Simultaneous Determination of Glycerol, Fructose and Glucose for Quality Control of Wines

Glycerol is the major by-product of ethanol fermentation and could be associated with wine quality. A simple method of assaying glycerol, glucose and fructose in wine by highperformance liquid chromatography using a modified polar bonded NH2-silica phase column and a refractive index detector was used to determine their concentrations in highand standard-quality wines. Results show glycerol. fructose, and glucose levels ranged from 1.65 to 9.54, non-detect to 2.57 and non-detect to 1.63 g/L, respectively, for high-quality wines. For standard-quality wines, these levels ranged from 2.67 to 8.43, 1.29 to 9.12, and 0.62 to 3.60 g/L, respectively. Fructose and glucose levels (76.2±30.8 and 43.9±18.0 g/L, respectively) were much higher in sweet and sparkling wines. Introduction Glycerol is predominant among several polyols usually found in wine, which also contains erythritol, arabitol, mannitol, sorbitol, mesoinositol and 2,3-butanediol. Glycerol is the major by-product of ethanol fermentation and results from the reduction of dihydroxyacetone phosphate to glycerol 3-phosphate concomitant with NADH oxidation by NAD-dependent glycerol 3-phosphate dehydrogenase (Gancedo et al. 1968). Several factors are involved in the final content of glycerol in wine: ripeness of the grapes, the micro-organisms present, the type of cellar equipment, the pH and temperature of fermentation, the source of nitrogen and the strain of yeast used in fermentation (Cronwright et al., 2002). The glycerol concentration in wine is rarely higher than 12 g/L unless the grapes are massively infected by the fungus Botrytis cinerea, when the concentration can be as high as 25 g/L (Ribereau-Gayon et al.,1998). Several authors have determined the glycerol concentration in investigating its correlation with wine quality: the results were inconclusive (Nieuwoudt et al., 2002; Sehovic et al., 2004). However, the presence of glycerol in wines can acquire new interest in wine control also considering that this ingredient is used as an adulterant to improve the sweetness, body, and fullness of wines. In this research we simultaneously determine glycerol, fructose and glucose, comparing their concentrations in high and standard-quality wines. The analytical technique used is a simple liquid-chromatography which uses a polar bonded silica (-NH2 modified) stationary phase connected to a refractive index detector (Moro et al. 2007). Material and Methods Chemicals and Reagents Glycerol, fructose and glucose of analytical grade were purchased from Carlo Erba (Milan, Italy). Wines Analysis was performed on certified (EC Regulation 2002) and uncertified wines listed below. High-Quality Red Wines Standard-Quality Red Wines Barbera d’Alba DOC 2004 (BA-04) Bricchello Rosso (BRR) Barbera del Monferrato DOC 2003 (BM-03) Cabernet del Veneto (CV) Barbera del Monferrato DOC 2005 (BM-05) Merlot del Veneto (MV) Castel del Monte Rosso DOC 2003 (CMR-03) Tavernello Rosso 1 (TR1) Chianti Colli Senesi DOCG 2002 (CS-02) Tavernello Rosso 2 (TR2) Montepulciano d’Abruzzo DOC 2003 (MA-03) Vigna dei Colli Rossi (VCR) Rosso di Puglia ITG 2003 (RP-03) Rosso di Sardegna DOC 1998 (RS-98) Sangiovese Umbria ITG 2004 (SN-04) Sassoalloro ITG 2004 (SS-04) Sudtiroler Lagrein Rosso DOC 2000 (SUL-00) Teroldego Rotaliano DOC 2004 (TR-04) Velletri Rosso DOC 2000 (VR-00) High-Quality White Wines Standard-Quality White Wines Bianco di Custoza DOC 2003 (BC-03) Bianco Frizzante (BF) Frascati Superiore DOC 2002 (FS-02) Bricchello Bianco (BRB) Frascati Superiore DOC 2003 (FS-03) Chardonnay Frizzante Secco (CFS) Lugana Superiore DOC 2003 (LS-03) Garzellino (GZ) Sudtiroler Sauvignon DOC 2003 (SS-03) Tavernello Bianco 1 (TB1) Tavernello Bianco 2 (TB2) Others Asti Spumante DOCG 2005 (AS) Bracchetto d’Acqui (BA) Fragolino (FR) Moscato (MS) Moscato d’Asti (MSA) Vin Santo (VS) High Performance Liquid Chromatography An 880-PU Liquid Chromatographic Pump (Jasco Corporation, Tokyo, Japan) connected to a refractive index (RI) detector RI-2031 (Jasco Corp.) was used for the analysis of the wines. The analytical column was a 250x4 mm LiChrosorb (Merck, Darmstadt) packed with a 5-μm diameter NH2-silica phase and was protected by a RP 18 guard column (Merck). The mobile phase was acetonitrile−water (85:15, v/v) pumped under constant flow at 1.0 mL/min at room temperature. Samples were injected via a Model 7125 injector valve equipped with a 20 μL loop (Rheodyne, Cotati, CA, USA). On-line data acquisition and calculations were performed by a Data Jet Integrator (Thermo Separation Products, Riviera Beach, FL, USA) and Winner/386 Autolab software (Thermo Separation Products). Wine samples were injected as such. Statistical analysis The significance of the difference between the mean values was calculated by analysis of variance (ANOVA) and then by Bonferroni multiple comparison test. Results Figure 1A shows a typical chromatogram of a standard solution containing glycerol 5.3 g/L (peak 4), fructose 2.5 g/L (peak 5) and glucose 3.0 g/L (peak 6). The calibration graphs calculated for glycerol were linear over the concentration range 0.1−12 g/L, with correlation coefficients r>0.999. Similar good linearity was obtained for fructose and glucose in the concentration range 0.1−150 g/L. In all cases, the limit of detection and quantification was 0.1 g/L.