Solomon Mormor N e w Mexico Highlands University Los Vegas
Column Chromatography Experiment Using Unknowns
The separation of mixtures, especially those containing products of a reaction being studied, is a problem often encountered by the practicing chemist. Column chromatography is still a very useful separation method, despite the advent of thin-layer chromatography. Therefore, it is of value to include column chromatography techniques as part of the undergraduate laboratory program. Several of the popular laboratory manuals for organic chemistry include an exercise that deals with the principles and methods of column chromatography. The separation of the components of a dye solution, which can be followed visibly, is one such simple exercise described in some manuals,' while the separation of solid, colorless2 3 or colored3 organic compound mixtures is the subject of the exercises in others. Two laboratory texts4 have discussions of the techniques and underlying theory of column chromatography, but no specific experiments are given. Although the exercises in the references cited accomplish the purpose of demonstrating the techniques and utility of column chromatography, they are lacking in two other respects. As is the case in most such laboratory exercises the outcome is not a t all in doubt. Furthermore, the difficulties associated with the separation of a mixt,ure of colorless solids, whose exact composition is unknown-as is most often the situation in which a chemist finds himself-are omitted entirely in the dye separation exercise, and minimized in the others for which complete directions are given. An experiment that demonstrates the principles and at the same time incorporates an element of the unknown has been devised and used with considerable success. The students are assigned a problem of attempting the separation of an unknown mixture consisting of two colorless solids. To facilitate the completion of the experiment in a reasonable period of time a com~romiseis made between havine the work
' (a) ADAMB,ROGER,JOHNSON, JOHNR., AND WILCOX, CHARLES F., JR., "Laboratory Experiments in Organic Chemistry," 5th ed., The Macmillan Co., New York, 1963, p. 124. R. Q., VANDERWERF, C. A., AND MCEWEN, (b) BREWSTER, W. E., "Unitized Experiments in Organic Chemistry," 2nd ed., D. Van Nostrand Co., Princeton, 1964, p. 36. "a) FIESER,L. F., "Experiments in Organic Chemistry," 3rd ed., D. C. Heath and Co., Boston, 1957, p. 90; (b) FIESER, L. F., "Organic Experiments," D. C. Heath and Co., Boston, 1964, p. 102. ' VOGEL,A. I., "A Textbook of Practical Organic Chemistry," 3rd ed., Longmans, Green and Co., London, 1956, p. 944. H., "Laboratory Text in "a) CABON,J., AND RAPOPORT, Organic Chemistry," 2nd ed., Prentice-Hall, Englewood Cliffs, S., "Laboratory Guide for 1962, pp. 31S29; (b) MILRMOR, Organic Chemistry," D. C. Heath and Co., Boston, 1964, pp. 54-9. 272
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Iournal o f Chemical Education
become a research problem a t one extreme and being a simple cookbook procedure a t the other. The adsorbent to be used is specified, as is the solvent system, although one or both of these may not be satisfactory for every mixture. The intent of the experiment is to illustrate the method and its advantages and disadvantages, and not to effect a complete separation of the mixture. The procedure to be described is the simplest one consistent with the aims of the experiment. There are many possible variations of the theme, some of which will be suggested. The Experiment
A polyethylene or glass chromatography tube having a diameter of about 20 mm and a column length of about 180 mm is clamped to a support. If the column does not have a stopcock assembly, a short piece of clean rubber tubing with a pinchclamp or screwclamp is affixed to the narrow end of the tube. A plug of glass wool is inserted and firmly seated in the constricted portion of the tube and a layer of clean sand, about 5 mm thick, is placed over the glass wool. Approximately 25 g of adsorption alumina (e.g., Fisher Scientific Company A-540), which has been intimately mixed with two or thrre grams of a filter aid is added. The sides of the tube are lightly tapped to settle the column of adsorbent. A circle of filter paper of the same diameter as the inner part of the tube is placed on top of the adsorbent. The column of adsorbent is washed with benzene, the solvent being added continually so that the top of the column never runs dry. When the solvent meniscus comes within 1 mm of the top of the column of adsorbent a solution of 0.5 g of the unknown in 3 ml or less of benzene is added quickly. (The solution is prepared by adding 1-ml increments of benzene to the solid in a small beaker. If all the solid has not dissolved when 3 ml of solvent have been added the mixture is warmed on the steam bath.) The solution is allowed to run through the column until the liquid level is about 1 mm from the top of the packing. The elution process is begun by adding 10 ml of benzene to the column and collecting in a small beaker 10 ml of eluate. A second 10-ml portion of benzene is used to elute the second fraction, which is collected in another beaker. The third and fourth elutions are carried out with methanol. Each eluate is evaporated to dryness in the hood. The process may be speeded up by warming the solutions on the steam bath in the hood. When each residue is thoroughly dry its melting range is determined. The melting range of the original mixture is also determined.
Discussion
I n the author's course in organic chemistry the students are required to submit a written report of the experiment. On the basis of their observations they are asked to reach a conclusion concerning the effectiveness of column chromatography as a separation method. They are further required to recommend modifications of the procedure in order to effect a more complete and perfect separation. This may involve suggestions of a greater number of elutions with each solvent, use of gradient solvent systems, change of adsorbent, etc. (In several instances, some students actually carried out t,heir own recommendations, a t their own insistence, to see if they were right.) The results of a number of such experiments as carried out by students during the past three years are summarized in the table. There are various ways in which the experiment may be conducted, depending on class size and the ambition and industry of the instructor. Each student may be given a different unknown mixture, or three or four unknowns may be distributed among the students. Possibly, one group of student,^ may use one adsorbent, such as alumina, while another group tries the separation of the same unknown with silica gel. Or, the type
of solvent may be varied. Whatever the system actually employed, the experiment will he more experimental, providing an opportunity for a more realistic scientific investigation, rather than a mere exercise. Partial Separation of Mixtures bv Column Chromatoara~hv Mixture
"".
Melting range of
Components 1mnw
rni*l,,,d
1 biphenyl (69-711 aoetanilide (114) b i ~ h e n y l(69-71) benranilide (161)
Melting range of eluate resid~eo
*
1
65-102
04-69
69-70
lO%lll
111-113
2
62-151
6&70°
70-72
158-164
159-162
3 pdimethoxybensene (66) benzhnilide (161) 4 aeensphthene (95) benranilide (161) 5 p-dimethoxyben-
53-142
6
160-162
160-162
rene i,i R ~ i ~ , aoetanilide (114) 6 benropbenone (49-50) biphenyl (09-71) 7 seetanilide (114) p-dibiomobenzene (87) ~~
100-162
85-149
d
93-95
9s-125
160-161
53-93
d
53-56
53-57
114-116 d
~
4449
45-54
42-40
d
73-82
43-51.
86-89
108-109
110-112
a Mirturea were prepared by dissolvingequal weights01 t h e components in acetone and then euaooratim off the solvent.
Volume 42, Number 5, Moy 1965
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