J. M. Bohen, M. M. Joullii, and F. A. Kaplan University of Pennsylvonio Philadelphia, 19104 and B. Loev Smith Kline & French Laboratories Philadelphia, Pennsylvania 19101
"Dry-Column" Chromatography A new technique for the undergraduate laboratory
Most chemical reactions yield mixtures of products which must be resolved and analyzed. Thus, in a modern undergraduate laboratory, chromatographic techniques should be introduced early in the term. At the University of Pennsylvania, "dry-column" chromatography ( 1 4 ) has been used very successfully, in conjunction with thin-layer chromatography, to separate and identify organic compounds. These chromatographic techniques are fist iutroduced to the students in a n experiment designed t o separate,and isolate the components of a dye mixture. Later in the term, the same methods are employed in a preparative experiment t o separate ferrocene, acetylferrocene, and 1,l'-diacetylferrocene (5). Separations ohtained by "dry-column" chromatography are directly related to those obtainable on thin-layer chromatographic plates. The relationship is extremely valuable because it permits the direct transfer of conditions from a thin-layer plate to a "dry-column." In addition, the dimensions of a column required for a preparative separation can he predicted ( 1 4 ) . In contrast with liquid-filled column chromatography, "dry-column" chromatography uses a deactivated adsorbent. The 'degree of separation ohtained with such an adsorbent is, in many instances, superior to that ohtained with highly activated adsorbents. Mixtures which cannot be separated on liquid-filled columns can often he easily and rapidly resolved on "dry-columns." From the standpoint of teaching chromatographic techniques to a large number of undergraduate students (150-ZOO), "dry-column" chromatography presents several advantages over the usual liquid-filled column chromatography. In addition to its direct relationship to thin-layer chromatography, "dry-column" chromatography may be carried out in nylon columns.l These columlls are cheaper and easier to use than glass columns for large numbers of students. They are relatively inexpensive to make and require far less solvent for the development of the chromatogram. The smaller volume of solvent and the use of Nylon instead of glass provides a n added measure of safety. Since Nylon is transparent to ultraviolet light, the "dry-column" technique can readily be used with both colored and colorless materials. Finally, "dry-columns" can he prepared and developed rapidly, permitting the experiment to be completed within even very short laboratory periods. Once the resolution of a mixture has been accomplished, the components need not be eluted; they can he isolated by sectioning the column with a knife and extracting each section with a n appropriate solvent. Experimental Procedure In this experiment, thin-layer chromatography is used to determine the number of compounds in a prepared dye mixture and "dry-column" chromatography is used to separate the mixture into its components: p-dimethylaminoazobenzene(I), a yellow dye, N-(p-dimethylaminophenyl)-1,4-uaphthoquinoneimine(II), a hlue dye, and a n impurity of unknown structure, also hlue in color. A fourth component, which seems to be a decomposition
product, is occasionally observed. After the separation of the individual components, thin-layer chromatography is used again to evaluate their purity, thus ascertaining the effectiveness of the separation.
The dye mixture is prepared before the experimentz and preweighed samples are distributed. A dye solution3 is made available for the preliminary analysis of the dye mixture by thin-layer chromatography. Thin-Layer Chromatography Silica Gel slides4 are used for the thin-layer chromatography experiments. Applicators are prepared from 90-mm long melting point tubes which have been drawn out to -175 mm to provide fine capillaries. Two or three applicators can he obtained by dividing the capillary into 25 mm lengths. A capillary is filled by immersing one end of it in the dye solution. A small amount of this dye solution is then deposited on the Silica Gel slide by touching the capillary to the surface of the ad'Nylon tubing, % in. diameter, 1 in. flat diameter, was purchased from Walter Coles and Company Ltd., P.O. Box No. 42, Plastic Works, 47-49 Tanner St., London, S. E. 1.. England. (1 in. Copol 8 (yk), 160 gauge Nylon tubing). Other suppliers include Kontes, Vineland, N. J. (No. K420170) and Waters Associates, 61 Fountain St., Framingham, Mass. ZThe dye mixture was prepared by dissolving 2.5.g of finely pulverized p-dimethylaminoazohenzene (K and K Labs. ~4978) and 5 g of finely pulverized N-(p-dimethylaminopheny1)-1.4-naphthoquinaneimine (Eastman, Technical grade, T-478) in 1 I of anhydrous ether. Alumina (37.5 g) was added to the dye mixture and mine" a water the solvent was removed an a rotarv* evamrator . bath at 40" C. The mixture was dried in air, pulverized, and stored in a desiccator. Each student received 0.2 g of the dye mixture. 3The dye solution was prepared by dissolving 1 g of N - @ dimethylaminophenyl)-1,4-naphthoquinoneinand 1 g of pdimethylaminoazabenzene in 200 ml of chloroform. It was dispensed in 4 oz dropper bottles. Caution: The dye solution should not he allowed to come in contact with skin or clothes. * Eastman Silica Gel chromagram sheets (6060 or 6061) were precut into 1 x 3 in. plates and stored in a desiccator. In "drycolumn" chromatography ( 1 4 , optimum conditions for a separation are normally determined on thin-layer plates and then transferred directly to a column adsorbent of the same composition. For this experiment, however, the Eastman silica gel chromagram sheets worked very well and offered the added advantage of introducing the student to the use of a second type of chromatography adsorbent. Volume 50, Number 5, May 1973
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Figure 1. Initial preparation of the Nylon tubing.
Figure 2. A filled umn" ready for use.
Figure 3. The "dry-column" chromatogram of the dye mixture at the beginning of the development process.
"dry-coi-
sarhent about 10 mm fram the bottom of the slide. The resulting spot should he no larger than 2 mm. The spot is allowed to dry and its position is marked in pencil on the edge of the slide to permit the calculation of R, values. The slide is then developed with benzene in the usual manner ( 6 ) . A beaker which has been lined with a piece of solvent-saturated filter paper and tightly covered with aluminum foil can be used as a development chamher. The position of each component in the dye mixture is marked on the developed slide, and the corresponding Rr values are calculated.
Figure 4. The completely developed chromatogram showing the separated components.
Figure 5. The sectioned Nylon column.
"Dry-Column" Chromatography on aluminaZ is then spread euenly on the top of the column and covered with a quarter lneh layer of clean sand. The column is developed with 50 ml of dry benzene. A small volume of solvent is allowed to flow down the side of the column so that the layers of sand and dye are not disturbed. The balance of the solvent is then added, under a constant liquid head of two inches, from a clean, dry, stoppered separatory funnel (Fig. 3). When the solvent front reaches the bottom of the column, the chromatogram is completed (Fig. 4). If, at this point, the blue dyes have not been resolved completely, more solvent must be passed through the system. Under these conditions, the yellow dye may he eluted from the column. The dye components are separated from each other by seetioning the column with a sharp knife (Fig. 5). The sections between the dye components must he cut aut and discarded. This precaution will prevent contamination of the dyes whieh might result fram minor band overlap in the interfaces. The sections containing the dyes are slit open and the alumina extruded into small beakers or flasks. Diethyl ether (approximately 5-10 ml) is added to each container. The contents are swirled briefly and the alumina is allowed to settle. The solutions are. then examined by thin-layer chromatography on Silica Gel slides with benzene as the developing solvent.
The column is prepared from a 20-in. strip of Nylon tubing which has been sealed at one end. The seal is made by folding over the bottom %-in. of Nylon tubing three times and stapling it. A small pad of glass wool is inserted in the tubing and packed firmly against the seal with a long glass rod. To prevent the formation of air pockets in the column when the adsorbent is added and to permit solvent drainage, several small holes are punched in the tubing between the seal and the glass wool (Fig. 1). The Nylon creases must be straightened out as much as possible by hand if satisfactory columns are to be obtained. A 15-in. x 314 in. column requires approximately 80 g of deactivated a l ~ m i n a Far . ~ best results, the alumina must he packed as solidly as possible. Packing of the column is extremely important. If an efficient separation is to be effected, the following directions must he carefully observed. One-third of the alumina is poured into the nylon tubing with the aid of a powder funnel. The column is tapped gently on the laboratory bench to settle the alumina. The tube is then held tightly between the fingers immediately above the alumina, and the column is tapped as firmly as possible to complete the packing. One hand should be used to guide the column and prevent it from buckling. More alumina is added and the process repeated until the column is completed (Fig. 2). A pre-prepared mixture (0.2 g) consisting of the dyes adsorbed
Literature Cited (11 Loev. BandSnader, K. M., Cham. Ind.. (hndonl, 15 119651. (21 Loev, B. and Goodman. M . M., Chem. Ind, (Londonl. 202s (19671. (31 h e " . B . and Gnodman, M. M., Inlm-Science Chemistry Reports, d. 283 11970). (41 Low. B. and Gmdman. M. M.. "Proqesn in Separations and Purifications." I m r seiencePubliahers. N w York, 1970,Vol. Ill. p.73. (51 Rabem, R. M.. Gilbert. J. C., Rodowald, L. B.. and Wingrove, A. S.. "An Infro-
The alumina was purchased fram Alcoa ("activated alumina," IF-20). The activity of this alumina ranged between I1 and I11 on the Brackmann scale and no further deactivation was required. For advanced work, however, the activity of the adsorbent is critical to the success of separations and the deactivation should be done as carefully as possible (2-4). Alternatively, the commereially deactivated material could be used. Woelm alumina for "drycolumn" chromatography is available from Waters Associates.
+ 368 /Journal of Chemical Education
duction to Modern Experimental Organic Chemistry." Holf. Rimhart, end Winsfon. Ine.. New York. 1969. p. 309 16) Roberts. R. M.. Gilbert. J. C., Rodeuald, L. B., and Wingmvo, A. 8.. "An Intraduetian fo Modem Experiments1 organic Chemistry." Hdf. Rimeha*, and winston, lnc.. New York.N . Y..1969.p.62.
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