S. Naff College Carlisle, Pennsylvania
M. Benton Naff and Anna Dickinson
TLC on Microscope Slides An organic chemistry experiment
Tllin layer chromatography (TIC), a micro method of adsorption chromatography, possesses distinct, advantages over paper chromatography. Specifically, the separations are faster and the developed spots are sharper. With inorganic adsorbents it is possihle to use detecting reagents which are destructive to paper, e.g., cone. sulfuric acid, basic permanganate, etc.' The following experiment illustrates the general utility of thin layer chromatography as a method of separation; all techniques commonly employed are introduced.
Figure 1.
Applicator and opplicotor boord.
The labomtory work consists of three short experiments: (1) Separation of three mixtures of dyes, the con~ponentsof which a.re known, and an unknown dye mixture to identify: (2) Separation of a known mixture of seven phenols and an unknown phenol to identify; and (3) Separation of dyes and impurities present in each of three fluorescein dyes. It is advantageous to show a t least one of the following films in the intrn-
' STARL, E., "Dunnschicht-Chromatographie," Springer, Berlin, 1962; English edition, Academic Press, in press.
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lournol of Chemical Education
duction of thin layer chromatography to a class: "Thin Layer Chromatography" produced by E. H. Ahrens, Jr., Rockefeller Institute. Playing time: 10 minutes. "Thin Layer Chromatography" produced by 0. S. Privett, Hormel Institute. Playing time: 45 minutes. This film requires z Bell and Howell 302 Magnetic Sound Projector.
The essential pieces of equipment are an applicator and applicator board (see Fig. I), a plastic ruling bar for channeling the plates, Peerless wooden applicator sticks2 which are used as a substitute for micropipets, a 4 ox. developing jara with a plastic cap, an all-glass spray bottle that delivers a fine mist, a short-wave ultraviolet light, and microscope slides. The applicator may be fashioned from a 3 in. length of 1 X 1-in. aluminum or plastic bar. The front edge is undercut 0.25 mni and machined uniformly. Plexiglas sheet, thick, is satisfactory for the construction of an applicator board. All the chemicals were Eastman white label except as noted. The dyes and phenols are listed i11 the table. In the preparation of solutions methanol was used as the solvent. The known phenol solntion4 contained 3% of each of the seven different phenols; and the unknown solution, 1% of the phenol. All other solutions mere 0.1% with respect t o each dye, except rhodamine B, which was 0.0.5s and Budan IV, which was 0.03%. It was necessary to dissolveSudan IV in hot methanol. The Experiment
Eleven slides were aligned on the applicator board. G A N I ~F., M., A N D STOTZ,E. H., ACS Ab~tmcta,Sept,, 1962, p. 13F. B T h e jar measured 2'/,in. diameter x 3'/rin. high, No. 10375, Central Scientific, Chicago, Illinois. Stable for 24 hrs.
Results of Se~arationson Silica Gel Coated Microsco~e Slides
Color Known mixture I Fluorescein Rhodamine Ba Malachite green Known mixture I1 Sudan yelloe Crystal violets Methylene blueb Known mixture 111 Sudan IVE o-Cresolsulphanephthalein Victoria. blue
Direct light R,
Rr
yellow red green
0.85 0.43 0.12
orange violet blue
0.83 0.20 0.02
UV light Rr
red-brown 0 .83 yellow blue-gray
0.65 0.30 0.00 Developing solvent: methyl ethyl ketone, acetic acid, isopropyl alcohol 2:2: 1 Develo~ine:time: 20 min
Phenols o-Nitrophenol 0.73 o-Phenylphenol 0.60 m-Cresol 0.53 m-Nitrophenol 0.45 o-Hydroxyhenzaldehyde 0.30 Resorcinold 0.23 Phloroglucinold 0.07 Developing solvent: toluene, dioxane 50:8 Develnnins time: 10 rnin Fluorescein dyes Fluoresceind 0.27 (yellow) Dichlorofluarescein 0.50 (orange) 0.75 (pink)
n
fi7
(pink) Developing solvent: toluene, acetic acid 65:35 ~evelo$ng time: 10 min
0.47 0.27 0.00 0.67 0.50 0.40 0.26 0.75 0.67
n fin 0.27
*National Aniline Division of Allied Chemical, New York, N. Y. b 5. T. Baker Chemical Co., PhilIipsburg, N. 3. Fiaher Scientific Co., Pittsburgh, Penna. * Eastman Kodak,,practical, Rochester, N. Y. Hartman-Leddon Co., Philadelphia, Penna.
Five ml of water and 2.5 g Brockmann silica gel G6 were mixed thoroughly. This mixture was applied to the slides with the applicat,or. The coated slides were activated for 10 min a t 105". To mark the solvent front, a heavy line was drawn cm from the top of the plate; 6 cm below this line the starting points were marked lightly and spaced according to the number of spots to be developed on the slide. By channeling the coated slides, it was possible to separate four different mixtures on one slide. Four ml of the developing solvent were pIaced in the jar. The composition of the solvent systems is listed in Table 1. To saturate the atmosphere in the container, the jar was capped and swirled vigorously for approximately 20 sec. The spotted plate was lowered carefully into the solvent and the jar capped. When the solvent reached the 6 rm mark, the plate was removed from the container and air-dried a t least 10 Brinkmanu Instmmenta Inc., Great Neck, New York.
min. The intrinsic color of the dyes makes the developed spots visible. After developing and airdrying, the phenol plate mas sprayed lightly with 0.1% solution of 2'7'-dichlorofluorescein in methanol.' Under ultraviolet light the phenols appeared as purple spots on a lavender or fluorescent yellow background. The developed fluorescein dye plate was dried 10 min a t 105O then viewed in direct light and under ultraviolet light. Heating the developed plate intensified the fluorescence of the spots. The distance traveled by each component was measured and the Rr value calculated. The Rr values determined for the dyes, phenols, and fluoresceins are summarized in Table 1. A tracing of each separation is shown in Figure 2. The dichlorofluorescein and tetrabromofluorescein dyes each contain impurities which probably are nnreacted fluorescein and the corresponding mono-, di-, tri-, or tetrahalogenated fluorescein. Separations on these coated slides are characterized by the same degree 01 sharpness obtained with larger chromatoplates. Since the Rr values determined by thin layer chromatography are not entirely reproducible, it is essential to run a known mixture and an unknown on the same plate. Saturation of the atmosphere in the developing chamber, prior to developing the plate, reduces the developing time and improves the quality of the spots.
Figure 2. Tracing of developed shramotoplates Dyer, IAI, known mixtures 1, 2. 3, and u n k n o w d r o m bottom to top the colors are: blue, violet, yellow, and orange. The components of the unknown ore: methylene blue, crystal violet, o-cresol%ulphonephthaIein, ond Sudan yellow, Phenols, IN, known mixture and unknown-phloroglusinol. Fluoretcein dyer, ICI, 11) Fluorescein, (21 Dichlorofluorercein, (31 Tetrabromofluorescein IEorin Y1. The shaded spots ore risible in dired light while the cirdes reprerent spots visible only under ultraviolet light.
I n practice thin layer chromatography on coated microscope slides is a rapid method for following the course and, qualitatively, the rate of a number of organic reactions, e.g., esterification of an alcohol, ammonolysis of an ester, and nitration of aromatic compounds. This technique is especially useful in determining optimum coqditions for separating compounds by column ~hromatography.~~~ A solution of 0.1'% 2'7'-dichlorofluorescein in methanol is a general reagent for detecting aromatic comuounds. A commercial aerosol container of the reagent was unsatiafactory, became the spray was too coarse. ' DUNCAN,G. EL., J . Chromatog., 8.37 (1962). 8 MILLER, J. M., AND KIRCHNER, J. G., Anal. Chem., 24, 1480 (1952). Volume 40, Number 70, October 1963
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