fractionation method for phenolic compounds in red. Their contribution to the color, flavor, and aging behav- wine, which would make possible the analysis of ...
Research Note
Fractionation of Phenolic Compounds in Red Wine J. OSZMIANSKI', T. RAMOS~,and M. BOURZEIX3' A simple analytical method was developed for the fractionation of phenolic compounds of red wine into phenolic acids (Fraction I), catechins, procyanidins, and anthocyanin monomers (Fraction II), flavonols (Fraction Ill), and anthocyanin polymers (Fraction IV) by passing wine through a preconditioned C,, Sep-pak cartridge and eluting sequentially with methanol. 16% acetonitrile at pH 2.0, ethyl acetate, and methanol. Each fraction was then injected onto a C,, HPLC column using a gradient of 3% to 100% acetonitrile in water at pH 2.6. This fractionation
technique is the first step of quantitative analysis of total compounds in every fraction by a simple and rapid method: spectrophotornctry. KEY WORDS:phenolic compounds, red wine, HPLC, C,, Sep-pak cartridge
The phenolic compounds of wines are of undoubted importance and have been exhaustively reviewed (6). Their contribution to the color, flavor, and aging behavior of red wines is well known; however, these components have similar chemical characteristics, and some problems of separation appear in red wine analysis. High performance liquid chromatography (HPLC) using reversed phnse has been able to separate the different phenolics (3,4,7); nevertheless, numerous compounds present a maximum absorption in a range of 280 to 320 nm, and a preliminary fractionation is necessary for a good resolution on the HPLC column. Fleuriet and ,Macheix (1)and Salago'ity-Auguste and Bertrand (5) separated neutral and acidic phenolic compounds of red wine by changing the pH prior to extraction by ethyl acetate; however, the extraction efficiency for procyanidins was very low. Jaworski and Lee (2) passed grape juice through n preconditioned C,, Sep-pak column in order to fractionate into ncidic and neutral phenolics. This fractionation technique showed a high resolution, but the methanol was not a selective solvent 'for the procyanidin coinpounds and flavonols, and anthocyanins were eluted with them.
The object of this study was to develop a simple fractionation method for phenolic compounds in red wine, which would make possible the analysis of total phenolic compounds in every fraction by spectrophotometry.
Materials and Methods Syrah red wine from the INRA Experimental Station, Pech Rouge-Narbonne, France, obtained in 1986 by classic fermentation, was used in this study. T e n milliliters wine added to 10 mL distilled water was concentrated to original volume by rotary evnporation a t 30°C to remove the alcohol without destroying the phenolic compounds (5). For the fractionation of neutral phenolic compounds, a preconditioned C18Sep-pak cartridge (Waters Associates) was used. This cartridge was preconditioned following the method of Jaworski and Lee (2). The fractionation of phenolics is shown in Figure 1. The medium was adjusted to pH 7.0 with 5 N NaOH, and 0.5 mL of this sample was passed through the preconditioned neutral C18Sep-pak cartridge to'absorb the neutral phenolic compounds. Phenolic acids were not fixed, and vine
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Fig. 1. Fractionation of phenolic
compounds in red wine.
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after washing the cartridge with water at pH 7-0,
259
passed to this solvent. After the pH of this effluent was lowered 2.0 with 0.1 N HCl, phenolic acids were fmed into a second preconditioned acidic C18Sep-pak cartridge and eluted using 2.5 mL methanol for injection onto the HPLC column.
Am. J. Enol. Vitic.. Vol. 39, No. 3, 1988
PHENOLIC COMPOUNDS - 261
The fraction containing the catechins, procyanidins, and anthocyanin monomers (Fraction XI) was eluted with 2.5 mL 16% acetonitrile a t p H 2.0 from the neutral Seppak after acidic preconditioning. The flavonols (Fraction 111) and the anthocyanin polymers (Fraction N)were eluted using 10 mL ethyl acetate and 2.5 mL methanol, respectively. The orgnnic solvents (16% acetonitrile and ethyl acetate) were removed with a rotary evaporator, and the fractions were diluted in 2.5 mL methanol for HPLC analysis. HPLC analysis: T h e standards of (+) catechin, (-) epicatechin, quercetin, myricetin, quercetin and myricetin glycosides, and phenolic acids were commercial products. Procyanidins B1, B2, B3, and B4 were furnished by Drs. Jerumanis and Derdelinkx (Univ. of Louvain, Belgium). A high performance liquid chromatograph (WatersMillipore), equipped with 6000A and EM 45 pumps, uv-v detector (model 490), solvent programmer (model 720), and data module (model 730), was used. Separation of phenolics was camed out on a C,,Rrownlee Labs column (220 mm X 4.6 mm) a t 32°C using a linenr gradient of
Table 1. Solvent gradient used in HPLC analysis.. Time % Acetonitrile % Water Curve (rnin) Initial
(pH 2.6) 97
3
10 24
90 86 60 Q
10 14 40 100
44
45
6 6
6 6
acetonitrile in water (pH 2.6, flow rate 1 mL/min) (Table 1).
Results and Discussion The chromatograms shown in Figures 2, 3, and 4 were obtained using red wine without treatment (part A) and under the operating conditions described above (part R). Components were identified by spiking with stnndnrds under the same conditions and by their retention times. The solvent gradient used in HPLC analysis was efficient to distinguish different phenolic acids, procyanidins, and flavonols; for anthocyanin monomers, i t was not adequate. Interestingly, anthocyanin polymers were
40
min
20
A
280
40
nrn
nlin
20
40
20
II~~II
B1 B2 Fig. 4. HPLC chromatograms of (A) Syrah red wine without treatment and (B,) Fraction I I and (B2) Fraction IV after fractionation with Sep-
pak cartridge. 1 procyanidin B1; 2
-
- catechin: 3 - procyanidin 84; 4 - procyanidin 82; 5 - epicatechin: and 6 - anthocyanin monomers. Am. J.
Enol. Vitic., Vol. 39, No.
3, 1988
r
- PHENOLIC COMPOUNDS
min
30
40
40
.
rnln
10
B
A
Fig. 2. HPLC chromatograrns of (A) Syrah red wine without treatment and (B) Fraction 1 after fractionation with Sep-pak cartridge. 1 cis-caffeoyl tartrate: 2 trans-caffeoyl tartrate: 3 cis-pcoumaroyl tartrate: 4 trans-pcoumaroyl tartrate: 5 cis-caffeic acid: 6 transcaffeic acid: 7 cis-pcoumaric acid: 8 trans-pcoumaric acid: and 9 ferulic acid.
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-
-
-
-
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-
#
2
Fig. 3. HPLC chromatograrns of (A) Syrah red wine without treatment and (6)Fraction Ill after fractionation with Sep-pak cartridge. 1 rnyricetin 3-glucoside: 2 quercetin 3-glucoside: 3 unidentified: 4 myricetin: and 5 quercetin.
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-
Am. J. Enol. Vitic.. Vol. 39. No. 3, 1988
Table 2. Extraction efficiency of various phenolic compounds by different methods. '
Phenolic compounds
Extraction efficiency (%) (a) (b) (C)
Catechin 92.3 90 91 Epicatechin 91.4 93 91 118.0 35 85 Procyanidin 83 trans-pCoumaroyl tartrate 97.3 95' pcoumaric acid 79 94 73. 93 Ouercetin (a) Jaworski and Lee (2): (b) Salagony-Auguste and Bertrand (5): and (c) obtained by us. I
excluded during fractionation from the monomers. The extraction efficiency of different phenolic corn!pounds was determined using a synthetic solution under the same operating conditions. Results are shown in Table 2. The percentages recovered were higher than those recovered by ethyl acetate extraction (5) and in accordance with the results of Jaworski and Lee (2). : Separation of phenolic acids from neutral phenolics .has been documented (1,2,5), but it was not possible to 'separate the flnvonols from the phenolic acids (1) or Iprocyanidins (2,5), and interferences in HPLC analysis appear. The current fractionation technique is able to separate flavonols from other phenolic compounds with a high recovery, and their total determination can be made by spectrophotometry. It had not been possible to. separate anthocyanin monomers from procyanidins nnd catechins in red wine; 'that is not a problem in white wines, where anthocyanins are not present. Different solvents for frnctionnting these components are currently being investigated.
Conclusions In conclusion, this fractionation technique is the first step of quantitative analysis of total compounds in every fraction by a simple and rapid method (spectrophotometry). I t facilitntes the individual collection of peaks and makes possible their identification. I t is a very easy fractionation, which permits separation of the principal phenolic groups with a high recovery.
Literature Cited 1. Fleuriet. A.. and J. J. Macheix. Separation et dosage par chromatographie en phase gazeuese de I'acide clorogbnique et des catbchines des fruits. J. Chromatogr. 74:339-45 (1972). 2. Jaworski. A. W.. and C. Y. Lee. Fractionation and HPLC determination of grape phenolics. J. Agric. Food Chem. 35249-51 (1987). 3. Lea. A. G. H.. P. Bridle. C. F.-~imberlake.and V. L. Singleton. The procyanidins of white grapes and wines. Am. J. Enol. Vitic. 30:289-300 (1979). 4. Ong. 8. Y.. and C. W. Nagel. High-pressure liquid chromatography analysis of hydroxycinnamic acid-tartaric acid esters and their glucose esters in V. vinifera. J. Chromatogr. 157:345-55 (1978).
5. Salagony-Auguste, M. H.. and A. J. Bertrand. Wine phenolics-analys~s of low molecular weight components by high performance liquid chromatography. J. Soc. Food Agric. 351241-7 (1984). 6. Singleton. V. L.. and P. Esau. Phenolic Substances in Grapes and Wine and Their Significance. Adv. Food Res. Suppl. 1. Academic Press, New York (1969).
7. Wulf. L. W.. and C. W. Nagel. High-pressure liquid chromatographic separation of anthocyanins of Vitis vinifera. Am. J. Enol. Vit~c.29:42-9 (1978).
Am. J. Enol. Vitic.. Val. 39, No. 3, 1988
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