In the Classroom edited by
JCE DigiDemos: Tested Demonstrations
Ed Vitz
Kutztown University Kutztown, PA 19530
Colorful Column Chromatography: A Classroom Demonstration of a Three-Component Separation submitted by:
checked by:
Daniel E. Blanchard Department of Physical Sciences, Kutztown University, Kutztown, PA 19530
J. Chem. Educ. 2008.85:524. Downloaded from pubs.acs.org by MOUNT ALLISON UNIV on 01/02/19. For personal use only.
Lars V. Heumann Department of Chemistry, University of Utah, Salt Lake City, UT 84112-0850;
[email protected] Silica gel chromatography is a reliable and commonly employed method for the separation of compounds from mixtures and the purification of individual substances. A mixture of compounds is loaded onto a stationary phase of powdered silica gel and eluted with a solvent or mixture of different solvents. Silica gel for chromatographic applications generally consists of porous, finely powdered sodium silicate with particle sizes ranging between 40 and 200 μm (1). The small particle size provides a large exterior area (540–640 m2/g) with a polar surface owing to the presence of silicon oxides (Figure 1) (2). During the elution process, the solute is continuously partitioned between the stationary and mobile phases, and as a result of different interactions of the individual constituents with the mobile and stationary phases, the chemical entities move at different rates through the column. A separation can be achieved if the eluant is collected as separate fractions. Thin-layer chromatography (TLC) can be used to analyze each collected portion for its content (3, 4). In addition, initial TLC analysis is generally used as a tool to gain information about the polarity and separation of the substances, and some rules have been established for applying the knowledge gained from TLC analysis to column chromatography (4). In an advanced organic laboratory experiment, students were given the task of purifying and identifying the colorless components of a binary mixture of unknown chemicals. To aid the students, the steps involved in separating a mixture of
Na O O
Si
Na O O Si
Na O O
Si
O
ONa
O Si ONa ONa Si O O O ONa O Si Si O ONa ONa O O O Si Si ONa O ONa Si O O Si O O Si O O O Si O O O ONa Si O O Si ONa O Si Figure 1. Simplified and enlarged, partial cross section of a silica gel particle including the surface and interior (1).
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organic compounds by silica gel column chromatography were demonstrated during a lecture. Only the equipment and techniques available in the undergraduate laboratory were used. The mixtures of unknowns were designed, in general, to be separable by silica gel column chromatography with solvent systems consisting of EtOAc/hexanes, Et2O/hexanes, or acetone/hexanes (3, 5). Some of the mixtures, however, also required the use of a higher polarity solvent such as MeOH to elute a more polar constituent such as a carboxylic acid, amide, or amine. These mixtures of unknown compounds were intended to resemble purification challenges that often arise in organic synthetic research laboratories when one or several components need to be separated and obtained in pure form. With this in mind a demonstration using colored compounds was developed so that the progress of the separation could easily be followed by the audience. A mixture of ferrocene and acetylferrocene, after a literature search (6, 7), and bromocresol green, after a number of trial chromatographies, were determined to have the right properties for the lecture demonstration (Figure 2). It should be emphasized, that bromocresol green was the only suitable dye of the more than thirty in stock to serve as the highly polar component of the ternary mixture. Ferrocene and acetylferrocene can be separated using EtOAc/hexanes and pass through the column as yellow and orange bands, respectively (Figure 3). Once the first two compounds have been collected, bromocresol green can be eluted with MeOH as a dark blue band. Test tubes were arranged in a row using several racks during the demonstration to allow the audience to follow the elution of each compound and the collection process. The order in which the compounds were eluted—first ferrocene, followed by acetylferrocene, and then bromocresol green—can be rationalized by examination of their chemical structures (Figure 2). Owing to the presence of the methylketone, acetylferrocene is more polar than ferrocene, and the presence of the sulfonate and two phenols results in the comparatively high polarity of bromocresol green. The strong interaction of the phenols with silica gel dictates the need for a polar eluant such as MeOH, which competes with bromocresol green for silica gel contacts and thus facilitates its migration through the column. It should be noted that MeOH is known to dissolve small quantities of silica gel; these can be removed from a concentrated sample if necessary by filtration through a micro-plug of silica gel with a less polar solvent lacking hydroxyl groups.1 While the compounds used in the column chromatography demonstration were colored and the product-containing frac-
Journal of Chemical Education • Vol. 85 No. 4 April 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Classroom
tions could be identified in the test tubes and on the unstained TLC plate by their color, the substances used in the unknown binary mixtures to be separated by the students were colorless. Thus the TLC plates, which were generated during the final test chromatography prior to the in-class-demonstration, were attached to a piece of cardboard and confined in a sheet protector to serve as an example for the analysis of the individual fractions (Figure 3) (3, 4). The simultaneous presentation of collected fractions and corresponding TLCs allowed the students to visualize and understand the relation between the Rf obtained by TLC analysis and point of elution for the column chromatography for the different compounds. The procedure can easily be reproduced following the protocol given below. The 15 minute demonstration prepared the students for their upcoming separation experiment and enhanced their understanding of column chromatography. In fact, the majority of students were able to prepare the column and perform the separation with little additional instruction.
Me Fe
ferrocene
acetylferrocene
OH
Br Br
HO
Br Br Me
O S
Me O
O
bromocresol green
Equipment and Chemicals Equipment
O
Fe
Figure 2. Structures of the colored compounds.
Five 500 mL Erlenmeyer flasks or beakers Glass rod, 30 cm disposable plastic syringe, 1 mL one-hole rubber plug (4.5 cm diameter) fritted glass column (2.3 i.d. × 38 cm) Waterproof marker Three 20 mL scintillation vials Three 5 mL vials
25% EtOAc/ hexanes
100% MeOH ferrocene
Air hose
acetylferrocene
Twenty-five 30 mL test tubes (18 × 150 mm)
bromocresol green
Test tube racks Pipets and pipet bulbs
Chemicals
test tube:
5
10
15
20
45 g dry silica gel (gravity silica gel, 60–200 μm) 3.0 g washed and dried sea sand 90 mL Hexanes
25% EtOAc/hexanes
10 mL CH2Cl2 250 mL 5% EtOAc/hexanes 250 mL 25% EtOAc/hexanes 300 mL MeOH 75 mg Ferrocene 75 mg Acetylferrocene 10 mg Bromocresol Green
Experimental Details Reagent grade solvents were used without further purification. Ferrocene, acetylferrocene, and bromocresol green were purchased from Aldrich; alternatively, acetylferrocene can easily be prepared following the published procedure (6). Thin-layer chromatography was performed on Merck Kieselgel 60 F254 plates eluting with a solvent mixture of 25% EtOAc/hexanes, visualized by a 254 nm UV lamp, and stained with a 6% solu-
spot: 5
10
15
20
25
Figure 3. Arrangement of test tubes with collected eluant during demonstration and corresponding TLC plates (spots visualized by UV and staining with ethanolic 12-molybdophosphoric acid).
tion of 12-molybdophosphoric acid solution in 95% EtOH; TLC plates for single spots with all three compounds were 2 × 6 cm in size, and TLC plates for spots from 5 test tubes were 4 × 6 cm. Column flash chromatography was performed with Silicycle grade 70–230 mesh, 60–200 μm, 60 Å silica gel (gravity silica gel). The following solvents were eluted by applying air or nitrogen pressure using a one-hole rubber plug (4.5 cm diam-
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 4 April 2008 • Journal of Chemical Education
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In the Classroom
eter) as an adapter for the column (2.3 cm i.d.); the diameter of rubber stopper was intentionally chosen to be larger than the column inner diameter (Figure 3) to prevent a possibly unsafe pressure build-up as a result of a jammed rubber plug. The hose was connected to the rubber stopper with the barrel of a 1 mL disposable plastic syringe from which about 0.5 cm had been removed with a utility knife at the end that the plunger usually slides in (Figure 3).
Acknowledgments
Hazards
1. A proven method used to remove trace quantities of silica gel is to filter the sample (10–25 mg; after chromatography with MeOH, collection, and concentration) dissolved in 1:1 EtOAc/hexanes, EtOAc, or THF through a 3 mm plug of silica gel in a 2 mL glass pipet (layers: sand, silica gel, sand, glass wool). 2. This procedure was tested on a smaller scale by the demonstration tester, Daniel Blanchard. Here is a compacted description of the details provide by the reviewer: A buret (1.2 cm i.d.) with a plastic frit placed in the bottom was packed with a slurry of “flash silica gel” (230–400 mesh; 12 g) and hexanes (>25 mL) to provide a 27 cm column that was capped with sand (2 g). A red–orange solution of ferrocene (20 mg), acetylferrocene (20 mg), and bromocresol green (3 mg) in CH2Cl2 (1 mL) was added and eluted with 5% EtOAc/hexanes (50 mL), 25% EtOAc/hexanes (55 mL), and MeOH. The eluant was collected in 8 mL fractions in which ferrocene was contained in fractions 3 to 5, acetylferrocene in fractions 11 to 13, and bromocresol green in fractions 17 to 19. In addition, it was noted by the tester that “even with the smaller column the bands were easily seen at a distance”.
Hexanes, EtOAc, MeOH, and ferrocene are highly flammable. Hexanes, EtOAc, CH2Cl2, and ferrocene are harmful or irritating. Acetylferrocene and MeOH are toxic. 12-Molybdophosphoric acid is oxidizing and corrosive. The presenter should wear gloves, eye protection, and appropriate clothing while handling these chemicals. All chemicals should be handled in a fume hood, and the audience protected with a shield. Experimental Procedure2 A sample of dry gravity silica gel (45 g) in a 500 mL Erlenmeyer flask was combined with hexanes (90 mL), and the resulting slurry was added with the help of a glass rod to a fritted glass column (2.3 × 38 cm) that was previously marked 20 cm above the frit with a waterproof marker; the glass rod was held, so that its upper part touched the rim of the Erlenmeyer flask and its other end touched the inside glass wall of the column, to facilitate the transfer of the slurry into the column. After the slurry was transferred, excess hexanes was added and eluted through the column by applying air or nitrogen pressure until the silica gel level reached 20 cm. Washed and dried sea sand (3.0 g) was added from a 20 mL scintillation vial through a funnel, and the solvent level was lowered until the sand layer (ca. 0.4 cm) was dry. Samples of ferrocene (75 mg; Rf = 0.77, 25% EtOAc/hexanes), acetylferrocene (75 mg; Rf = 0.32, 25% EtOAc/hexanes), and bromocresol green (10 mg; Rf = 0.00, 25% EtOAc/hexanes; Rf = 0.95, 100% MeOH) in separate 5 mL glass vials were combined in a 20 mL scintillation vial and dissolved in CH2Cl2 (1 mL). The black solution was transferred onto the column with the help of a 2 mL pipet equipped with a bulb, and the liquid level was lowered until the sand layer was dry. A solution of 5% EtOAc/hexanes (2 mL) was added, and the liquid level was again lowered until the sand layer was dry. An additional solution of 5% EtOAc/hexanes (2 mL) was added and the remaining mixture of 5% EtOAc/hexanes was added (210 mL) in portions down the glass rod to minimize spilling and disturbance of the column packing. The eluted solvent was collected in 30 mL test tubes as ca. 29 mL fractions; the test tubes were lined up in a single row using several test-tube racks. Test tubes corresponding to fractions 3 to 5 contained a yellow solution of ferrocene, and this finding was corroborated by TLC analysis. A mixture of 25% EtOAc/hexanes was added (240 mL) in portions down the glass rod and eluted into 30 mL test tubes; fractions 10 to 13 contained an orange solution of acetylferrocene. Portions of MeOH (300 mL) were added down the glass rod and eluted into 30 mL test tubes; fractions 18 to 20 contained a blue solution of bromocresol green.
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Charles B. Grissom is gratefully acknowledged for providing the inspiration for this demonstration. Gary E. Keck is gratefully acknowledged for his support during the groundwork for this presentation. Notes
Literature Cited 1. (a) Chuang, I.-S.; Maciel, G. E. J. Phys. Chem. B 1997, 101, 3052–3064. (b) Peri, J. B.; Hensley, A. L., Jr. J. Phys. Chem. 1968, 72, 2926–2933. (c) Klein, P. D. Anal. Chem. 1962, 34, 733–736. (d) Klein, P. D. Anal. Chem. 1961, 33, 1737–1741. 2. Hoffmann, R. L.; McConnell, D. G.; List, G. R.; Evans, C. D. Science 1967, 157, 550–551. 3. (a) Dickson, H.; Kittredge, K. W.; Sarquis, A. M. J. Chem. Educ. 2004, 81, 1023–1025. (b) Levine, S. G. J. Chem. Educ. 1996, 73, A4. (c) Nash, J. J.; Meyer, J. A.; Everson, B. J. Chem. Educ. 2001, 78, 364–365. (d) Gordon, A. J.; Ford, R. A. The Chemist’s Companion: A Handbook of Practical Data, Techniques, and References; John Wiley & Sons: New York, 1972; pp 369–407. 4. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–2925. 5. Feist, P. L. J. Chem. Educ. 2004, 81, 109–110. 6. (a) Davis, J.; Vaughan, D. H.; Cardosi, M. F. J. Chem. Educ. 1995, 72, 266–267. (b) Vogel, G. C.; Perry, W. D. J. Chem. Educ. 1991, 68, 607–608. (c) Wade, L. G., Jr. J. Chem. Educ. 1978, 55, 208. (d) Gilbert, J. C.; Monti, S. A. J. Chem. Educ. 1973, 50, 369–370. (e) Bozak, R. E. J. Chem. Educ. 1966, 43, 73. 7. Hwa, R.; Weizman, H. J. Chem. Educ. 2007, 84, 1497–1498.
Supporting JCE Online Material
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Journal of Chemical Education • Vol. 85 No. 4 April 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
