuehsin, Methyl Red, Rhodamine B, Bramocresol Green, Fluorescein. Caution: Rhodamine B is a suspected carcinogen. Solvents: iso-Propanal and Glacial Acetic Acid (for ehromatograms); Absolute Ethanol (for dissolving dyes) Plates: 20 x 20 cm glass plates pre-coated with silica gel 60 (0.25 mm thickness)-EMScience Chamber: Glass TLC Developing Tank (27.5 x 27.5 x 7.5 cm) Spotters: 1 pL micropipets Preparation of Dye Mixtures Table 1 lists the 25 dye solutions that would be required to obtain all the letters of the alphahet. Although this table is extensive, in practice most words used in a demoustration require a relatively small number of solutions to be freshly made. Preparation of the dye solutions is the most time-consuming part of the experiment and might take one to two hours depending on the number of solutions required. Each solution is prepared by dissolving the indicated constituent dyes i n ethanol to the concentrations listed in the dye legend. Note: A fresh or thoroughly cleaned spatula should be used when weighing each dye to avoid cross-contaminating solutions. All dyes are available from Aldrich Chemical Company and only the highest purity dyes were purchased. How-
Figure l. The entire alphabet created ustng the dye combinations described in Tables 1 and 2. Table 1. Dye Solutions Required for the Complete Alphabet
Solution 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Dyes 1 Methylene blue, 1 mglmL 2 New Methylene blue, 2 mg1mL 3 Rhodamine B, 0.5 mg1mL 4 Basic fuchsin,0.5 mg1mL 5 Methyl red, 0.5 mglmL 6 Bromocresol green, 2 mg1mL 7 Fluorescein. 2 mglmL 2,6
21 22 23 24
2.7 3.5 3,6 4.5 4.7 5,6 6.7 2-4 2,3,7 2,4,7 2,5,7 2.67 3A7 24.7 4-7 2.47
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LETTER
Spotting the Plates The quickest and most efficient method for spotting i s to construct a template that can he used over and over each time a silica plate is prepared. Each 20 x 20-cm plate can accommodate up to five letters. Although a five letter template will he described, four- and three-letter templates are similarly prepared.
lorn
Figure 2.Five-letter template (not to scale),
DEVEMP -16 MIN
Figure 3.Forming the letter "A' Table 2. Dye Solutions and Spotting Pattern Required for the Complete Alphabet
Letter
Dye Solutiona Lane 1
A B
C D
E F G
H I J
K L M N 0 P
Q R
S
T U V W X
Y
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25 25 25 25 25 25 25 25 9 17 25 25 25 25 25 25 25 25 24 7 25 23 25 20 23
Lane2 13 18 9 9 18 13 9 4 9 9 4 2 14 14 9 13 9 13 18 7 2 3 3 10 4
Lane3 13 18 9 9 18 13 9 4 25 25 10 2 4 4 9 13 17 13 18 25 2 2 4 4 16
Journal of Chemical Education
Lane4 13 18 9 11 18 13 18 4 9 7 8 2 14 3 9 13 25 21 18 7 2 3 3 10 4
~~~~
4
3
.
ever, dyes of high purity from other sources can he used. I t should be noted that for the most part these solutions can be stored for months but, depending on the source of purchase, methyl red will develop a faster moving spot on sitting. Ideally, all solutions should he freshly prepared and spotted the day of use. It is recommended that each demonstration be planned in advance so that the minimum number of solutions will need to be prepared.
Lane5 25 25 9 12 18 13 22 25 9 7 7 2 25 25 25 23 1 24 22 7 25 23 25 20 23
Preparing the Template Cut out a 2 x 20-cm piece of paper or cardboard, and along the 20-cm edge, using a pencil, mark the lo-, 48-, 86-, 124-, and 162-mm marks. These points will define the beginning (lane one) of each letter. Each letter is then completed hv consecutively adding four more spots (lanes 2-5, spaced 7 mm apart) after each initial spot, making each letter five spots (or lanes) and 28 mm in width. For example, starting a t the 10-mm mark, four additional marks would he made a t 17,24,31, and 38 mm to define the five lanes for the first letter. As described, the final template would have one-centimeter right and left m'lrgins, a one-centimeter gap Iwtween each letter, and 25 lane markers, fiw Der letter. Set! Fig. 2. Thli firrure is not drawn to scale)
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Preparing the Plate
First gently pencil-mark the 20 x 20 cm plate a t the right and left edges 1.5 cm from the bottom. Carefully draw a light pencil line across the plate connecting these points. Center the template along this line, and using the spots on the template, mark the corresponding spots on the TLC plate. Once the plate has been marked a t each position to be spotted, i t is finally spotted with the appropriate dye solutions using a separate l-pL micropipet for each solution (see Table 2 for a listing of the dye solutions used to make the complete alphabet). A micropipet is gently touched to the surface of a prepared solution, and the dye is drawn into the micropipet via capillary action. The dye-containing micropipet is then touched to the appropriate spot on the plate and the dye solution is transferred to the plate. As a n example, the initial pattern for the letter "A" would he obtained by spotting five lanes with the dye solutions 25 and 13 as shown in Figure 3. After the chromatogram has been run i t would look essentially as shown in Figure 3. A word the size of "CHEMISTRY' would take approximately 20 min to spot. Alternatively, aluminum-hacked plates can be used instead of glass. These plates offer the advantage of being unbreakable and are reasonably stable unless purposely bent. These plates can be easily cut in half, which allows the experimenter to use two 10 x 20 cm plates for separate demonstrations. Thus, these plates are more cost effective than the glass plates. In another modification, individual
the experimenter to use two 10 x 20 cm plates for separate demonstrations. Thus, these plates are more cost effective than the glass plates. In another modification, individual letters can be prepared on aluminum-backed silica gel plates available from VWR Scientific (cat no. EM 5534-3). The 5- x 20-cm plates can be cut to a 5- x 10-cm size and spotted 1.5 cm from the bottom of the plate as described above. These plates are approximately 48 mm in width. Thus, if the first spot is placed 8 mm from the left edge and the remaining four spots spaced every 8 mm, the letter will have 8 mm right and left margins. For longer words such as "CHEMISTRY', it would be more cost effective and simpler to develop individual letters in separate jars or in a large developing chamber. Thin-Layer Chromatography Experiment The developing chamber should be prepared in a hood by pouring 100 mL of glacial acetic acidliso-propanol (215, vlv) into the tank and shaking to saturate the tank atmosphere with solvent vapors. The pre-spotted plate is placed gently into the tank, spotted end down. After 4 0 4 5 min the word on the plate is readable, and the plate can be removed to a well-ventilated area to dry. After drying, the pencil marks and spotting guideline may be erased. As a teaching tool, teachers also could run a second plate with each of the pure dye solutions (or mixtures) that were used
to form the letters that are made. Students could then be asked to predict which mixtureslpure dyes had been used in each lane to produce the letter. This exercise would show them a t least one way scientists really use thin-layer chromatography. During such a discussion it is important to show the students that the relative position of each dye is consistent between plates and that each runs to about the same position when run for 4 0 4 5 min. This discussion leads naturally to the principle of Rfand how important TLC is as a characterization tool in a number of scientific fields. Discussion Our demonstration focuses on thin-layer chromatography, one of the primary tools available to an organic chemist for the isolation and purification of target compounds. Dye writing is highly recommended as a medium to help captivate the audience. As such, this demonstration is a colorful and memorable method to show what separation means to an organic chemist. This technique also can be used to help make a mundane presentation covering the fundamental concepts of thin-layer chromatography more exciting to a student audience. While we have had excellent results with the above experiment as written, we do not rule out that a number of other dyes and combinations may be used to get improved results.
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