Quantification of Catechins in Tea Using Cyclic ... - ACS Publications

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

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Quantification of Catechins in Tea Using Cyclic Voltammetry Alice H. Suroviec,* Katarina Jones, and Grace Sarabia Department of Chemistry & Biochemistry, Berry College, Mt. Berry, Georgia 30149, United States

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S Supporting Information *

ABSTRACT: Flavonoids have been studied extensively for their antioxidant properties. Specifically, the flavonoids in tea, such as catechin and epigallocatechin gallate, have been shown to have positive health effects. These compounds are also oxidizable, so the concentrations of these compounds in a given kind of tea can be determined using cyclic voltammetry. Green, black, oolong, and herbal teas were each diluted using pH 7.0 phosphate buffer, analyzed using cyclic voltammetry, and compared to the standards of epigallocatechin gallate, catechin hydrate, and catechol. The effect of brewing temperature on the concentration of catechins was also examined. Students learned about the common flavonoids in tea and the effect of brewing temperature on the concentration of catechins in solution.

KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Electrochemistry, Food Science

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INTRODUCTION Tea and coffee have been popular beverages for hundreds of years and are also able to provide a significant source of phenolic compounds for the diet.1 Most common kinds of tea are produced from leaves of the Camellia sinenisis bush. Tea leaves from the Camellia sinenisis are hand picked, washed, fermented and then withered. The amount of fermentation (oxidation) leads to the different types of tea. Black tea is fully oxidized and oolong tea is partially oxidized while green tea is only dried. Tea is a complex mixture of components containing flavonoids, caffeine, and chlorophyll, as well as amino acids. Drinking tea has been associated with inhibition of certain types of cancer,1−3 prevention of heart disease,2,3 as well as increased muscle endurance.4 The health benefits of drinking tea have been correlated to the flavonoid components as these are natural antioxidants.5−7 Most of the flavonoid components found in tea belong to the catechin family, which can be as much as 30% w/w of the tea solid.2 Catechin monomers have been shown to have radical-scavenging abilities.1 The most common of which are four pairs of stereoisomers: catechin and epicatechin; gallocatechin and epigallocatechin; catechin gallate and epicatechin gallate; and gallocatechin gallate and epigallocatechin gallate. For the purpose of this lab we examined only catechin (Figure 1B) and epigallocatechin gallate (Figure 1C) as well as the related molecule catechol (Figure 1A). Molecules such as catechin can be oxidized into a quinone through a semiquinone intermediate. This is a two-electron oxidation. The potential at which this oxidation occurs can help identify the molecule. In addition, the current produced from this oxidation can be used to determine the concentration of the molecule in solution. © XXXX American Chemical Society and Division of Chemical Education, Inc.

Catechins are found naturally in many foods such as tea, fresh fruit (apples, apricots, and cherries), wine, and chocolate.2,5 Research has shown that extraction of catechins from teas is greater with stirring and at higher water temperatures.2 It has been shown that, during the brewing process of tea, considerable amounts of catechins are epimerized at the 2-position.8 This would mean, for example, that any epicatechin in the tea sample would convert into catechin (Figure 2). These can be distinguished as they have different oxidation potentials. So, as the brewing continues, the current at the peak potential for the catechin increases. Students brewing the tea samples at different temperatures will see the concentration of catechin increase with increasing brewing temperature by measuring the current at the appropriate peak potential. In this paper, we describe a rapid method for identification and quantification of catechins in green, black, oolong, and herbal teas. While there have been a few experiments published in the Journal of Chemical Education to analyze catechins by HPLC,3,9,10 spectrophotometry,9 and TLC,11 this presents the first electrochemical determination of catechins. The use of electrochemistry in the undergraduate laboratory is often missing, and this lab provides a “real-life” experiment that will appeal to students. The use of cyclic voltammetry also requires no sample preparation as would be needed for other spectrophotometric techniques. The tea samples can be brewed and tested within minutes. This allows the students to Received: June 28, 2018 Revised: November 9, 2018

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DOI: 10.1021/acs.jchemed.8b00503 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

ments and their applicability to food sciences. There are 5 learning objectives: 1. Students will demonstrate understanding of oxidation and reduction. 2. Students will use cyclic voltammetry to determine Ep (peak potential) and ip (peak current) for a given flavonoid. 3. Students will use electrochemical measurements to prepare a calibration curve and determine regression lines, correlation coefficients, and uncertainties associated with their plot. 4. Students will identify the type(s) of flavonoids found in tea and calculate average concentrations from different types of tea. 5. Students will demonstrate the heat epimerization of flavonoids through measuring the effect of brewing temperature on concentration.

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EXPERIMENTAL PROCEDURES

Equipment and Materials

An electrochemical workstation was used to acquire data and analysis. A CH instrument (CHI 630) was used to acquire cyclic voltammograms. A scintillation vial was used to accommodate the three-electrode experimental configuration (Figure 3). A Teflon cap with appropriately sized holes was Figure 1. Chemical structure of catechins found in green, black, and oolong teas: (A) catechol, (B) catechin, and (C) epigallocatechin gallate.

Figure 3. Three-electrode setup for analysis of flavonoids in tea. The green lead is attached to the working electrode. The white lead is attached to the reference electrode, and the red lead is attached to the platinum wire counter electrode. The cell is a 20 mL scintillation vial.

used to hold the electrodes in place. A ring-stand and clamp were used to support the vial. Care must be taken to ensure that the electrodes are not touching each other in solution and that the alligator clips are not touching on the outside of the vial. The standard three-electrode system used in this paper included a glassy carbon working electrode, a Ag/AgCl reference electrode, and a platinum wire counter electrode. The buffer used was 50 mM phosphate buffer at pH 7.0. A buffer must be used to provide the ions needed in solution for an electrochemical experiment. The resistance of the solution would be too high in deionized water for meaningful results. The buffer of pH 7.0 is chosen as that is the approximate pH of water. The teas analyzed were consumer grade black, green, oolong, and chamomile. The standards used were epigallocatechin gallate, catechin hydrate, and catechol (all purchased

Figure 2. Chemical structures of (A) (−) epicatechin (2R, 3R) and (B) (+) catechin (2R, 3S). The epimerization occurs at the 3position of epicatechin to become catechin.

quickly analyze many samples in the given lab time. This lab can be completed in two 3 h laboratory periods by upper-level undergraduates in courses such as analytical chemistry or instrumental analysis.

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LEARNING OBJECTIVES The laboratory exercise presented in this paper provides students with experiences related to electrochemical measureB




Figure 4. Cyclic voltammogram of (A) 0.5 mM epigallocatechin gallate, (B) 0.05 mM catechin, and (C) 0.05 mM catechol standards measured at a 2 mm glassy carbon electrode at 0.1 V/s in 50 mM phosphate buffer pH 7.0 with Ag/AgCl reference electrode.

standard, 100 mL of approximately 1.0 mM was prepared in advance. These should be prepared in amber bottles and stored at 4 °C. Enough standard was made by the instructor so students can make serial dilutions from the stock. Students prepared their samples by brewing a tea bag for 3 min in 200 mL of stirred deionized water. The temperature effects of the brewing were examined by brewing tea at 50, 60, 70, 80, 90, and 100 °C. Once the tea was brewed, 1.00 mL of tea was diluted in to a final volume of 50.0 mL of 50.0 mM phosphate buffer pH 7.0. During the two 3 h laboratory periods, a pair of students analyzed 4 types of tea: black, green, oolong, and herbal (chamomile). Students worked in pairs to make the standards and tea samples. After preparing the standards the students identified the Ep of their standard using cyclic voltammetry. After identifying the Ep for the standard, the students prepared a calibration curve using a range of concentrations (0.05−0.5 mM). The cyclic voltammetry was performed using a 2 mm glass carbon electrode (GCE), a platinum wire counter electrode,

from MilliporeSigma). Detailed solution preparation, CAS numbers, and electrode cleaning information are provided in the Supporting Information. Analytical Procedure

The laboratory procedure consists of (1) preparing standards and a calibration curve that familiarizes students with cyclic voltammetry and (2) an analysis of brewing temperature on catechin concentration in common tea samples. The experiments were carried out in groups of two to three students in two 3 h lab periods, and discussion questions and calculations were assigned within the laboratory report due 1 week after the lab concludes. The experimental protocol has been run several times. Specific questions on the lab reports demonstrated that students understood oxidation and reduction and were able to successfully calculate uncertainties associated with their calibration plots. Prior to the laboratory session, concentrated standards epigallocatechin gallate, catechin hydrate, and catechol (purchased from MilliporeSigma) were prepared for the students in 50 mM phosphate buffer at pH 7.0. For each C




Figure 5. Representative student calibration data for the analysis of epigallocatechin gallate (EGCG), R2 = 0.961, and catechin, R2 = 0.969.

100 °C. In a solution as complex as tea it can be expected that there will be many compounds that can be oxidized. However, only flavonoids are expected to produce a current at potentials less than 400 mV.2 As can be seen, the Ep is at 0.280 V which matches well with the catechin standard and with the reference cyclic voltammograms of Earl Gray.2 The cyclic voltammograms of green and oolong teas have peak potentials that most closely match epigallocatechin gallate which is expected to be the major flavonoid present in those teas.2 Students compared the ip of the tea sample with the calibration curves and, adjusting for dilution, calculated the concentration of the appropriate standard in their sample.12 Students were also able to calculate the uncertainties associated with their catechin concentration by propagating the error from their calibration curve. Of the teas examined by the students, Earl Gray had the highest catechin concentration and chamomile had the lowest. It has been shown that during the brewing process of tea considerable amounts of catechins are epimerized at the 2position.8 This would mean for example that any epicatechin in the tea sample would convert into catechin which is measurable by the difference in the peak potentials for the two compounds. Students brewed the tea samples at different temperatures and saw the apparent concentration of catechin increase with increasing brewing temperature. Figure 7 shows a typical cyclic voltammogram for Earl Gray. The students were able to see increasing apparent catechin concentration with brewing temperature. Students were then able to calculate the concentration of catechin for each of the brewing temperatures for a standard serving size (1 tea bag/8 oz water).

and a silver−silver chloride reference electrode. A scintillation vial containing 10.0 mL of sample is used as the electrochemical cell for these experiments. These samples were analyzed at a scan rate of 0.1 V/s over the range 0.05−0.65 V. The samples were all run in triplicate to ensure consistency of results. The electrodes were polished between each run to prevent electrode fouling.

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HAZARDS There are no significant hazards in this lab. Lab coats, gloves, and protective eyewear should be worn at all times during the experiment. Catechol, epigallocatechin gallate, and catechin are all skin and eye irritants and would be harmful if swallowed. At the end of the experiment all waste should be disposed of properly.

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RESULTS

Figure 4 shows a typical cyclic voltammogram of the epigallocatechin gallate standard. Epigallocatechin gallate has two oxidation peaks; for this experiment the more predominant peak at 0.180 V was used. From this the student identified the Ep (peak potential) and recorded the ip (current at the peak potential). After identifying the Ep for the standard, the students prepared a calibration curve using a range of concentrations (0.05−0.5 mM) as shown in Figure 5. Students used the linear least-squares method of determining regression lines, correlation coefficients, and uncertainties associated with their plot. Table 1 lists the expected peak potential of the components of the study.2 Figure 6 shows a typical cyclic voltammogram of Earl Gray tea (a black tea blend flavored with bergamot oil) brewed to

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CONCLUSIONS This laboratory experiment introduced students to basic cyclic voltammetry for the purpose of quantitative analysis of commercial teas. Students had the opportunity to learn about flavonoids, heat epimerization, and the importance of instrumental analysis in the field of agriculture and food sciences.

Table 1. Expected Peak Potentials (V) of Flavonoids Studied Compound

Ep,a (V)

Catechin Epigallocatechin gallate Catechol

0.250 0.08, 0.180 0.330 D




Figure 6. Cyclic voltammogram of dilute Earl Gray tea brewed at 100 °C and then cooled to room temperature. A 2 mm glassy carbon electrode at 0.1 V/s in 50 mM phosphate buffer pH 7.0 with Ag/AgCl reference electrode was used.

Figure 7. Earl Gray brewing temperature effect on catechol concentration. A 2 mm glassy carbon electrode at 0.1 V/s in 50 mM phosphate buffer pH 7.0 with Ag/AgCl reference electrode was used.

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ASSOCIATED CONTENT

ACKNOWLEDGMENTS Financial support for this work was generously provided by the Faculty Development Fund at Berry College.

* Supporting Information S

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00503. Notes for instructors and student procedure (PDF, DOCX)

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REFERENCES

(1) Sharangi, A. B. Medicinal and therapeutic potentialities of tea (Camellia sinensis L.) − A review. Food Res. Int. 2009, 42, 529−535. (2) Kilmartin, P. A.; Hsu, C. F. Characterisation of polyphenols in green, oolong, and black teas, and in coffee, using cyclic voltammetry. Food Chem. 2003, 82, 501−512. (3) Freeman, J. D.; Niemeyer, E. D. Quantification of Tea Flavonoids by High Performance Liquid Chromatography. J. Chem. Educ. 2008, 85 (7), 951−953. (4) Murase, T.; Haramizu, S.; Shimotoyodome, A.; Nagasawa, A.; Tokimitsu, I. Green tea extract improves endurance capacity and increases muscle lipid oxidation in mice. American Journal of Physiology-Regulatory, Integrative, and Comparative Physiology 2005, 288 (3), R708−R715. (5) Gardner, E. J.; Ruxton, C. H. S.; Leeds, A. R. Black tea, helpful or harmful. Eur. J. Clin. Nutr. 2007, 61, 3−18.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alice H. Suroviec: 0000-0002-9252-2468 Notes

The authors declare no competing financial interest. E




(6) Arts, I. C. W.; Hollman, P. C. H. Catechin intake might explain the inverse relation between tea consumption and ischemic heart disease. Am. J. Clin. Nutr. 2001, 74, 227−232. (7) Alkhalidy, H.; Wang, Y.; Liu, D. Dietary Flavonoids in the prevention of T2D: An overview. Nutrients 2018, 10 (4), 438−471. (8) Ikeda, I.; Kobayashi, M.; Hamada, T.; Tsuda, K.; Goto, H.; Imaizumi, K.; Nozawa, A.; Sugimoto, A.; Kakuda, T. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate. J. Agric. Food Chem. 2003, 51, 7303−7307. (9) da Queija, C.; Queiros, M. A.; Rodrigues, L. M. Determination of flavonoids in wine by high performance liquid chromatography. J. Chem. Educ. 2001, 78, 236−237. (10) Curtright, R. D.; Rynearson, J. A.; Markwell, J. Fruit anthocyanin: Colorful sensors of molecular milleau. J. Chem. Educ. 1994, 71, 682−684. (11) Curtright, R. D.; Rynearson, J. A.; Markwell, J. Anthocyanins: Model compound for learning more about pH. J. Chem. Educ. 1996, 73, 306−309. (12) Henning, S. M.; Fajardo-Lira, C.; Lee, H. W.; Youssefian, A. A.; Go, V. L. W.; Heber, D. Catechin Content of 18 teas and a green tea extract supplement correlates with the antioxidant capacity. Nutr. Cancer 2003, 45, 226−235.

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Quantification of Catechins in Tea Using Cyclic ... - ACS Publications

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