Activity pubs.acs.org/jchemeduc
Plant Pigment Identification: A Classroom and Outreach Activity Kathleen C. A. Garber,†,§ Antoinette Y. Odendaal,†,§ and Erin E. Carlson*,†,‡ Departments of †Chemistry and ‡Cellular and Molecular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States S Supporting Information *
ABSTRACT: Anthocyanins are a class of pigments responsible for the bright colors of many flowers, fruits, and vegetables typically resulting in shades of red, blue, and purple. Students were asked to perform an activity to enable them to identify which anthocyanin was present in one of several possible plant materials through a hands-on activity. Students extracted the pigments from the biological sources and obtained color profiles by altering the pH of the biological extracts. These extracts were compared to standards of anthocyanin pigments so that students could deduce which pigment was present in the plant material that they had selected. This activity interrelates several chemical concepts and provides a link between chemical and biological sciences. KEYWORDS: Elementary/Middle School Science Outreach, Public Understanding/Outreach, Inquiry-Based/Discovery Learning, Acid/Base, Dyes/Pigments, Molecular Properties, Natural Products, pH lives.4,5 From the colors of flowers and the smell of citrus to the agents used as drugs, these compounds perform functions that students can recognize and relate to.6 Plants use a host of natural products to interact with their environment, for example, as insect attractants or chemical warfare agents.7 Many of these processes are highly recognizable even to young children, making them ideal subject matter for a scientific outreach activity. Many plant-produced compounds are colored or pigmented. Anthocyanins (Figure 1), members of the flavonoid family of
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umerous experiences ranging from chemistry magic shows to accelerated classes have shown that the future generations of scientists will be born through an introduction to chemistry that is interesting, exciting, and “cool”. Students, be they the ever-excitable 8-year olds or jaded 16-year olds, are curious and open to being surprised. The chemistry of explosions and light shows can be a thrilling introduction, but it is the next step, illustration of how chemistry plays a role in the world around them, that makes science, and particularly chemistry, both approachable and relevant.1 The science classes offered in most elementary and middle schools teach students the theories and laws of science. The field is presented as a collection of facts and rules and all too often does not convey the creativity, experimentation, and curiosity so fundamental to the scientific world. As a result, many students shy away from science at a young age. These early impressions have a major impact on the number of students that go on to earn a degree in science. In fact, eighth grade children (approximately age 13 years) that identified that they wanted to pursue a career in the sciences were considerably more likely to receive a sciencerelated bachelors degree (1.9× in life science and 3.4× in physical sciences and engineering).2 Accordingly, it is critical that new programs are developed to promote the interest of students at a young age. Interest-arousing classroom activities and demonstrations have the power to function as valuable preludes for chemical education.3 Some of the benefits of these activities include keeping students engaged, promoting discussion, and helping students form connections between observations and learned chemical concepts.
Figure 1. Basic structure of common anthocyanins and their aglycones (anthocyanidins). The color profiles of anthocyanidins and their corresponding anthocyanins are comparable.
natural products, are responsible for the colors of many flowers, fruits, and leaves yielding a range of colors from orange to blue.8,9 Botanists and chemists alike have long been fascinated by flavonoids. The aglycone forms of these pigments are known as anthocyanidins. These compounds are most often found as the glycosylated derivatives, which are called anthocyanins.10,11 The biosynthesis of these pigments9 and their biological and ecological significance12,13 have been studied for more than 90 years.14 Geissman15,16 and Huntress17−19 have published
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ANTHOCYANINS: FAMILIAR NATURAL PRODUCTS Natural products provide the ideal model with which to ignite an interest in science in general and chemistry in particular as their functional diversity influences nearly every aspect of our © XXXX American Chemical Society and Division of Chemical Education, Inc.
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Figure 2. Cyanidin undergoes dramatic color changes upon exposure to solutions of varying pH values.20
several articles in this Journal describing these compounds in great detail. The color of these pigments varies greatly depending upon the number of hydroxyl group substitutions and the pH of the solution. Figure 2 illustrates how the pH of the environment affects the observed color of an anthocyanidin.20 Several interesting activities and laboratory experiments have been developed using plant pigments to discuss chemistry concepts. Anthocyanins in particular have been used most often in the discussion of acid−base chemistry.21−25 Anthocyanins have also been utilized for the introduction of chemical equilibrium26 and chromatography.23,27 Other topics that could be related to these pigments include aromaticity, conjugation, molecular architecture, functional groups, constitutional isomers, resonance structures, the hydrophobic effect, and metal chelation. We became interested in developing a new application of anthocyanins, however, and their use in the introduction of natural product isolation and identification from biological sources. Herein, we present a natural products activity in which students of all age groups analyzed extracts from botanical sources to determine the pigments responsible for the colors in several common fruits and vegetables.10,28 In this hands-on activity, students extracted pigments from a botanical source. Upon acid−base treatment of their extract, the students obtained distinct pigment profiles. Students identified which anthocyanin was present in their biological material by comparison of the acid−base profile of their extract to those of several commercially available pigment standards. The experiment functioned most efficiently when students worked in small groups of two or three and is well suited for middle school children. This activity requires 30−60 min to complete depending on the number of biological materials analyzed by each group.
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Table 1. Botanical Sources and Their Major Anthocyanins Source Red Rose Cranberry (Vaccinium erythrocarpum) Red Cabbage (Brassica oleracea var. capitata f. rubra) Radish (Raphanus sativus) Blackberry Concord Grape (Vitis labrusca)
Major Anthocyanin Present
Approximate Aglycones (%)
Cyanidin-3,5-diglucoside29 Peonidin-3-galactoside30
90−10029 46−5128,31
Cyanidin-3-galactoside30 Cyanidin-3-diglucoside-5-glucoside28,32
43−4728,31 10031
Pelargonidin-3,5-diglycoside28,33
10028,31
Cyanidin-3-glucoside34 Delphinidin-3-glucoside35
10028,31 5931
Cyanidin-3-glucoside34
2031
glycosylated derivatives of these anthocyanidins are prohibitively expensive or unavailable. The color profiles of anthocyanidins and their corresponding anthocyanins are comparable. Equipment and Chemicals
MATERIALS FOR THE ACTIVITY
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Botanical Sources
• Small plastic test tube (5 test tubes needed for each biological sample) • Wood or plastic stir stick to pulverize the botanical sample • Pipet or dropper to divide solutions • Water • Water/ethanol (1:1) solution • 1 M aqueous HCl (pH 0) • 0.5 M phosphate buffer (pH 7.4) • 1 M aqueous NaOH (pH 14) • Small scissors • Vegetable peeler
PREPARATION OF STANDARD PIGMENT PROFILES A small quantity of each pigment (approximately 0.5 mg) was dissolved in 2 mL water/ethanol (1:1) and the solution was divided into four test tubes (0.5 mL each). The concentrations of the standard pigment solutions were approximately 0.25 mg pigment/mL. Each test tube was treated with one of the following solutions: Test tube 1: 0.5 mL water and one drop 1 M HCl (∼50 μL) Test tube 2: 0.5 mL water Test tube 3: 0.5 mL 0.5 M phosphate buffer, pH 7.4 Test tube 4: 0.5 mL water and one drop 1 M NaOH (∼50 μL) The test tubes were agitated and their colors recorded. The instructors collected digital images for each standard, which
Several common biological samples were chosen including red cabbage, red radish, cranberry, red rose, concord grape juice, and blackberry, all of which are readily available at grocery stores. There are ample literature sources citing anthocyanins in many common flowers, fruits, and vegetables, and this information can be used by instructors to create their own lists of botanical sources.11,28 The anthocyanins in our biological sample set are listed in Table 1. The instructors purchased standards (cyanin chloride, delphinidin chloride, pelargonidin chloride, and malvin chloride) for which standard pigment profiles were prepared. The compounds produced by the biological sources are the glycosylated forms of the pigments (anthocyanins), whereas two of the standards used in our activity (delphindin and pelargonidin) are the aglycone forms (anthocyanidins); the B
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Figure 3. The pH profiles of the materials. Students compared their results (A) with those of the profiles generated by instructors from a pure standard of the major pigments present in these plants (B).
tube set. The obtained pigment profiles were compared to those of the known pigment standards to assign the represented pigment in each biological sample. As mentioned above, most biological samples contain multiple anthocyanin derivatives and the pigment profiles obtained by the students may not exactly match the profiles of the commercial standards. With this in mind, students were asked to pick the pigment that best represented their sample. Digital images of the pH profiles for these sample materials can be found in Figure 3. For instructors working with young children outside of a laboratory setting and who are concerned with using the above acid and base solutions, the activity can be modified by using common kitchen ingredients: distilled white vinegar (pH ∼3) or filtered pure lemon juice (pH ∼3), and a saturated aqueous baking soda solution (pH ∼9). Please refer to the Supporting Information for additional information regarding these suggested modifications.
were included in the demonstration rubric for students (see the Supporting Information). The stability of anthocyanins in aqueous solution, particularly at high pH, is poor,11,36 so it is important to record observations and take pictures to document the colors immediately. The final pH values for each solution were approximately 1, 5, 7, and 13 for test tubes 1, 2, 3, and 4, respectively. The pH of these solutions were determined using colorpHast pH-indicator strips with a pH range of 0−14.
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PREPARATION OF BOTANICAL SOURCE PIGMENT PROFILES Approximately 0.5−1 g of each selected botanical source was required to conduct an experiment. Rose petal samples were harvested by carefully cutting each petal from the flower without including the sepal. A single petal provided sufficient material. The cabbage was cut into small strips (two 1-in. strips per student). Adequate pigment could also be obtained from the skin of one radish, the peel of four cranberries, one-half of a blackberry, or 1 mL of grape juice. The chosen biological sample was placed into a plastic test tube and 2 mL of the water/ethanol (1:1) solution was added. The sample was carefully crushed until the solution took on the color of the sample; this was done with a wooden applicator stick (any blunt plastic or wooden object will do). The resulting solution was aliquoted into four test tubes (approximately 0.5 mL each). As before, the following solutions were added to each test tube (each test tube will contain one of the four solution described above): Test tube 1: 0.5 mL water and one drop 1 M HCl (∼50 μL) Test tube 2: 0.5 mL water Test tube 3: 0.5 mL 0.5 M phosphate buffer, pH 7.4 Test tube 4: 0.5 mL water and one drop 1 M NaOH (∼50 μL) The test tubes were agitated and the observed colors recorded. This can also be done by collecting a digital image of the test
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SAFETY AND HAZARDS Safety goggles and gloves should be worn during the entire activity. Acid and base solutions are corrosive and should be prepared with caution to avoid skin and eye contact. Ethanol is a flammable liquid and should be kept away from heat sources. Good safety standards should be practiced during the activity and students should be urged to be careful when pulverizing the samples to avoid splashing the solution mixture. Caution must be taken when handling acid and base droppers to avoid skin and eye contact. Even when diluted, acid and base solutions can cause severe irritation. Provide liquid and solid waste containers for the proper disposal of all samples. Solutions can be neutralized and discarded as aqueous waste.
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DISCUSSION The described activity can be tailored toward a number of different educational levels. This experiment was performed successfully with 200 Girl Scout Brownie Troop members C
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materials, demonstrating the utility of this activity to promote deductive reasoning. Having multiple biological samples available had several advantages: students that preferred working in groups each picked a different sample, and were able to compare outcomes. Also, students were able to validate each others’ results and working together in small groups encouraged discussions. Most of the participants, particularly those in the younger age groups, appreciated the hands-on aspect of the experiment. They enjoyed being able to perform solution additions and sample mixing, and to directly effect the color changes they observed in their samples. Furthermore, comments collected from the rubric indicated students were engaged and thinking about their observations during the activity. It was evident from students′ questions and discussions that several chemical concepts mentioned earlier could be successfully tied into a lecturedbased activity or demonstration.
ranging in age from 7 to 10 years. The activity was also tested during our annual chemistry department open house day, which involved a wide age range of attendees from 5 to 18 years old. The instructors provided worksheets to allow the participants to tabulate their observations during the experiment and to record their hypotheses about the pigments present in their biological samples. To give the participants the opportunity to express their understanding in their own words, an assessment questionnaire was also provided.37 Results obtained through these questionnaires were used to determine the efficacy of the activity. They could also be used to modify or strengthen the activity for future use. On the basis of these questionnaire results, two major conclusions were drawn: (1) the pigment composition of some of the botanical sources proved to be easier for the participants to assign than others, and (2) they found the activity exciting and engaging. As expected, the pigment profiles of some of the biological samples did not exactly resemble those of the commercial standards (Figure 3). For example, although all four of the cabbage pigment profile test tubes were very similar to the cyanin standard, only test tubes 1−3 for the radish sample resembled the pelargonidin standard, and for the rose sample matched the cyanin standard. It was expected that the blackberry, grape, and cranberry samples to be hardest to identify, as only 1−2 of the test tubes matched their respective standard samples and, in the case of grape and cranberry, consist of mixtures of more than one anthocyanin. As expected, students were readily able to correctly identify cyanin as the major pigment in red cabbage (87% identified the correct pigment). Although cranberries have both peonidin and cyanin pigments, most students were able to identify the presence of cyanin in the extracted profiles (78%); due to high cost, a peonidin standard was not obtained for our demonstration. Fewer students correctly identified the major pigments in the blackberry (27% correct) and concord grape juice (21% correct) samples; the red rose and radish samples proved most difficult to ascertain (no one correctly identified either of these samples). In all of these samples, at least one and up to three of the test tubes did not match the standard test tubes, presumably leading to confusion. It is unclear why, in particular, students had trouble identifying the presence of pelargonidin in the radish sample; it is possible that the students had trouble with sample preparation which led to colors being more unlike the standard samples than those that were obtained as illustrated in Figure 3. Encouraging students to mix their final solutions completely should help them better identify their pigments, as the single drops of the acid and base solutions added to the biological extracts can adhere to the test tube sides, giving an inadequate pH to obtain the desired colors. Furthermore, a simple filtration step can be added to assist students having difficulties identifying the pigments due to cloudiness. Filter paper (or a coffee filter outside of a laboratory environment) can be used to filter the extracted solutions prior to addition of the acid and base solutions. Students with potential color vision deficiencies could be paired with a partner to help them identify the pigment of their biological source and colorblind charts can also be used to assist these students. Several students recognized that there were one or more colors that did not match exactly; these students selected the pigment that best resembled the pigment profile of their sample. Many participants provided clear rationale for the anthocyanins that they did select based upon the results they obtained versus the data that they were given for the standard
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ASSOCIATED CONTENT
S Supporting Information *
Digital images for the pigment profiles of the standards and the biological samples. A sample rubric is also included. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions §
K.C.A.G. and A.Y.O. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Amanda Mertsching (AXE, Chemistry Department, Indiana University, Bloomington, IN) for organizing the volunteers for the Brownie Math and Science Day Event, and AXE members for volunteering. We thank James S. Clark (Chemistry Department, Indiana University, Bloomington, IN) for arranging a laboratory in which the activity was held during the ACS Chemistry Week open house at Indiana University. Daniel S. Meyers is thanked for volunteering as an assistant during this activity (Chemistry Department, Indiana University, Bloomington, IN). We thank the Research Corporation for Science Advancement (Cottrell Scholar Award) and an NSF CAREER Award for funding.
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