Irving 8. Ruppel, Jr., Frederick 1. Cuneo, and Josef G. Krause Niagara University Niagara University, New York 14109
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Column and Thin-Layer Chromatography A combined experiment for the undergraduate organic laboratory
W e have been using the product mixture obtained from the nitration of ~ h e n o lto demonstrate the techniques of column and thin-layer chromatography with excellent results. We feel the experiment has several advantages over the column chromatographic experiments usually described in undergraduate laboratory manuals and, therefore, would like to make our experience with the procedure described herein known to others. Standard nitration of phenol with concentrated nitric acid produces a mixture of o-nitrophenol, the main product, p-nitrophenol, a minor product, as well as small amounts of 2,4-dinitrophenol and 2,4,6-trinitrophenol. If the crude product mixture is applied to an alumina column and eluted with methylene chloride, individual fractions containing each of these products can be collected and identified using thin-layer chromatography. The advantages of this experiment are 1. The use of methylene chloride as the eluting solvent eliminates the fire hazard associated with use of common eluting solvents such as petroleum ether, benzene, and diethyl ether. 2. Elution time is 45 to 60 min so that the column can be set up and the operation completed well within the space of a three hour laboratory period. In fact, we find it possible to complete the column separation and run the thin-layer determinations on all fractions in one period. 3. The procedure combines two important chromatographic techniques in one experiment which can be completed in the course of three hours and illustrates one of the extremely useful applications of thin-layer, i.e., rapid analysis of column effluents. 4. Since the o-nitrophenol is intra-molecularly hydrogen bonded, it has less associ&m with the ha5ic column packing than does the p-nitrophenol. Consequently, the ortho isomer is first to he eluted from the column. This serves to illustrate important principles of organic ohemistry and of adsorption chromatography. 5 , The procedure demonstrates the separation of a relatively complex reaction mixture containing four products rather than performing an apparently simpler and less interesting (to the student at any rate) separation of an unreacted starting material from a, single product as is the case with several column chromatography experiments appearing in popular hhoratory manuals.
Customarily, we tell the student how to perform the nitration and prepare the product mixture for application to the column. He is also told to elute the column with methylene chloride and collect 10-ml fractions. Thin layer analyses can be made using silica gel-coated slides or commercially available pre-coated sheets
which have been cut into 10 em lengths. Benzene is the solvent used to develop the chromatograms, and iodine vapor is used to visualize the spots. We leave it to the student to determine the total number of products possible, order of elution from the column, and positive identification of the components in each fraction collected. Experimental Nitration Procedure
Add 3 ml of concentrated nitric acid to 7 ml of wster and cool to 5°C. Add this to 3 g of phenol in s 50.ml flask and, with stirring, maintain the temperature between 20 and 25 degrees for 15 min then between 30 and 35 degrees for 15 min by cooling in an ice bath. Add 7 ml of ice water and extract with two 10. ml portions of methylene chloride. Wash the combined organic layers with two portions of water, dry over anhydrous sodium sulfate, and evaporate the solvent on a. steam bath. Preparation of the Column
Twenty-five grams of dumina are slowly poured into s 50-ml buret or similarly sized column containing a plug of glass wool and a laver of sand and filled with methvlene chloride. Another sand is added, and the solvent run down to the head portion of the column. The entire product mixture from the previous step (dissolved in a small amount of methylene chloride) is added to the column and eluted with methylene chloride. A yellow band is clearly visible as it migrates down the column. When the first yellow drops are eluted, begin saving fractions and changing receivers every 10 ml until a total of eight to ten fractions are collected.
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Thin Layer Examination of the Column Effluent
Each fraction obtained above is spotted on silica. gel thinlayer plates (either microscope slides dipped in a silica gel G slurry-35 g silica gel in 100 ml of chloroform/methanol in the ratio 2 to 1-or pre-coated plastic sheets can be used), developed using henaene a s the solvent, and visualized in a. chamber of iodine vapor. Typical RR,values are
Typical column fraction analysis fractions 1 and 2; a-nitrophenol fraction 3; o- and p-nitrophenols fractions 4 and 5; p-nitrophenol fraction 6 ; 2,4-dinitrophenol fraction 7; 2,4-dinitra and 2,4,6trinitrophenols fraction 8; 2,4,6trinitrophenol
Volume 48, Number 9, September 1971
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