Use of Pseudophase TLC in Teaching Laboratories Daniel W. Armstrong,' Khanh H. Bui, and Regina M. Barry Georgetown University. Washington, DC 20057 The chemistry student's initial (and often only) contact with liquid chromatography involves the use of thin-layer chromatography (TLC). TLC is popular in teaching laboratories because of its simplicity, low cost, and wide applicability. Many organic laboratory courses use TLC to follow syntheses or to monitor the success of purification procedures. In addition there are some separations that are devoted exclusively to TLC (e.g., separation of dye mixtures) and not used in conjunction with other experiments. Most TLC separations necessarily utilize organic solvents or solvent mixtures. Most of these solvents are flammable, many are toxic and/or carcinogenic (benzene, for example), and almost all oresent disoosal oroblems in this safetv conscious era. These problems can hekliminated by using the recently developed' techniaue of ~seudonhaseliauid chromatoeraohv (PLC) (1-8). PLC utiiizes aqueous skfactant solutio& ( 5 6 . 4 ' ~ ) mobile phases rather than organic solvents. These solutions are biodegradable, nonflammable, and of very low toxicity. Aqueous surfactant solutions can be used to separate a variety of water insoluble or sparingly soluble compounds. This is ~ o s s i h l ebecause surfactants spontaneouslv form micelles when dissolved in water at s~~ffiriently hi& concentrations [ I , .I) Generdly, micelle formation and structure is studied only briefly in organic chemistry lecture (usually in conjunction with fats, fatty acids, and/or soaps). Using inexpensive micellar solutions in the laboratory n i t only enhances safety, but also allows the student to observe and use some of the micelle's unusual properties (e.g., soluhilization of organic compounds, apparent change of a compound's pKa, etc.) (9). There are manv reasons to increase the student's familiaritv with surfactants and micelles. They are used increasingly ih research and industry ( l o ) ,and they provide one with an excellent example of entropy in a chemical system since micelle formation is entrooicallv controlled (9.10). . . . Surfactants are not thk only substances one can dissolve in water to obtain an unusual mobile ~ h a s eAqueous . solutions of cyclodextrins can also produce varietyof unique TLC separations (6-8). Cyclodextrius are cyclic compounds composed of linked glucose units (11). Like micelles, cyclodextrins can be used to solubilize certain organic compounds in water. Because cyclodextrins have a finite diameter, only molecules of certain sizes and shapes can form inclusion complexes with them (7.8). It has been found that TLC separations with a-cyclodextrin mobile phases are unsurpassed for the separation of most ortho-, meta-, and para-substituted phenylcompounds (e.g., substituted phenols, anilines, and benzoic acids). The main shortcoming of using cyclodextrins in teaching laboratories is that they are relatively expensive (whereas surfactants are inexoensive). ' The pseudophase TLC sep&ations described below all have been developed in our laboratory. Many of the compounds separated were chosen on the basis of their current use in modem organic lahoratorv textbooks. It should be noted that PLC is ~esBeffective than traditional methods in separating some compounds (plant pigments and derivatized amino acids, for example). Experimental
a
Materials Most pseudophase TLC separations require a polyamide stationary phase. Silica-Gel" has never been used successfullysince it tends to bind the surfactant andlor cyclodextrin too tightly. Flexible, plas-
tic-backed polyamide plates can be obtained from Brinkmann or Baker. Each 20 X 20-cm plate should be cut into eight 5 X 10-emstrips (an adequate size for most laboratory separations).One can obtain 200 chromatographic TLC strips per box of TLC plates. Any jar of adequate size can he used as a development tank. Sodium dodecylsulfate(SDS) is the mast commonly used anionic surfactant in PLC. Separations vary with the purity of surfactant used. SDS from Bio Rad Laboratories has been found to give the best results. Cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC) are the most commonly used eationie surfactants. They can he purchased from Sigma, Eastman, or Fisher. a-Cyclodextrin can he purchased from Advanced Separation Tech., Inc.2 Methods When spotting the plate it is important to keep the solution as dilute as is consistent with easy visualization and the spots as small as possible. Spotting too much material in too large an area will overload the plate and result in poor resolution (i.e., streaking). Water insoluble materials are dissolved in methanol or chloroform for spotting. It is important that the spots be 1 to 11' 2 cm from the plate edge. This eliminates the "edge effect" which can result in anomalous separations.
nC Separations Utilizing Micellar Mobile Phases (1) Separation of McCormick's food colors (i.e., erythrosin B, sunset yellow, indigo carmine, tartrazine, and amaranth) Stationary Phase: polyamide Mobile Phase: 0.4 M SDS Visualization Method: compounds visible Comments: The food colors (which can be obtained in most local supermarkets)should be diluted (1 drop coloring115drops HzO) before spotting on TLC plate. The red food color contains two dyes. The green food color consists of.yellow and blue dyes. Standard dyes can be purchased from Pfaltz and Bauer. Rfs: Erythrosin B = 0.10, sunset yellow = 0.40, indigo carmine = 0.19, tartrazine = 0.50, amaranth = 0.30 (2) Separation of caffeine in coffee from impurities (e.g., chloro~hvll . . dearadation uroducts. etc.) Statiunary Phase: polvamide with tluorrzrrnt indicator Mubile I'harc: 1,d5 31 3)sur LIUS 3I CTAC
5 min, cool, and filter solution. Extract the filtered solution in a 500-ml separatnry funnel with 30 ml of chloroform. Gently swirl the heterogeneous mixture during extraction. Vigorous shaking causes emulsions ( 1 2 ) . RPs: with SDS, caffeine = 0.72, impurities = 0.02; with CTAC, caffeine= 0.75, impurities = 0.39
(3) Separation of phenol, resorcinol. and .. ~vroaallol
Stationary Phase: polyamide with fluorescent indicator Mohile Phase: 0.4 M SDS Visualization Method: fluorescence quenching Comments: Phenol can be replaced with catechol which has a similar Rf. RPs: phenol = 0.50, resorcinol = 0.23, pyrogallol = 0.36
(4) Separation of methylene hlue, bromthymol hlue, methyl orange, and fluorescein Stationary Phase: alumina or polyamide Mobile Phase: 0.05 M SDS Visualization Method: compounds visible
' Present address: Texas Tech University, Lubbock. TX 79409. 37 Leslie Ct., Box 297, Whippany. NJ 07981.
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Comments: Methylene blue can he separated from fluorescein on alumina hut not on polyamide. Rfs: on polyamide, methylene blue = 0.56, bromthymol blue = 0.47, methyl orange = 0.35, fluorescein = 0.57; onalurnina, methylene blue = 0.06, bromthymol blue = 0.47, methyl orange = 0.72, fluorescein = 0.86 (5) Separation of benzamide, @-naphthol, and biphenyl Stationary Phase: polyamide with fluorescent indicator Mobile Phase: 0.1 M SDS or 0.1 M CTAC Visualization Method: fluorescencequenching Comments: The SDS mobile phase gives better resolution. Rfs: with SDS, benzamide = 0.0, &naphthol = 0.07, biphenyl = 0.30; with CTAC, benzamide = 0.76, @-naphthol= 0.87, biphenyl = 0.61.
(6) Separation of l-naphthol, P-naphthyl benzoate, 2naphthalene sulfonic acid, and 1,4-naphthaquinone Stationary Phase: polyamide with fluorescent indicator Mobile Phase: 0.3 M SDS Visualization Method: fluorescence quenching Rfs: l-naphthol = 0.19,P-naphthylbenzoate = 0.41,2-naphthalene sulfonic acid = 0.63, l,4-naphthaquinone = 1.0.
(7) Separation of t h e polynuclear aromatic hydrocarbons: naphthalene, fluorene, anthracene, and perylene
(2) Separation of o-nitroaniline from p-nitroaniline Stationary Phase: polyamide Mobile Phase: 0.1 M u-cyclodextrin Visualization Method: compounds are visible (yellow). Rfs: o-nitroaniline = 0.13, p-nitroaniline = 0.85
(3) Separation of most ortho-, meta-, and para-substituted isomers of aniline, phenol, a n d benzoic acid (6-8) Stationary Phase: polyamide with fluorescent indicator Mobile Phase: 0.1 M 0-cyclodextrin Visualization Method: fluorescencequenching Comments: The para isomers always have the highest Rf values, while the ortho isomers have the lowest. Meta-substituted compounds have intermediate values (6-8).
Acknowledgment . Support of this work by the National Science Foundation (CHE-8119055) and Whatman Chemical S e ~ a r a t i o nInc. . is gratefully acknowledged.
Literature Cited
Stationary Phase: polyamide with fluorescent indicator Mobile Phase: 0.4 M SDS Visualization Method: fluorescence quenching RFs: naphthalene = 1.0, fluorene = 0.46, anthracene = 0.36, perylene = 0.17 TLC Separations Utilizing ff-Cyclodextrin Mobile Phases ( I ) Separation of syn-azobenzene from anti-azobenzene Stationary Phase: polyamide or polyamide with fluorescent indicator Mobile Phase: 0.1 M a-cyclodextrin
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Journal of Chemical Education
(1981).
(9) Rcsen, M. J.,"Surfaefsn~and Interfacial Phenomena: John Wiiey Br Sons,h e . , NY,
1978.
H..and Fendler, E. J.,:'Cafalysis in Mieellarand Msrromokcular Systems," Academic Press. NY, 1975. (11) Bonder,M. L., m d Komiyams, M.."CydodextrinChemistry,"Springer Verlag. Berlin, (10) Fondler, J. 1978.
(12) Mohrig, J. R., and Neckers, D. C., "Laboratory Experiments in Organic Chemistry: D.Van Nostrand and Co., NY, 1979. , .,,
