Extraction, Purification, and Spectroscopic Characterization of a

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

Extraction, Purification, and Spectroscopic Characterization of a Mixture of Capsaicinoids Carl E. Wagner,* Thomas M. Cahill, and Pamela A. Marshall Division of Mathematical and Natural Sciences, Arizona State University at the West Campus, Glendale, Arizona 85306, United States

bS Supporting Information ABSTRACT: This laboratory experiment provides a safe and effective way to instruct undergraduate organic chemistry students about natural-product extraction, purification, and NMR spectroscopic characterization. On the first day, students extract dried haba~nero peppers with toluene, perform a pipet silica gel column to separate carotenoids from capsaicinoids, and perform thin-layer chromatography. On the second day, students characterize their mixture of capsaicinoids, approximating the relative quantities of the two primary capsaicinoids, capsaicin and dihydrocapsaicin, by NMR. Additionally, students are introduced to 2D NMR analysis. The effectiveness of the laboratory is assessed by two methods: a qualitative postlab student self-assessment and a quantitative pre- and postlab test. For the qualitative assessment, students stated that they acquired an improved understanding of NMR from the experiment. Accordingly, the total average on the quantitative prelab and postlab 8-point test increased from 3.43 ( 1.62 (SD) to 4.25 ( 1.48 (SD) and demonstrated a statistically significant improvement (p value of 0.001), as did the average for the 5-points of NMR-related questions in the prelab- and postlab 8-point test: 1.61 ( 1.21 (SD) to 2.13 ( 1.11 (SD), with p value of 0.004. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Chromatography, IR Spectroscopy, NMR Spectroscopy, Natural Products Capsaicin, 1 (Figure 1), has many structural features (phenol, ether, amide, alkene, and alkyl) that make it ideal for demonstrating a wide range of 1H NMR shift values. The shift values fall within the expected range based on typical NMR tables in organic chemistry texts.3 The chemical also demonstrates the spin spin coupling principle well and it can be used to determine which functional groups are adjacent to each other as well as the substitution pattern on the aromatic ring. Capsaicin lacks functional groups and features that give rise to a complex spectrum, such as multiple fused ring structures, heterocyclic hydrocarbons, and highly branched alkanes present in other traditional natural-products laboratories such as caffeine extraction from tea. The experiment described herein represents a complete natural-product laboratory exercise that takes the students through the extraction, isolation, purification, and NMR analysis of capsaicinoids from haba~nero peppers. Students benefit from the experience of isolating the capsaicinoid mixture from other compounds and by being able to follow through an extraction from pepper all the way to purification and purity analysis. If students are careful in their purification of the capsaicinoids, they can approximate the relative quantities of capsaicin, 1, to dihydrocapsaicin, 2 (Figure 1), which are the two major constituents of the capsaicinoid extract,4 by 1H NMR. This experiment has been

T

he concept of extracting capsaicinoids from hot chili peppers to instruct students in basic organic natural-productextraction techniques and in isolation and characterization of organic compounds is not new; this idea has been the central concept in many experiments described in this Journal.1 Additionally, structure-activity-reationship (SAR) discussions concerning capsaicin and related capsaicinoids have been published in this Journal to highlight the useful medicinal qualities of these compounds and their relevance to the undergraduate chemistry curriculum.2 Several of the laboratory exercises1 characterize the capsaicinoid extract using HPLC UV. However, this lab is novel because the focus of the laboratory experiment is not only the extraction of capsaicinoids, but also the purification and isolation of the capsaicinoids by standard chromatographic methods and the characterization of those capsaicinoids by NMR. In many ways, capsaicin is an ideal model chemical for naturalproduct chemistry from both the “student interest” and NMR spectroscopy points of view. Most students are familiar with hot peppers and spicy foods, so this gives the students experience investigating an everyday chemical they have encountered. The capsaicinoids are also the active ingredients in self-defense pepper sprays, which gives this chemical a forensics interest. Additionally, capsaicin is the active ingredient in some topical arthritis creams and thus is a model pharmaceutical chemical for students interested in the medical fields. The NMR spectral qualities of the capsaicinoids are ideal for demonstrating many of the characteristics of NMR analysis. Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

Published: August 26, 2011 1574

dx.doi.org/10.1021/ed1006025 | J. Chem. Educ. 2011, 88, 1574–1579

Journal of Chemical Education implemented in the organic laboratory curriculum over a 2-day period with 102 students, and the lab has been assessed through quantitative pre- and postlab tests, as well as a qualitative postlab student self-assessment. The assessments demonstrate that this approach is effective in achieving student mastery of NMR spectroscopic methods. This experiment can also be implemented in the advanced organic or the undergraduate analytical laboratory curriculum with a focus on 2D NMR techniques.

’ EXPERIMENTAL DETAIL There are four parts to this experiment: (I) the toluene extraction of dried peppers and purification of the capsaicinoids by a silica gel column, (II) the thin-layer chromatography (TLC) of the column fractions, (III) the isolation of the capsaicinoids by evaporation of the column eluent, and (IV) the NMR characterization of the capsaicinoid mixture. In part I, students grind 5 dried, stemless haba~ nero peppers with a mortar and pestle in a fume hood, taking care to avoid exposure to the dried powder. Students then extract the powder in a 15 mL centrifuge tube by shaking it in 8 mL of toluene and separating the solids from the toluene by centrifugation. Toluene was chosen as the preferred solvent for this experiment because it is more selective in the

Figure 1. Structures of capsaicin (1) and dihydrocapsaicin (2), the major components of capsaicinoids extracted from haba~nero peppers.

LABORATORY EXPERIMENT

extraction of the capsaicinoids than more polar solvents such as acetone, thus giving a purer extract even though the mass of capsaicinoids extracted is lower than other solvents. Gravity filtration-chromatography, through a 5 3/4 in. length Pasteur pipet plugged with glass wool and filled with chromatographygrade silica gel up to its neckband in 3:1 hexanes/ethyl acetate solvent, is used to separate the components of the organic toluene extract: capsaicinoids and carotenoids. Students collect two 5 mL fractions followed by a 10 mL fraction collected in tared glass test tubes. Lab instructors may also find it beneficial to provide an in-class demonstration of a larger column for largescale material separations. In part II, students conduct TLC analysis of the three fractions to determine which fraction contains the capsaicinoid mixture. Students spot a TLC plate with a small volume of capsaicin (1) standard and the three fractions in different lanes and then develop the TLC plate in a 1:1 hexanes/ethyl acetate solution in a TLC chamber. The students observe that the first fraction contains carotenoids that appear between Rf values of 0.80 and 0.85. The second fraction contains little to no capsaicinoids, and the third fraction contains the bulk of the capsaicinoids, with the dihydrocapsaicin co-eluting with the capsaicin at an Rf of 0.25 to 0.30. After the solvent has evaporated from the developed TLC plate, students visualize the spots on the plate by a UV lamp. This part of the experiment is an excellent place for students to practice TLC spotting skills and learn about monitoring column fraction composition by TLC. Because both the column and TLC plate use silica gel as the stationary phase, this exercise reinforces the principles of chromatography in that the chemicals that elute first from the column will migrate the farthest on the TLC plate. In part III, students evaporate the solvent of the capsaicinoid fraction. The manner in which the evaporation is accomplished can be left to the discretion of the instructor. The use of a rotary evaporator (rotavap) can potentially demonstrate the principles of distillation under reduced pressure.

Figure 2. 1H NMR spectrum of capsaicin (1). The insets show more spectral detail. 1575

dx.doi.org/10.1021/ed1006025 |J. Chem. Educ. 2011, 88, 1574–1579

Journal of Chemical Education


Figure 3. 1H NMR spectrum of dihydrocapsaicin (2). The insets show more spectral detail.

Figure 4. Student acquired 1H NMR spectrum of capsaicinoid mixture (1/2 ∼ 55:45). The insets show more spectral detail.

Part IV is the NMR analysis of the student extracts and it is conducted in the second lab period. Students dissolve their capsaicinoid product (5 20 mg) in approximately 1 mL of deuterochloroform in a fume hood, place this mixture in an

NMR tube, and acquire a proton NMR spectrum. As a prelab exercise on the second day of lab, students are asked to assign the protons of capsaicin to the proton resonance peaks in the 1 H NMR spectrum of capsaicin (Figure 2). The 1H NMR and 1576




13

C NMR spectra of capsaicin have been thoroughly analyzed and documented.5 To complete the rigorous assignment of protons in the capsaicin 1H NMR spectrum, students need the COSY of capsaicin, and this is an ideal opportunity for instructors to introduce 2D NMR techniques to their undergraduate students. The COSY of capsaicin shows that the methine proton of the isopropyl group resonance is buried under the methylene triplet resonance from the protons on the R-carbon adjacent to the amide carbonyl. Students are also provided with a 1H NMR spectrum of dihydrocapsaicin, and they are asked to approximate the relative quantities of 1 and 2 in their capsaicinoid mixture from their 1H NMR mixture spectrum. The ratio of capsaicin to dihydrocapsaicin was observed to be approximately 60:40 for most haba~ nero peppers tested in this laboratory exercise. The 1H 13 NMR, C NMR, COSY, and HSQC spectra for 1 and 2 are included in the Supporting Information. The opportunity to teach students about long-range W-coupling in aromatic rings, not only in prelab NMR lectures, but also as students acquire the 1H NMR spectrum of their capsaicinoid mixtures, can be exploited in this exercise. Often, the 2.0 Hz W-coupling in the aromatic rings of both capsaicin and dihydrocapsaicin could be clearly resolved in the capsaicinoid mixture. Additionally, the COSY spectra of capsaicin and dihydrocapsaicin corroborate the structural information students glean from the assignment of aromatic proton position from the W-coupling. Clearly visible in the 1H NMR spectrum of capsaicin (1) is the doublet of the isopropyl group at 0.94 ppm. In dihydrocapsaicin (2), this doublet occurs at 0.84 ppm (Figure 3). Additionally, the isopropyl methine proton resonance (a nonet with only seven of the nine peaks apparent) is visible in the spectrum of 2 at 1.50 ppm, whereas it is hidden in the spectrum of 1 at 2.20 ppm. Importantly, students can see both doublets in 1H NMR spectrum of their capsaicinoid mixture and approximate the relative quantities of 1 to 2. Shown in Figure 4 is a representative student 1H NMR spectrum of the capsaicinoid mixture (1/2 ∼ 55:45). Students may also take the Fourier-transform infrared (FTIR) spectrum of their capsaicinoid mixture of 1 and 2 and compare it to a pure spectrum of capsaicin (1) (see the Supporting Information). Although FTIR spectroscopic analysis is not a necessary component of this laboratory, FTIR analysis shows the presence of the functional groups. FTIR and 1H NMR data for capsaicin (1) are also available at the Sigma-Aldrich Web site, though the NMR spectrum more closely resembles the one shown in Figure 4 depicting a mixture of capsaicin and dihydrocapsaicin.6 Spectra of the starting material (1) and product (2) are included in the Supporting Information.

’ HAZARDS Dried haba~ nero pepper powder contains capsaicin and other capsaicinoids that will irritate the eyes and skin and may be toxic if inhaled or ingested. Warnings have also been published in this Journal concerning students performing experiments with capsaicin.7 Toluene, hexanes, and ethyl acetate are volatile, flammable organic solvents. All students and the instructor should be wearing goggles, gloves, and lab coats or aprons. Grinding the haba~nero peppers should be carried out in a well-ventilated room and the extractions should be carried out in a fume hood. The TLC plate should be handled with tweezers, not by hand. Finally, preparation of the NMR sample, or any work with deuterochloroform, should be conducted in a fume hood as chloroform-d is a carcinogen, is toxic by inhalation, and can cause respiratory irritation.

Figure 5. Comparison of student scores on prelab test (n = 61), postlab test (n = 89), and final exam (n = 101).

Figure 6. Comparison of student ability to correctly answer the five NMR-related questions on the prelab test (n = 61), postlab test (n = 89), and final exam (n = 101).

’ ASSESSMENT OF STUDENT LEARNING Exemption from human subjects approval was granted by the Arizona State University Institutional Review Board as pursuant to Federal regulations, 45 CFR Part 46.101(b)(1)(2). This lab was assessed by two means: comparison of pre- and postlab scores on an in-class test (included in Supporting Information) and by a postlab anonymous student self-assessment. The prelab test was given immediately before the lab and the postlab test was administered one week after the lab.8 Both prelab and postlab tests consisted of eight multiple-choice questions, five of which concerned NMR, the central learning theme of the experiment. Additionally, a final examination was held three weeks after the lab and the eight questions from the prelab and postlab test were re-asked on the exam. The total average on the quantitative prelab and postlab test increased from 3.43 ( 1.62 (SD) to 4.25 ( 1.48 (SD), respectively, and demonstrated a statistically significant improvement (p value of 0.001), as did the average for the five NMR-related questions in the pre- and post-test: 1.61 ( 1.21 (SD) to 2.13 ( 1.11 (SD) with p value of 0.004. Analysis 1577




Table 1. Postlab Assessment of the Laboratory Student Response Mean ( SD (n = 89)a

Statement

4.10 ( 1.06

I enjoyed this lab. I would recommend that this lab exercise be kept in the chemistry lab curriculum.

4.11 ( 1.08

This lab exercise made me think.

4.39 ( 0.87

This lab exercise fit in well with the curriculum of the lecture.

3.84 ( 1.25

I hated this particular lab.

2.26 ( 1.27

This lab was an active process for me.

3.97 ( 0.98

I learned something from this lab.

4.32 ( 0.85

This lab made me ask questions, such as, “How would I determine which peaks in the 1H NMR spectrum of capsaicin correspond to the benzylic protons by applying the classical (n+1) splitting rule?”

4.02 ( 1.13

a

The answer scale is agree strongly = 5, agree slightly = 4, neither agree nor disagree = 3, disagree slightly = 2, and disagree strongly = 1. Excel was used to determine mean and standard deviation.

revealed that there is a statistically significant difference between prelab and postlab scores (using an unpaired homoscedastic t test, the p values were found as stated above). Furthermore, the average on the 8-point test increased to 4.69 ( 1.51 (SD), as did the average for the 5-points of NMR-related questions to 2.54 ( 1.14 (SD), on the final exam. Graphical analysis included in Figure 5 indicates that most students were able to perform better on the postlab test than the prelab test, as indicated by the overall increase in test scores from the prelab to the postlab test, and further that most students were able to perform better on the final exam than the postlab test. Further analysis of the five NMR concept questions in the prelab test, postlab test, and final exam (Figure 6) indicated that most students were able to perform better on the postlab test than the prelab test and even better on the final exam. Although the increase in the ability to correctly answer the NMR concept questions from the prelab test to postlab test reasonably may result from the performance of the laboratory experiment, the increase observed on the final exam may be attributable to additional factors such as instructor review and student study. Nevertheless, the laboratory activity may have helped prime students to understand NMR better when they encountered it in the lecture, thus raising their performance on the final exam. Finally, students were given the option to complete a postlab anonymous self-assessment. These surveys are reported in Table 1. In the postlab survey, students reported that they learned something and also indicated that they enjoyed the exercise. Thus, by the assessment measurements, students learned from this laboratory.

’ EXTENSION OF LABORATORY EXPERIMENT If instructors prefer to use this exercise as a more open-ended or hypothesis-driven lab, the procedures are easily modified. Students can bring in their own peppers or choose from a variety brought by the instructor (such as Thai hot, jalape~ no, cayenne, and Bhut Jolokia peppers). Students can then develop and test hypotheses about which peppers will have the most capsaicinoids based upon what they know about pepper flavor. The ratio of capsaicin to dihydrocapsaicin can also be compared between different pepper varieties. This lab has been used in an upper-division instrumental analysis class in which the students are only given the spectra (both 1H- NMR and COSY) for pure capsaicin. The students are informed that there is an additional, unknown chemical in natural pepper extracts. The students then must elucidate the structure of the unknown chemical (e.g., dihydrocapsaicin) based on the

NMR of their extract and the NMR spectra of pure capsaicin. The students also need to determine the proportion of these two chemicals in the extract.

’ CONCLUSION An easy extraction, purification, and characterization of capsaicinoids from dried haba~nero peppers are reported where students practice column chromatography, TLC, and characterization by NMR spectroscopy. Prelab and postlab analysis indicates that this lab increased student understanding of the central NMR concepts. ’ ASSOCIATED CONTENT Information bS 1 Supporting 13

H NMR, C NMR, COSY, and HSQC spectra for 1 and 2; FTIR spectrum of pure capsaicin; in-class test; student selfassessment; student handout. This material is available via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We thank the Chemistry Division of the National Science Foundation for financial support of this work through a grant for the 400 MHz NMR machine at ASU West (Grant CHE0741978). ’ REFERENCES (1) (a) Betts, T. A. J. Chem. Educ. 1999, 76, 240–244. (b) Batchelor, J. D.; Jones, B. T. J. Chem. Educ. 2000, 77, 266–267. (c) Huang, J.; Mabury, S. A.; Sagebiel, J. C. J. Chem. Educ. 2000, 77, 1630–1631. (2) (a) Rusterholz, D. B. J. Chem. Educ. 2006, 83, 1809–1815. (b) Kimbrough, D. R. J. Chem. Educ. 1997, 74, 861–862. (3) (a) Crews, P.; Rodríguez, J.; Jaspar, M. Organic Structure Analysis; Oxford University Press: New York, 1998: Chapter 3. (b) Marshall, J. L. Carbon-Carbon and Carbon-Proton NMR Couplings: Applications to Organic Stereochemistry and Conformational Analysis (Methods in Stereochemical Analysis); VCH: New York, 1983. (c) Vollhardt, K. P. C.; Schore, N. E. Organic Chemistry Structure and Function, 5th ed.; Freeman: New York, 2007: Chapter 10. 1578




(4) Thomas, B. V.; Schreiber, A. A.; Weisskopf, C. P. J. Agric. Food Chem. 1998, 46, 2655. (5) Sigma-Aldrich Home Page. http://www.sigmaaldrich.com/ (accessed Aug 2011). (6) (a) Vorndam, P. E. J. Chem. Educ. 2000, 77, 444. (b) Jones, B. T. J. Chem. Educ. 2000, 77, 444. (7) (a) Kobata, K.; Todo, T.; Yazawa, S.; Iwai, K.; Watanabe, T. J. Agric. Food Chem. 1998, 46, 1695–1697. (b) Lin, L. Z.; West, D. P.; Cordell, G. A. Nat. Prod. Lett. 1993, 3, 5–8. (8) The prelab and postlab tests for the lab were voluntary and anonymous, students could withdraw from the tests at any time, and a varying number of students participated. The results of the prelab and postlab tests, as well as the final exam, were thus compared with statistical methods that accounted for variation in the number of participants.

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Extraction, Purification, and Spectroscopic Characterization of a

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