Instructor Information
JCE Classroom Activity: #76
I Screen, You Screen, We All Screen for Phenolics Michael B. Sady Department of Chemistry, Western Nevada Community College, Douglas Campus, Minden, NV 89423;
[email protected] In this Activity, students use a colorimetric visualization test to screen grape juice for phenolic content. Highly-colored complexes form in neutral aqueous solutions between iron(II) and the hydroxyl groups of phenolic compounds. Students use the test to examine differences in phenolic content of juices prepared with different processing methods.
Integrating the Activity into Your Curriculum This Activity allows simple and rapid screening of grape juice for phenolic content and comparison of different grape varieties and processing methods. It could be adapted for use with other fruit juices. It might be included in a biology or nutrition class. A trip to a farm or vineyard could be a delightful venue for field analysis of ripe grapes.
About the Activity
Juice samples are evaluated using a modified colorimetric visualization test normally used to estimate phenolic content in white grape juice and wine (3). Most materials are available in local stores. One pound of grapes is enough for about 10 student groups, depending on the grape variety. Backache pain reliever tablets are available in drug stores. Quercitin may be found in health food stores. Ammonium iron(II) sulfate hexahydrate and gallic acid may be purchased from a chemical vendor such as Fisher (4). Instructors need to prepare three solutions in advance and store each in a labeled brown glass dropper bottle: (1) A saturated aqueous solution of sodium bicarbonate (baking soda): mix 4–5 g of baking soda in 60 mL of distilled water. (2) A 1% (w/v) aqueous ammonium iron(II) sulfate reagent: mix 0.6 g of ammonium iron(II) sulfate hexahydrate in 60 mL of distilled water. (3) At least one of the following phenolic standards: magnesium salicylate tetrahydrate (found in backache pain reliever tablets, ref 5), quercitin, or gallic acid. For quercitin or gallic acid, prepare a concentration of 1 mg/mL: mix 0.06 g of the standard in 60 mL of 70% (v/v) 2propanol (rubbing alcohol):water. For magnesium salicylate tetrahydrate, obtain a backache pain reliever tablet containing 580 mg magnesium salicylate tetrahydrate. Break the tablet in half and crush into a powder using a spoon. Dissolve the powder in 30 mL of 70% rubbing alcohol. Mix 10 mL of the resulting solution with 50 mL of 70% rubbing alcohol. These solutions have a shelf life of at least one year. Instructors may also wish to have students perform the calculations for appropriate preparation of the first two standards.
perforated
Answers to Questions 1. Acids in the grape juice react with the sodium bicarbonate (a base) to produce carbon dioxide gas. The pH of grape juice tends to be acidic. 2. Ref 3 refers to shades of brown, red, and purple as indicative of a positive test for phenolic compounds. Testing standards simultaneously might enable students to infer the type of phenolic compounds present in a juice sample. 3. Differences in the intensity and shade of color may be observed for differently processed juices. Grape seeds contain higher concentrations of phenolic compounds than other parts of the fruit, so pressing seeds and keeping them in contact with the juice can increase phenolic content (6). 4. The color and intensity of the saved swab can be compared with results from color tests performed in the future. 5. A standard is a control that may be compared with experimental results to check that a test is working properly. A standard contains a known phenolic compound, which gives color-test results in a known color/intensity.
This Classroom Activity may be reproduced for use in the subscriber’s classroom.
Phenolics are important in the diet. They contribute to the taste of foods and are reported to have antioxidant properties that delay or inhibit cardiovascular deterioration, cancer, and aging (1). Flavonoids (which includes the fruit-skin pigments known as anthocyanins), hydroxycinnamate, stilbene, and hydroxybenzoate derivatives all have at least one phenolic OH group (see structural examples in Student Activity WWW ref 1 ). An Activity that illustrates the chemistry of plant and berry-skin pigments (the anthocyanins) has been published previously in this Journal (2). photo by Michael B. Sady
fold here and tear out
Background
References and Additional Related Activities (all sites accessed Sep 2005) 1. Waterhouse, A. L.; Walzem, R. L. Nutrition of Grape Phenolics. In Flavonoids in Health and Disease; Rice-Evans, C. A., Packer, L., Eds.; Marcel Dekker: New York, 1998; pp 359–385. 2. JCE Staff. Anthocyanins: A Colorful Class of Compounds. J. Chem. Educ. 1997, 74, 1176A–1176B. 3. Zoecklein, B. W.; Fugelsang, K. C.; Gump, B. H.; Nury, F. S. Wine Analysis and Production; Aspen: Gaithersburg, MD, 1999; pp 115–151, 459. 4. Fisher Scientific, Chicago, IL. http://www.fishersci.com. 800/766–7000. 5. Wright, S. W. The Oxidation of Iron in a Gel Using Consumer Chemicals. J. Chem. Educ. 2005, 82, 1633–1635. 6. Field Test to Rapidly Screen Grape Juice for Phenolic Content. http://www.wncc.edu/~mbsady/grape.htm. Acknowledgment: The author thanks Alice Sady (8th grade) and Astrid Sady for testing the procedure.
JCE Classroom Activities are edited by Erica K. Jacobsen and Julie Cunningham
www.JCE.DivCHED.org •
Vol. 82 No. 12 December 2005 •
Journal of Chemical Education
1808A
JCE Classroom Activity: #76
Student Activity
I Screen, You Screen, We All Screen for Phenolics
Try This
photo by J. J. Jacobsen, E. K. Jacobsen, J. L. Harris
Take a couple of bites from an apple or banana, put it down for a while, then go back and look at it. Is it brownish where you bit into it? Phenolic compounds (phenolics) in some fruits undergo oxidation in the open air causing the color change. In the presence of oxygen, enzymes in the fruits convert phenolics to quinones, which appear brown. Phenolics contribute to the flavor, acidity, and color of most fruits. There are many different phenolics and they have diverse chemical structures. Many are nutritious and healthful. The brown color in oxidized fruit is usually a sign that phenolics are present and will act in your body to prevent harmful oxidation. Color tests are often used to find out whether particular chemicals are present (to “screen” for these chemicals). For example, test strips that change color when dipped into a solution with a high sugar content are used by diabetics to test their urine. This Activity uses a color test to screen for phenolics in several samples of grape juice prepared in different ways. When phenolics combine with iron(II) in neutral aqueous solution, a colored substance forms. The more phenolics there are in the juice sample, the more intense the color is—a color test result might be light brown or dark brown. Different types of phenolics also give different colors in the test.
You will need: grapes (any variety), hand-held garlic press, four polystyrene cups, marker, coffee filter, measuring spoon, dropper, cotton swabs, distilled water, juice sample (see step 8), solutions from your instructor: saturated solution of aqueous sodium bicarbonate (baking soda), 1% aqueous ammonium iron(II) sulfate reagent, phenolic standard. Be Safe! Goggles should be worn during this Activity. __1. Prepare 25 mL of grape juice from grapes. (If using red- or blue-skinned grapes, first remove the skins. Why might the skins interfere with the test results?) Place a peeled None of the food products, grape and any seeds in a hand-held garlic press and gently squeeze the juice from the including grapes and their grape into a polystyrene cup. Then discard the pulp and seeds and continue the process juice, should be eaten. with another grape. Be prepared for squirting grape juice and sticky hands! __2. If the juice from step 1 contains any additional matter such as seeds or pieces of pulp, use a coffee filter to filter the juice into a new polystyrene cup. __3. Label two polystyrene cups “sample 1” and “sample 2”. Using a measuring spoon, transfer 2 teaspoons (10 mL) of the juice prepared in steps 1–2 into the cup labeled “sample 1”. Repeat with the cup labeled “sample 2”. __4. To neutralize the juice, add 1/2 teaspoon (2–3 mL) of a saturated solution of aqueous sodium bicarbonate (supplied by your instructor) to each sample. Swirl the cup to mix. Add additional saturated solution until there are no more escaping bubbles. __5. While swirling the cup, add 1–2 drops of 1% aqueous ammonium iron(II) sulfate reagent (supplied by your instructor) to each neutralized sample. Observe and record the color, intensity (darkness), and any other changes such as presence of a precipitate (insoluble solid). __6. Dip a separate cotton swab into each sample and let each swab dry. Label and save each swab (such as in a closed notebook) for future reference. __7. Mix 1 mL of a phenolic standard (supplied by your instructor) with 9 mL of distilled water in a dry, clean polystyrene cup. Repeat steps 5–6 with the standard sample (Note: the standard does not need to be neutralized, as in step 4.) Compare the intensity and color obtained with the first two juice sample results. __8. Repeat steps 4–6 with a new sample of grape juice prepared with a different processing method (use bottled or frozen concentrated grape juice, or juice extracted while pressing harder with the garlic press). Does the processing method affect the phenolic content? __9. Dilute the test solutions with tap water and discard according to your instructor’s directions.
Questions 1. 2. 3. 4.
When sodium bicarbonate is added to a sample, what causes bubbles? What does this suggest about a sample’s pH? Describe the results of the color test. Include the color, the intensity (darkness), and whether a precipitate formed. Does the processing method appear to influence the phenolic content? Explain. Dried cotton swabs from step 6 can retain the same color you originally observed in the color test. How do the swabs provide a guide for testing done at a later date? 5. What is the purpose of the phenolic standard in step 7?
Information from the World Wide Web (accessed Sep 2005) Phenolic compounds. http://www.biologie.uni-hamburg.de/b-online/e20/20d.htm Color your way to 5 a day: blues/purples. http://www.cdc.gov/nccdphp/dnpa/5ADay/campaign/color/blues.htm MedlinePlus: Antioxidants. http://www.nlm.nih.gov/medlineplus/antioxidants.html This Classroom Activity may be reproduced for use in the subscriber’s classroom.
1808B
Journal of Chemical Education •
Vol. 82 No. 12 December 2005 •
www.JCE.DivCHED.org
