Anal. Chem. 2002, 74, 2157-2161
Determination of Total Polyphenolic Content in Red Wines by Means of the Combined He-Ne Laser Optothermal Window and Folin-Ciocalteu Colorimetry Assay Otto´ Do´ka*,† and Dane Bicanic‡,§
Department of Physics, University of West Hungary, P.O. Box 90, 9201 Mosonmagyaro´ va´ r, Hungary, and Laser Photoaocustic Laboratory, Biophysics Division, Department of Agrotechnology and Food Sciences, Wageningen University and Research Centre, Dreijenlaan 3, 6700 HA Wageningen, The Netherlands
The He-Ne laser (632.8 nm) and the concept of optothermal window (OW), a variant of the open photoacoustic cell, were combined with the Folin-Ciocalteu colorimetry assay to quantitate phenolics in four red wines. The total polyphenolic content in selected red wines varied between 786 and 1630 mg/L gallic acid equivalent (GAE) as determined by OW-Folin-Ciocalteu colorimetry, which compares well to 778 and 1614 mg/L GAE obtained for the same wines by means of classical spectrophotometry. The originality and merit of OW colorimetry used here is that, unlike what is encountered in conventional spectrometry, no intermediate dilution step is required when total polyhenolics are determined in red wine. The precision, defined as the closeness to each other of 256 replicate readings of the OW signal, is generally better than 2%. Phenolic compounds in wines contribute to their characteristic color, flavor, and aroma1,2 and also act as the antioxidants.3,4 Various papers report on the beneficial effects of wine phenolics on human health; for example, increased dietary intake of phenolic antioxidants correlates with a reduced incidence of diseases such as coronary heart disease5 and certain forms of cancer.6 The amount and the composition of phenolics in wine depend on factors such as grape variety, vinification, and chemical processes occurring during the aging of wine.7-9 The total phenolic content in red wines varies typically between 2 and 3 g/L, which is double * Corresponding author. E-mail:
[email protected]. Fax: 36-96-566 620. † University of West Hungary. ‡ Wageningen University and Research Centre. § E-mail:
[email protected]. Fax: 31-317-482725. (1) Lee, C. Y.; Jaworski, A. W. Am. J. Enol. Vitic. 1997, 48, 277-280. (2) Karagiannis, S.; Economon, A.; Lanaridis, P. J. Agric. Food Chem. 2000, 48, 5369-5375. (3) Pellegrini, N.; Simenotti, P.; Gardana, C.; Brenna, O.; Brighenti, F.; Pietta, P. J. Agric. Food Chem. 2000, 48, 732-735. (4) Testolin, G. J. J. Agric. Food Chem. 1997, 45, 1152-1155. (5) Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B.; Kromhont, D. Lancet 1993, 342, 1007-1011. (6) Huang, M.-T.; Ferraro, T.; Ho, C.-T. In Food phytochemicals; Huang, M.-T., Osawa, T., Ho, C.-T., Rosen, R. T., Eds.; American Chemical Society: Washington DC, 1994; pp 2-16. (7) Andrade, P.; Seabra, R.; Ferreira, M.; Ferreres, F.; Garcı´a-Viguera, C. Z. Lebensm. -Unters. -Forsch. A 1998, 206, 161-164. 10.1021/ac011001s CCC: $22.00 Published on Web 03/22/2002
© 2002 American Chemical Society
that found in rose wines and ∼10 times higher than the concentration of total phenolics in white wines. Yeast may modify the composition of phenolics during the fermentation of must.10 The same applies to the solubilization and extraction of phenolics (due to ethanol either generated during the processing or released from the wall of wooden barrels).11 Analytical methods used to determine total phenolic content in wines are classified into two major categories. Folin-Ciocalteu colorimetry12,13 (with conventional spectrophotometric detection at 750 nm), presently the recommended official method, measures the absorbance caused by a specific reaction following the oxidation of polyphenolic compounds with phosphomolybdic and phosphotungstic acids in a basic medium; the resulting color intensity is directly proportional to the concentration of polyphenols.14,15 The “total polyphenolic index” is derived from the calibration curve constructed using a series of standard solutions that contain gallic acid. Folin-Ciocalteu colorimetry12,13 provides satisfactory results for total phenolic content of the red wines, while the interaction between sulfur dioxide and o-dihydrophenols is a cause of gross interference in the standard determination of phenolics in white wines.16 The second approach is that of HPLC, which, unlike colorimetry, detects the individual polyphenols.17-19 The above-stated Folin-Ciocalteu colorimetry requires red wines to be diluted before their absorbance can be measured using a traditional spectrophotometry. However, the photothermal (8) Go´mez-Plaza, E.; Gil-Mun ˜oz, R.; Lo´pez-Roca, J. M.; Martı´nez, A. J. Agric. Food. Chem. 2000, 48, 736-741. (9) Castellari, M.; Matricardi, L.; Arfelli, G.; Galassi, A.; Amati, A. Food Chem. 2000, 69, 61-69. (10) Ghiselli, A.; Nardini, M.; Baldi, A.; Scaccini, C. J. Agric. Food Chem. 1998, 46, 361-367. (11) Vivas N. J. Sci. Tech. Tonnellerie 1995, 1, 9-16c. (12) Folin, O.; Ciocalteu, V. J. Biol. Chem. 1927, 73, 627-650. (13) Lowry O. H.; Rosenrough, N. J.; Farr A. L.; Randall R. J. J. Biol. Chem. 1951, 193, 265-275. (14) Singleton, V. L.; Rossi, J. A. Am. J. Enol. Vitic. 1965, 16, 144-151. (15) Celeste, M.; Toma´s, C.; Cladera, A.; Estela, J. M.; Cerda`, V. Anal. Chim. Acta 1992, 269, 21-28. (16) Somers, T. C.; Ziemelis, G. J. Sci. Food Agric. 1980, 31, 600-610. (17) Pen ˜a-Neira, A.; Herna´ndez, T.; Garcı´a-Vallejo, C.; Estrella, I.; Suarez, J. A. Eur. Food Res. Technol. 2000, 210, 445-448. (18) Tinttunen, S.; Lehtonen, P. Eur. Food Res. Technol. 2001, 212, 390-394. (19) Garcı´a-Viguera, C.; Bakker, J.; Bellworthy, S. J.; Reader, H. P.; Watkins, S. J.; Bridle, P. Z. Lebensm. -Unters. -Forsch. A 1997, 205, 321-324.
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EXPERIMENTAL SECTION The experimental arrangement used in this study is shown in Figure 1. The 632.8-nm radiation (15 mW) from the He-Ne laser (Melles Griot) was periodically (11 Hz) modulated using the mechanical chopper (EG&G 165) and after reflection (90°) at the plane mirror directed toward the OW. This latter is actually a sapphire disk (0.3 mm thick, 13-mm diameter), the rear face of which is glued to a ring formed piezoelectric transducer made of lead zirconium titanate (PZT; PbZrx Ti1-x O3)). The sapphire disk is not only highly transparent to incident 632.8-nm radiation but in addition is also characterized by its large thermal expansion coefficient. The circle-shaped layer of silicon glue was affixed to the surface of the sapphire disk in such a way that its vertical symmetry axis coincides with that of the sapphire disk. In such a manner, one has created a well-defined volume (diameter 6 mm, height ∼5 mm) that served to accommodate sample (some 50 µL is pipetted directly on the OW) and to prevent spillover. At the same time, it also ensures that both the shape (a layer of liquid) and the volume of the wine sample taken for the analysis were always the same.
Since the concept of OW is relatively unfamiliar, its basic operational principle is outlined below.21,22 As in any other spectroscopy, the spectral coincidence between the wavelength of the incident radiation (632.nm of He-Ne laser) and the absorption wavelength of wine phenolics (analyte species) is an absolute necessity. Provided the above requirement is met, absorption of laser radiation leads to the production of heat (and of thermal waves) in wine, causing its temperature to rise. Due to good thermal contact between the wine and the sapphire disk, the temperature of disk will also change, causing its periodic expansion/contraction. The PZT crystal glued to a disk responds to such changes by generating a periodic voltage (so-called OW signal) that is detected at the modulation frequency by means of the-lock-in amplifier. In general, stronger absorption leads to a more intense heating and hence to a higher OW signal and vice versa. It is also worth mentioning the necessity for optimal alignment of the OW experimental setup. This implies the minimization of the background signal, i.e., the OW signal obtained when no wine is deposited on the sapphire disk. One effective way to bring this about is to prevent the incoming HeNe laser radiation from directly striking the PZT, thereby causing heating and hence the unwanted OW signal. Mounting the adjustable iris diaphragm coaxially with the sapphire disk OW (see Figure 1) proved an elegant solution to this potential difficulty. It is obvious that due to the above-mentioned sequence of steps (absorption of radiation, relaxation of excited state, generation of heat, thermal diffusion, expansion of sapphire disk, production of stress, and conversion to a voltage) that govern the generation of the OW signal, this latter will, at a given power density of the incident radiation, depend on the optical and thermal properties of the sample and the sensor, respectively. In general, both the amplitude and the phase of the OW signal depend on these parameters in a rather complex manner. However, if the sample is opaque (i.e., optical penetration depth defined as a reciprocal of the sample’s absorption coefficient at a given wavelength is very short, as is the case for red wine), there exists a simple relationship between the magnitude of the OW signal and the product of the sample’s thermal diffusion length (that itself depends on the modulation frequency f as f -1/2) and the optical penetration depth.24 It is for the scientist involved in the OW measurement to select experimental conditions that justify the validity of this less complicated relationship mentioned above. Since the optical penetration length, an intrinsic property of the sample itself, is constant, one has to choose the appropriate thermal diffusion length by selecting the corresponding modulation frequency. Then the OW signal becomes dependent solely on the sample’s optical properties and the OW concept allows determination of the absorption coefficient and, hence, also the assessment of analyte concentration. Since it is only the sample layer, the thickness of which equals one thermal diffusion length, that contributes to the OW signal, it follows that the actual physical thickness of the sample is not of relevance for quantitative OW measurements. In the OW experiment described in this paper, all measurements were carried out at 11 Hz at which thermal diffusion length in wine is ∼68 µm.
(20) Rosencwaig, A. Photoacoustic and Photoacoustic Spectroscopy; Robert E. Krieger Publishing Co.: Malabar, FL, 1990. (21) Helander, P. J. Appl. Phys. 1986, 59, 3339-3343.
(22) Helander, P. Meas. Sci. Technol. 1993, 4, 178-185. (23) Helander, P. ACREO AB, Sweden, Personal communication. (24) Pelzl, J.; Chirtoc, M.; Bicanic, D. J. Mol. Struct. 1995, 348, 469-472.
Figure 1. Schematic diagram of the experimental setup for absorption measurement by means of hyphenated OW colorimetry.
method termed optothermal window (OW), or open photoacoustic cell,20,21 has recently emerged as a practical tool for investigation of samples that are either strongly absorbing or even completely opaque. Unique to the OW is the fact that, unlike conventional spectrophotometry, the absorbance is not derived from the measurement of transmitted radiation. (see below for more details about the operational principle). For this reason, it was of interest to explore the prospects of Folin-Ciocalteu colorimetry with OW detection at 632.8 nm for determining the total phenolic content in wines without the need for a dilution step. This report describes a hyphenated FolinCiocalteu OW colorimetry and its application in determining total phenolic content in four red wines. Quantification of phenolic content in, at this stage not more than four wines, was considered as adequate to demonstrate the usefulness and practicality of the OW approach. Data obtained by Folin-Ciocalteu OW colorimetry were compared to those acquired by a reference technique, i.e., Folin-Ciocalteu colorimetry using spectrophotometric detection.
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Figure 2. Absorption spectrum (400-800 nm) recorded after coloring reaction by the conventional spectrophotometer (1-cm cuvette) from of a solution containing 0.5 g of gallic acid in water. Symbol (/) indicates two different analytical wavelengths.
In conventional Folin-Ciocalteu’s colorimetry, detection is usually performed at 750 nm at which the absorbance is maximal as shown in Figure 2, acquired with the spectrophotometer (Cary 1E UV/visible equipped with a 1-cm standard cuvette) from an aqueous solution (0.5 g/L) of gallic acid (3,4,5-trihydroxybenzoic acid). A laser emitting at 750 nm is currently not available in our laboratory, and therefore, 632.8-nm radiation of He-Ne was used instead for excitation in the OW experiment. At 632.8 nm, the absorbance is (Figure 2) ∼90% of that at 750 nm. Prior to actual quantification of phenolic content in wines, we constructed the calibration curve for OW measurement using freshly made solutions of gallic acid (adopted as the reference standard since the measure of total phenolics is reported as “gallic acid equivalents”, i.e., mg/L GAE). The amount of gallic acid dissolved in 1000 mL of distilled water varied from 0.25, 0.5, 0.75, 1, 1.5, and 2, to 2.5 g, respectively; this range was chosen because it overlaps with the true polyphenolic content in the red wine. To produce a colorimetric reaction, 1 mL of each solution was initially mixed with 5 mL of Folin-Ciocalteu’s phenol reagent (SigmaAldrich Chemie GmbH, Merck, Darmstadt, Germany). Then 20 mL of aqueous solution (20 g of sodium carbonate in 1000 mL of distilled water) was added to the above mixture and made to a 100-mL volume with distilled water. RESULTS The calibration curve obtained via OW measurement from colored gallic acid standards is displayed in Figure 3. This latter is recorded at 11 Hz and shows the magnitude of the net lock-in signal S (µV) versus the concentration c (g/L) of the colored complex; the concentration range extends from 0.25 to 2.5 g/L. Each data point in Figure 3 is the average of the three measurements. Under the term “measurement”, one refers to a series of 250 consecutive readings (of the lock-in signal) obtained from one and the same wine sample; these 250 readings are then averaged. As a next step, the new wine sample is deposited on the OW and another series of 250 consecutive readings is recorded (under identical experimental conditions) and averaged. The same procedure is repeated for the last time with yet another wine sample; the arithmetic mean of the three averages is the data point shown in Figure 3. The relationship between S and c in Figure 3 is linear (R ) 0.99) and satisfies the equation S(µV) ) 6.25 c(g/L) - 0.46. The
Figure 3. OW signal (at 632.8 nm and 11 Hz) recorded from a series of concentration standards after completion of the coloring reaction.
reason the calibration curve intersects the positive x-axis is that the S values along the vertical axis represent the net OW signal, i.e., the lock-in signal that was corrected (by vectorial subtraction) for the background contribution (i.e., OW signal obtained under the same experimental conditions from the blank sample). The mixture consisting of 1 mL of distilled water, 5 mL of FolinCiocalteu reagent, and 20 mL of a 20% aqueous solution of sodium carbonate was used here as a blank. Extrapolation of the straight line gives S ) -0.46 µV for the zero concentration; hence, at 11 Hz, the background signal is 0.46 µV for the present setup. Figure 3 illustrates at the same time one important advantage of the OW method over conventional spectrophotometry and that is its ability to deal with optically thick samples. For comparison, if a 1-cm-long cuvette filled with 0.5 g/L of the above-mentioned colored complex is studied by conventional spectrophotometer, the absorbance value A obtained is 0.5. Linear scaling gives A ) 2.5 for 2.5 g/L; however, at this concentration level, the solution appears intensely blue, precluding direct measurement of absorbance by conventional spectrophotometry. As a next step, the hyphenated OW-Folin-Ciocalteu colorimetric assay was used to determine total polyphenolic content in four different red wines. Three wines (Cuvee Bean Solei, Cabernet Syrah, Sangre de Toro) were purchased in the local supermarket; the fourth sample (Visˇko Crveno Vino) was a homemade product originating from the Dalmatian island Vis in Croatia. All red wines are simultaneously both optically thick (their optical penetration depth is shorter than the physical thickness (approximately a few millimeters) and thermally thick (at 11 Hz, the thermal diffusion length in wine is ∼65 µm). The sapphire disk on the other hand is optically thin and thermally thick (at 11 Hz, the thermal diffusion length is 180 µm). Except for the reagent that was added, all four wines could be studied directly as they are, that is, without any dilution. The total polyphenolic content of wines ranges between 0.8 and 1.6 g/L (see Table 1) as obtained from measured OW signals and the calibration curve shown in Figure 3. It was of interest to find out how the performance of the proposed hyphenated Folin-Ciocalteu OW colorimetry compares (in terms of precision, reproducibility and accuracy) with that of Folin-Ciocalteu colorimetry with spectrophotometric detection. The calibration curve for spectrophotometric measurements at Analytical Chemistry, Vol. 74, No. 9, May 1, 2002
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Table 1. Total Polyphenol Content (Expressed in GAE Units) of Red Wines Measured by Conventional Spectrophotometry and by Hyphenated He-Ne Laser OW Colorimetry Cuvee bean solei polyphenol content (mg/L) mean of 3 samples ∑x ∑x2 standard deviation variance standard error of mean coefficient of variation degrees of freedom mean of 256 samples ∑x ∑x2 standard deviation variance standard error of mean coefficient of variation degrees of freedom
Visˇko crveno vino
Cabernet syrah
Results Acquired with the Spectrophotometer at 750 nm 812.01 779.35 1002.59 814.20 780.66 999.16 822.08 788.37 1009.82 816.10 782.79 1003.86 2448.3 2348.4 3011.6 1998120.2 1838360.4 3023268.9 5.3 4.9 5.4 28.04 23.732 29.626 3.06 2.813 3.142 0.65 0.622 0.542 3 3 3 Results Acquired by Optothermal Window Colorimetry at 632.8 nm 814.1 786.9 1023.6 208343.1 201411.2 267059.0 169666047.8 158474197.3 278685094.8 20.59 6.67 18.71 423.87 44.52 350.07 1.29 0.42 1.17 2.53 0.85 1.83 255 255 255
750 nm was produced in the same way as described above for the OW measurements. The only exception was the content of gallic acid that this time varied from 0.05 to 0.5 g/L, the major reason being the opacity of solutions with concentrations exceeding 0.5 g/L. Therefore, to make the absorbance values match the 0.05 to 0.5 g/L range specified above, all wines had to be diluted (5-fold) prior to actual measurement. The equation of the straight line that relates absorbance A to the concentration c (mg/L) of gallic acid in distilled water reads as follows: A ) 0.00113c 0.0006. The apparent total polyphenolic content in wine obtained when substituting a value for A (acquired from diluted wines by conventional spectrophotometry) into the A ) 0.00113c - 0.0006 calibration line must then be corrected (multiplication by 5) in order to assess the true phenolic content. The reproducibility, defined as a comparison of precisions achievable by Folin-Ciocalteu’s OW colorimetry (at 632.8 nm) and spectrophotometric detection (at 750 nm), was estimated statistically by performing an F test, which compares the variances of results measured by two different methods. The reason for using two different wavelengths for excitation was explained above. Since the result of the F test for all wine samples does not exceed the tabulated value (19.49), we conclude that there is no significant difference between precisions of the two methods at a 95% confidence level. As a next step, Student’s t test was employed at a 95% confidence level in order to compare the relative accuracies of the two methods. For all wines except the Sangre de Toro, the calculated t values are lower than the critical value for t (1.97); hence, the accuracies of the two methods are not different at the 95% confidence level. In the case of Sangre de Toro, the wine with the highest polyphenol concentration, the calculated t value (3.89) is very close to the critical t value, implying a significant difference between two analytical methods at this concentration level. CONCLUSION Total polyphenolic content in four red wines obtained by hyphenated Folin-Ciocalteu OW colorimetry at 632.8 nm varied 2160 Analytical Chemistry, Vol. 74, No. 9, May 1, 2002
Sangre de Toro 1614.76 1614.38 1618.25 1615.80 4847.4 7832444.4 2.1 4.553 1.232 0.132 3 1629.9 419032.7 685902091.4 6.26 39.22 0.39 0.38 255
between 786 and 1630 mg/L GAE. This compares well to 778 and 1614 mg/L GAE determined for same wines by means of FolinCiocalteu colorimetry (with the spectrophotometric detection at 750 nm), the official and preferred method for quantitative analysis of total phenolics in red wines. It is the way the absorption is being detected (the OW signal is generated due to absorption of radiation occurring within a wine layer 68 µm thick (one thermal diffusion length at 11 Hz) that allows the hyphenated FolinCiocalteu OW colorimetry to be used with the undiluted wines. The extent of the interferences in the OW measurement of phenolic content is the same as that in classical spectrophotometry. The precision, defined as the closeness to each other of 256 replicate readings of the OW signal, is less than 3% (highest value for coefficient of variation is 2.5%). The results obtained by hyphenated OW/colorimetry at 632.8 nm agree very well with literature data acquired by other methods at a different wavelength (750 nm).14,15 Besides the fact it does not require the intermediate dilution step, hyphenated Folin-Ciocalteu OW colorimetry for determination of polyphenolic content in red wines offers several additional attractive features among which are relatively low cost, speed, simple sample loading, and easy cleaning procedure. The concept of OW colorimetry that can easily be extended to other wavelengths (provided a disk remains highly transparent) forms a practical tool for quantitative studies of other strongly absorbing (semi)fluids, pastes, suspensions, etc. In addition to the optical characterization discussed in this paper, attempts are currently underway to use the OW technique also for the measurement of dynamic thermophysical parameters (such as thermal diffusivity and effusivity) of the above-mentioned samples. Although this study demonstrates the usefulness of OW colorimetry for measurement of substantially high concentrations, it is worth mentioning that the same concept can be used in the low concentration range. In this experiment, the output power of He-Ne laser was 12 mW and the diameter of an unfocused beam was 1.6 mm; the corresponding power density is therefore 0.6
Wcm-2. This value is ∼5 times above 0.13 Wcm-2 (equivalent to 1-mW power within a beam 1 mm in diameter) regarded as a threshold for an OW experiment.23 If, for example, a compact and a powerful excitation source is used (such as the laser diode emitting at 750 nm), and the overall thermal mass of the heated region is reduced (such reduction is easily achieved by taking a sapphire disk thinner than presently used, 300 µm), the hyphenated OW colorimetry could develop into a candidate method suitable for detection even at a trace level. As such it could also prove useful to assess the total polyphenolic content in edible oils.
ACKNOWLEDGMENT O.D. acknowledges the Bolyai Fellowship granted by the Hungarian Academy of Sciences and the Visiting Scientist Fellowship provided by Wageningen University and Research Centre, Wageningen, The Netherlands. Some parts of the study were performed within the frame of activities conducted under BRRTCT98-5041 European Thematic Network. Received for review September 19, 2001. Accepted January 30, 2002. AC011001S
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