Liquid CO2 in Centrifuge Tubes: Separation of Chamazulene from

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Liquid CO2 in Centrifuge Tubes: Separation of Chamazulene from Blue Tansy (Tanacetum annuum) Oil via Extraction and Thin-Layer Chromatography Bruce W. Baldwin* and Thomas S. Kuntzleman Department of Chemistry, Spring Arbor University, Spring Arbor, Michigan 49283, United States S Supporting Information *

ABSTRACT: The separation of chamazulene from hydrophilic contaminants present in blue tansy oil provides a visually engaging example of two common techniques: extraction and thin-layer chromatography (TLC). This application uses liquid CO2 as a lipophilic solvent to pull a brilliant blue hydrocarbon molecule, chamazulene, out of or through a common hydrophilic material, cellulose. Yellow-green, hydrophilic components remain in a cotton ball or at the lower end of a paper strip. In dramatic fashion, the separation of colored components of blue tansy oil is accomplished with an environmentally sustainable solvent, CO2, and an innocuous support material, cellulose. We believe this is the first reported use of liquid CO2 in centrifuge tubes to conduct TLC. KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Laboratory Instruction, Organic Chemistry, Chromatography, IR Spectroscopy, Natural Products, Separation Science, Thin Layer Chromatography, UV−Vis Spectroscopy

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INTRODUCTION Experiments involving liquid CO2 extractions in centrifuge tubes have been implemented throughout the chemistry curriculum from K−8 outreach events to upper-level undergraduate courses.1−5 Part of the interest in this experiment certainly stems from the fact that all three phases of CO2 can simultaneously be observed during the extraction process. Perhaps the most widely used of these experiments involves extraction of limonene from orange peels,1,2 although extractions of anethole from fennel seeds3 and eugenol from oil of cloves4 have also been reported. Gas chromatography,2 NMR spectroscopy,3 and the properties of antioxidants and anesthetics4 are all topics that have been explored when using liquid CO2 extraction. In an interesting twist, “stains” of essential oils introduced onto T-shirts have been “cleaned” by liquid CO2 extraction to introduce kindergarten to eighth grade students to some of the chemical principles involved in dry cleaning.5 Herein, this previous work is extended by showing how liquid CO2 can be used to extract chamazulene (Figure 1) from blue tansy oil, which is a dark, blue-green liquid. In contrast to the oil, chamazulene has a strikingly beautiful blue color. The contrasting colors allow students to observe the extraction easily in real time as it takes place. While the extracted fluid almost certainly contains a mixture of hydrocarbons, the chamazulene contained within is concentrated enough to identify using IR and visible spectroscopies. In addition, it is shown how the previously reported protocols for liquid CO2 extractions can be simply modified © XXXX American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Chemical structure of chamazulene, an inky-blue, hydrophobic compound.

to separate chamazulene from blue tansy oil using thin-layer chromatography (TLC). The speed with which the chromatogram develops coupled with the colors involved allow for a striking and rapid demonstration of a chromatographic separation. To our knowledge, this is the first reported use of liquid CO2 in centrifuge tubes to develop TLC.

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BACKGROUND Blue tansy oil is an essential oil with a strong, sweet, herbal odor. It is extracted from the leaves and flowers of Tanacetum annuum.6−9 Blue tansy oil is prized for its brilliant blue color, which arises from the high proportion of chamazulene it contains.6−9 However, the chamazulene content in blue tansy oil ranges from 3% to almost 30%.6−9 This variation is likely Received: August 8, 2017 Revised: November 14, 2017

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DOI: 10.1021/acs.jchemed.7b00610 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

due in part to the fact that azulenes oxidize in the presence of air, causing their blue color to change to green, yellow, or even brown.10 Similar to other essential oils,9,11 blue tansy oil also contains over 100 other compounds, mostly terpenes and terpenoids12 (Figure 2) such as camphor, sabinene, β-pinene,

Figure 2. Chemical structures of β-pinene (left) and camphor (right). β-Pinene is a terpene, while camphor is a terpenoid.

α-phellandrene, α-bisabolol, and limonene, to name a few.6−9 Due in part to their volatility, terpenes and terpenoids13 tend to impart pleasant aromas to essential oils. As with chamazulene, oxidation and other chemical changes can alter the makeup of these other compounds in essential oils during storage, which further contributes to variations in color and odor of essential oils.14−18 Because oxidation tends to convert hydrophobic compounds to hydrophilic ones, hydrophobic solvents can be used to separate the colorful and fragrant, lipophilic components from essential oils. In this experiment, liquid CO2 is used in this regard to concentrate such desired components from blue tansy oil.

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EXPERIMENT An aluminum wire is wound two or three times around a pencil to fashion a “holder” for a small piece of cotton. Both ends of the wire are oriented parallel to the pencil. The fashioned wire is removed from the pencil and placed in a centrifuge tube. Next, 10 drops of blue tansy oil are added to the cotton. The cotton is pushed into the centrifuge tube so that it rests on the wire holder. Powdered dry ice is added to fill the centrifuge tube which is immediately sealed tight. The tube is placed in about 300 mL of water at 60−70 °C contained in a clear plastic soda bottle with its top removed. Once in the water, the centrifuge tube is held in place so the tube is submerged in the water using a clamp screw attached to a ring stand (Figure 3). The CO2 usually liquefies in less than 45 s, and the liquid CO2 flows through the cotton. This process dissolves the bright blue chamazulene and other hydrocarbons, carrying them to the bottom of the centrifuge tube. The liquid CO2 takes on a bright blue color, and the cotton piece begins to appear dark green as the extraction takes place. The extract is concentrated enough in chamazulene to collect the IR spectrum, while a few drops of the extract are dissolved in isopropyl alcohol for the visible spectrum. More detail including a short video of the extraction is provided in the Supporting Information. For the parallel TLC experiment, the coiled aluminum wire is placed in the bottom of the centrifuge tube to ensure the strip of filter paper does not fall too far into the liquid CO2. A single drop of blue tansy oil is placed about halfway up a 1 × 5 cm2 strip of filter paper using a glass capillary tube. Water is heated to 60−70 °C and placed in a 12 oz PETE (polyethylene terephthalate) soda bottle with its top removed, ready to hold the dry-ice-charged centrifuge tube. About 6−7 mL of pulverized dry ice is added to the centrifuge tube, and the spotted TLC strip is positioned on top of the dry ice. The apparatus is sealed with a cap, placed into the warm water, and

Figure 3. Apparatus for extraction of blue tansy oil with liquid CO2. Note the difference in color between the dark green cotton ball and blue liquid.

secured with a screw clamp attached to a ring stand. As before, the CO2 usually liquefies in less than 45 s. The apparatus often needs to be joggled so that the end of the TLC paper remains submerged in liquid CO2. The solvent front is observed to move through the origin spot, carrying blue material with it. Once developed, the chromatogram appears to contain a graygreen spot at the origin and a bright blue band at the solvent front. The developed TLC strip can be removed from the centrifuge tube and placed in the air stream of a heat gun. Upon doing so, the blue band at the solvent front disappears, while the green spot at the origin takes on a yellow color. More detail including videos of the TLC development and heat treatment is provided in the Supporting Information.

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HAZARDS Eye protection must be worn during this experiment. Dry ice is extremely cold: use insulating gloves during handling. Blue tansy oil is an accepted essential oil in aromatherapy. However, as with all essential oils, it can be a skin, eye, and respiratory irritant and is harmful if swallowed in large quantities. Chamazulene, the principal product, is harmful if swallowed and can be a skin, eye, and respiratory irritant. The cotton ball with adsorbed components of blue tansy oil should be disposed of in an organic hazardous waste container along with the principal products. Isopropyl alcohol is flammable. The pressurized centrifuge tubes present projectile and tube-rupture hazards. Exposure of the polypropylene centrifuge tube to low temperature can cause crazing, which can potentially result in explosion. It is very important that the centrifuge tube be B




IR Spectrum

charged with dry ice only after the water bath, contained in a 12 oz clear PETE soda bottle and prewarmed to ∼60 °C, is immediately ready to receive the charged and sealed centrifuge tube. This precaution minimizes excessive cooling of the centrifuge tube, which has resulted in rupture of two centrifuge tubes in well over 200 student-run experiments.

During the experiment, students think about the intermolecular forces at play that cause chamazulene and other lipophilic components to be extracted by liquid CO2. The discussion focuses on the intensely blue chamazulene because it is easy to visualize. Because chamazulene is a lipophilic hydrocarbon, it does not interact strongly with the hydrophilic cellulose matrix of the cotton ball. Thus, when hydrophobic CO2 rinses through the cotton piece, chamazulene dissolves in the CO2 and is extracted (Figure 3). Other lipophilic components are extracted along with chamazulene, and this extracted material represents the portion of the essential oil with desired qualities. It is useful to compare the appearance and odor of the extracted material with those of the original blue tansy oil: a slight difference in odor is detected, and the extracted material is significantly more blue in color. Also, inspection of the cotton piece reveals a dark green residue that remains. This residue likely contains many unwanted, hydrophilic components of the blue tansy oil that are strongly attracted to the cellulose matrix of the cotton ball. However, it is likely that the green color of the residue results from some residual chamazulene mixed in with oxidized material (see the subsequent TLC discussion).

The neat liquid obtained by extraction is conveniently characterized using an ATR accessory, or the liquid can be placed between sodium chloride plates. ATR is by far the more convenient method. The spectrum obtained (Figure 5) is quite similar to a previously published IR spectrum of chamazulene.19 Given the aromatic and aliphatic groups of chamazulene, there are three types of peaks to analyze: sp2 CH, sp3 CH, and CC stretches (Figure 5). A small yet important peak at 3060 cm−1 indicates the presence of aromatic or alkene CH bonds. The prominent peaks at 1650 and 1450 cm−1 verify that the CH stretches must arise from aromatic CH bonds rather than alkene CH bonds because such peaks arise from aromatic CC stretches, not alkene CC stretches. Previous work has shown a broad OH peak at 3150 cm−1 due to the presence of residual solvent ethanol.18 The present approach has the advantage that liquid CO2 evaporates during the extraction process, eliminating the potential for contamination by solvent. In fact, IR spectroscopy is uniquely sensitive to the presence of carbon dioxide. Such a peak centered at 2360 cm−1 does not appear in the spectrum. However, a strong peak at 1740 cm−1 does appear for the blue tansy extract. This peak is probably not part of chamazulene; rather, it arises from the presence of camphor, which contains an angle-strained ketone group. An independent IR spectrum of camphor yields a strong peak at 1742 cm−1, very close to the peak in the extracted blue material. Camphor is a consistent, major component of blue tansy oil.6−9 The very strong peaks at 2950 cm−1 therefore represent alkane CH stretches on both chamazulene and camphor.

Thin-Layer Chromatography

Vis Spectrum

During its development, students observe the formation of two well-resolved regions with highly contrasting colors on the chromatogram (Figure 4). The blue region results from the

One drop of the native blue tansy oil and also one drop of the blue extract are dissolved into separate 10 mL portions of isopropyl alcohol and transferred to cuvettes, and the absorption spectrum of each is obtained (Figure 6). It is easy for students to understand that the brilliant blue color of the extract is due to the wide swath of light transmission in the blue region (425−500 nm) of the spectrum of the extract (Figure 6, solid line). Students also recognize that the window of transmission (450−540 nm) of the native blue tansy oil (Figure 6, dashed line) leads to a blue-green hue in the unextracted oil. Overlay of these two spectra allows students to see that the violet absorbing material that is present in the native oil is removed during the extraction process. The overall form of and location of peaks in the spectrum match remarkably well with previously reported spectra for chamazulene,10,19,20 allowing students to identify the presence of chamazulene in the extract.

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RESULTS AND DISCUSSION

Extraction

Figure 4. Chromatogram of blue tansy oil developed with liquid CO2.

hydrophobic chamazulene that travels with the liquid CO2 solvent front. The green region, which remains positioned at the origin, could be due to a combination of retained blue chamazulene and oxidized yellow material. The oxidized components remain at the origin due to strong interactions between these hydrophilic compounds and hydroxy groups on the cellulose matrix of the paper. A simple visualization of this state of affairs is performed by heating the developed TLC strip in the air stream of a heat gun. Upon doing so, the blue chamazulene (bp = 161 °C) at the solvent front and at the origin evaporates away. Thus, the blue band at the solvent front disappears, while the green spot at the origin simultaneously turns yellow. The latter color change is interpreted to mean that the simple heating process drives away residual chamazulene at the origin, leaving yellow, oxidized compounds behind at the origin. However, it should be noted that the heating process might also be driving away hydrophilic material that is blue and volatile remaining at the origin.

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LEARNING OBJECTIVES The experiments described herein have been used multiple times in a nonmajors general science course; a general, organic, and biochemistry (GOB) course; and an organic chemistry course. In addition, modifications of these experiments have been explored as small independent research projects as part of the organic laboratory curriculum. Students generally worked in groups of 2 or 3 in the lower division courses, but independently in the organic chemistry course. A variety of chemical topics have been introduced to students using this experiment. For example, students have been expected to apply the principles of molecular polarity to explain the results of the extraction and chromatographic separation. Students in the C




Figure 5. Student-acquired IR spectrum of isolated product obtained from the extraction of blue tansy oil with liquid CO2. Illustrated peaks picked at the following (cm−1): sp2 CH at 3060 (red), sp3 CH at 2950 (blue), CO at 1740 (green), and CC at 1650 and 1450 (magenta).

lipophilic components from blue tansy oil. During the process, the brilliant blue chamazulene was extracted from the essential oil, allowing students to visualize the process in real time as it occurs. While other hydrophobic components were extracted along with chamazulene, these components did not interfere with the identification of chamazulene by IR and visible spectroscopies. Also, a modification is reported in which TLC was carried out in centrifuge tubes using CO2 as the mobile phase. These chromatograms developed very quickly, allowing students to easily visualize the process as it was carried out. Finally, the work allowed students to learn about environmentally friendly extraction methods. Extraction and TLC techniques often require the use of volatile organic compounds, which technically add to the pollution burden of these techniques. The fifth of 12 principles of green chemistry states that solvents and separation agents should be innocuous when used.21 Carbon dioxide is a material that is harvested from the air and is then released back into the air after the experiment, making this a very sustainable solvent. The separation agents are innocuous cellulose materials. The third principle of green chemistry asks that the materials used possess little or no toxicity. Blue tansy oil is a natural product extract used extensively in aromatherapy. Finally, students added analyte directly to the cellulosic materials without solvent predilution. Thus, another subtle point regarding elimination of solvents in chemical processes was made. These provided students with unique examples of valuable chemical techniques that are friendly to the environment and safe to students, given that the liquefaction of CO2 was done with proper care as outlined in this paper and in other J. Chem. Educ. publications.1−5

Figure 6. Visible spectrum of isolated product from the extraction of blue tansy oil with liquid CO2 (solid line), and native blue tansy oil (dashed line). Spectra were acquired on student-collected samples.

GOB course have obtained the vis spectrum of both the blue tansy oil and the blue extract (Figure 6). The differences in the vis spectra were used to explain the differences in the colors observed between the blue tansy oil and its extracted counterpart. Organic chemistry students obtained the IR spectrum of the blue extract and identified various peaks in the spectrum consistent with the presence of chamazulene. Further commentary on learning objectives including student laboratory sheets, pretests, and post-tests can be found in the Supporting Information.

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CONCLUSION The experiment presented here extended the previously reported body of work involving liquid CO2 extractions in centrifuge tubes. First, this method was used to extract desired, D



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(14) Turek, C.; Stintzing, F. C. Stability of Essential Oils: A Review. Compr. Rev. Food Sci. Food Saf. 2013, 12, 40−53. (15) Schmidt, E. Production of Essential Oils. In Handbook of Essential Oils; Hüsnü, K., Baser, C., Buchbauer, G., Eds.; CRC Press: Boca Raton, 2010; p 101. (16) Sun, H.; Ni, H.; Yang, Y.; Wu, L.; Cai, H.-N.; Xiao, A.-F.; Chen, F. Investigation of Sunlight-Induced Deterioration of Aroma of Pummelo (Citrus maxima) Essential Oil. J. Agric. Food Chem. 2014, 62, 11818−11830. (17) Avonto, C.; Wang, M.; Chittiboyina, A. G.; Avula, B.; Zhao, J.; Khan, I. A. Hydroxylated Bisabolol Oxides: Evidence for Secondary Oxidative Metabolism in Matricaria chamomilla. J. Nat. Prod. 2013, 76, 1848−1853. (18) Mockutë, D.; Bernotienë, G.; Judentienë, A. Storage-Induced Changes in Essential Oil Composition of Leonurus cardiac L. Plants Growing Wild in Vilnius and of Commercial Herbs. CHEMIJA 2005, 16, 29−32. (19) Berger, S.; Sicker, D. Classics in Spectroscopy: Isolation and Structure Elucidation of Natural Products; Wiley-VCH: Weinheim, 2009; pp 153−168. (20) Cheplogoi, P. K. Extraction of Essential Oils and Active Compounds from Matricaria chamomilla L. and Their Application in Toilet Soap. M.Sc. Dissertation, University of Nairobi, 1997. (21) Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice; Oxford University Press: New York, 1998; p 30.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00610. Student laboratory instructions and additional instructor materials (PDF, DOCX) Videos of the extraction process, TLC process, and heating of TLC strip (ZIP)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Bruce W. Baldwin: 0000-0003-2547-6761 Thomas S. Kuntzleman: 0000-0002-2691-288X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank the many Spring Arbor University students that conducted this experiment. REFERENCES

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Liquid CO2 in Centrifuge Tubes: Separation of Chamazulene from

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