In the Laboratory
Determination of Flavonoids in Wine by High Performance Liquid Chromatography Celeste da Queija, M. A. Queirós, and Ligia M. Rodrigues* Departamento de Química-IBQF, Universidade do Minho, Campus de Gualtar, 4700-320 Braga, Portugal
Flavonoids are widely distributed in plants and almost 2000 flavonoids are known. Important dietary sources of these compounds are vegetables and fruits, and also beverages, which can account for 25–30% of our total daily intake (1). Flavonoids have possible antiinflammatory, antiallergenic, anticarcinogenic, and protective cardiovascular effects (2–4). The general structure of flavonoids is presented below. They commonly occur as O-glycosides with sugars bound to the C3 position. Flavonoids have been identified and quantified in fruits and vegetables (5), teas, fruit juices, and wines (4). Quercetin, myricetin, apigenin, and kaempferol are the most investigated as potentially anticarcinogenic. R1 3´
OH
2´ 4´ 8
HO
O
7
2 6
4 5
OH
6´
5´
3
R3
R2
Flavonoid
R1
R2
R3
Quercetin
OH
H
OH
H
OH
Kaempferol
H
Myricetin
OH
Apigenin
H
OH OH H
H
O
This paper describes a comprehensive experiment that analyzes flavonoids in wines. The laboratory work includes the hydrolysis of the O-glycosides to obtain the flavonoids, which are then identified and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection. All textbooks in instrumental analysis devote a section to chromatographic methods, which includes HPLC, a widely used technique available in most laboratories. The theoretical basis of HPLC is introduced as part of the curriculum in chemistry courses and the experiment described here gives the student the opportunity to practice and apply concepts previously learned. So, we feel that this work is appropriate for teaching classes in analysis and is also very stimulating because it makes the students aware of the interaction of chemistry with our daily lives (food and health). This experiment can also be adapted to other appropriate foodstuffs. Experimental Procedure
Instrumentation and Operating Conditions The HPLC system used comprised a Shimadzu LC-6A pump, a Rheodyne injector, a UV/V Shimadzu SPD-6AV detector set at 370 nm, a Shimadzu C-R6A recorder/integrator, and a Merck inox steel column (4 × 250 mm), Lichrospher 100 RP-18 (5 µm) in LiChroCART 125-4. Samples of 10 µL were injected at a flow rate of 0.9 mL/min, at 1.5 × 100 kg/cm2.
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Chemicals and Stock Solutions Reference flavonoids quercetin and kaempferol were purchased from Sigma Chemical Co.; myricetin and apigenin, from Aldrich Chemical Co. Acetonitrile and methanol (HPLC grade) and potassium dihydrogen phosphate were purchased from Aldrich Chemical Co. Portuguese red wines from five marked regions (Bairrada, Dão, Alentejo, Douro, and Minho) were purchased from local retailers. The mobile phase was acetonitrile/0.025 M dihydrogen phosphate buffer 30/70 (v/v) at pH 5.2 (adjusted with concentrated HCl). This solution was filtered through a Millipore filter (0.45 µm) and sonicated in an ultrasonic bath for 5 minutes. The solvent mixture used contained 50 mL of methanol, 30 mL of water, and 20 mL of 6 M HCl. A 1.00 × 10᎑3 M standard stock solution of the flavonoids was prepared by dissolving the appropriate mass of the flavonoid in the solvent mixture. This solution was stored at 4 °C, protected from room light with aluminium foil. The mobile phase should be collected in a labeled container for waste disposal.
Sample Preparation In a 250-mL round-bottom flask a 15.00-mL aliquot of wine is added to a mixture of 25 mL of methanol, 15 mL of water, and 15 mL of 6 M HCl. The mixture is refluxed for 4 hours. After cooling, the mixture is quantitatively transferred to a 100-mL volumetric flask and diluted to volume with the solvent mixture. The solution is filtered through a Millipore filter (0.45 µm) and stirred in an ultrasonic bath for 5 minutes. Hazards Acetonitrile must be handled with care to prevent inhalation and contact with skin. Results and Discussion This laboratory work has been carried out in two 4-hour sessions. At the beginning of the first session students prepare the unknown for analysis by hydrolyzing the wine sample supplied by the instructor. The instructor prepares standard stock solutions containing myricetin and quercetin in the appropriate solvent at 1.00 × 10᎑3 M each. The standard solutions have concentrations in the range of 1.0 × 10᎑4 to 2.0 × 10᎑6 M for myricetin and 7.0 × 10᎑5 to 5.0 × 10᎑6 M for quercetin. Each student is responsible for one unknown and the data for standards are shared. Standard solutions are injected directly into the chromatograph in duplicate. The mean retention time for each flavonoid is calculated and used
Journal of Chemical Education • Vol. 78 No. 2 February 2001 • JChemEd.chem.wisc.edu
In the Laboratory Table 1. Data for Standard Solutions of Myricetin and Quercetin Mean Peak Area (A)
Flavonoid t R /min C/(mol dm᎑3)
Figure 1. Chromatogram of a standard flavonoid mixture. (A) Myricetin (tR = 6.78 min). (B) Quercetin (tR = 12.30 min). (C) Apigenin (tR = 21.71 min). (D) Kaempferol (tR = 24.20 min)
Regression Coefficients (A = a + bC)
R2
6.78
2.00 × 10᎑6 1.00 × 10᎑5 1.50 × 10᎑5 5.00 × 10᎑5 7.00 × 10᎑5 1.00 × 10᎑4
2.39 × 103 6.01 × 103 9.47 × 103 3.31 × 104 4.31 × 104 6.18 × 104
a = 7.10 × 102 b = 6.16 × 108
.999
Quercetin 12.30
5.00 × 10᎑6 1.00 × 10᎑5 1.50 × 10᎑5 3.00 × 10᎑5 5.00 × 10᎑5 7.00 × 10᎑5
6.72 × 102 1.54 × 103 2.43 × 103 4.94 × 103 7.16 × 103 9.45 × 103
a = 3.31 × 102 b = 1.35 × 108
.990
Myricetin
Table 2. Myricetin and Quercetin Content of Portuguese Wines Figure 2. Chromatogram of a hydrolyzed wine sample. (A) Myricetin (tR = 6.78 min). (B) Quercetin (tR = 12.30 min).
Myricetin Region
Brand
Area a
Area a
C/ mg dm᎑3
Minho
Ponte de Lima
1.07 × 104
5.18
4.71 × 103
10.97
Dão
Grão Vasco
7.65 × 103
3.58
2.95 × 103
6.55
Alentejo
Vinho do Marco 6.10 × 103
2.78
3.78 × 103
8.61
Bairrada
Borlido
4.86 × 103
2.14
1.95 × 103
4.06
Scarpa
7.80 × 10
3.66
2.48 × 10
5.38
Douro
aAverage
for identification by comparison with available data provided by the instructor. Figure 1 displays a chromatogram obtained for a solution of flavonoids. From the known standard concentrations and mean peak areas obtained by each group a calibration graph for each flavonoid is plotted. The students are asked to determine the regression lines and correlation coefficients by the least-squares method. Typical analytical results are presented in Table 1. The second session is used to run each of the unknown samples in triplicate. Figure 2 shows a typical chromatogram of a hydrolyzed wine sample. The concentrations of the flavonoids are determined by interpolation from the corresponding regression lines. The concentration of each component in the wine is then calculated and expressed in mg/dm3. The results for wines from five regions are presented in Table 2. Having in mind the nature of the C18 reverse-phase column, students are asked to explain the chromatogram in terms of the polarity of the compounds analyzed and to predict the relative peak positions of apigenin and kaempferol. They are also requested to discuss the effect of changing the mobile phase composition and pH on the chromatogram.
Quercetin
C/ mg dm᎑3
3
3
of six determinations.
Conclusion The analysis of flavonoids in wines is very stimulating for the students because it deals with a common everyday product. Verification and quantification of compounds whose preventive and therapeutic effects have been reported helps to engage students in the discipline of analytical chemistry. This laboratory work provides a comprehensive experiment that gives students an opportunity to combine preparative work with more advanced procedures used in instrumental analysis. Literature Cited 1. Kuhnau, J. World Rev. Nutr. Diet 1976, 24, 117. 2. Pamuck, A. M.; Yalciner, S.; Hatcher, J. F.; Bryan, G. T. Cancer Res. 1980, 40, 3468. 3. Waterhouse, A. L. Chem. Ind. 1995, 338. 4. Hertog, M. G. L.; Hollman, P. C. H.; Putte, B. J. Agric. Food Chem. 1993, 41, 1242. 5. Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B. J. Agric. Food Chem. 1992, 40, 2379.
JChemEd.chem.wisc.edu • Vol. 78 No. 2 February 2001 • Journal of Chemical Education
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