Identification of the major drivers of 'phenolic' taste in ... - GWRDC

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AWRI: Identification Of The Major Drivers Of ‘Phenolic’ Taste In White Wines Selection of Column A number of HPLC columns with different separations chemistries were screened using the 2009 Chardonnay or Riesling hard pressings wine. The choice of column for further optimization was determined by the maximum number of resolved peaks (obtained using standardized integration parameters) at 280 nm (all phenyl-containing compounds) and the peaks in which the absorbance was higher at 320 nm than at 280 nm (hydroxycinnamate-containing compounds). The initial screening looked at different separation chemistries or column presentations: UDC Cholesterol (Cogent); Synergi Polar RP, Synergi Hydro RP, Gemini C6-Phenyl, Kintex PFP (Phenomenex); Supelco Ascentis Phenyl (Sigma) Spherisorb Phenyl, XBridge (Waters). Throughout the experiments, 20 µL of the same wine sample (2009 Riesling hard pressings wine), stored at -18°C was injected. Standardised integration parameters were used to assess the number of peaks resulting for the different columns or operating conditions: slope sensitivity = 1; peak width = 0.2 min; area reject = 50 mAu; height reject = 5 mAu. The total number of peaks at 280 nm was recorded as was the number of peaks where the absorbance at 320 nm was greater than that at 280 nm which represented the hydroxycinnamates. A.3.3 Results and Discussion The success of column selection was measured in terms of the total number of separate peaks observed at 280 nm and peaks arising from hydroxycinnamoyl-containing compounds. Over 100 separate peaks arising from phenolic compounds were detected. Of those columns studied, the Gemini C6-Phenyl and Kintex PFP columns appeared to provide the highest degree of separation of phenolic compounds (Table A-1). As further planned research will have require up-scaling for semi- preparative separations for sensory analysis, the former column was chosen as more packing options were available at the time of this work. Optimization of Chromatography Using Gemini C6-Phenyl Column. Further optimization was carried out on the formic acid concentration and effect of TFA concentration. In order to improve the resolution of early-eluting compounds, the pH was modified to be potentially below the pKa of some hydroxybenzoic or cinnamic acids so that they were predominantly in the unionized form. This was carried out by including trifluoroacetic acid into both mobile phases and also modulating the concentration of formic acid. In view of the complexity of the chromatogram with the number of peaks eluting, all dead volumes were minimized by fitting the shortest possible lengths of 0.12 mm i.d. stainless steel tubing from injector seat to detector and using a semi-micro flow cell. A binary pump was installed instead of the quaternary to minimize pressure fluctuations. An injection volume of 10 µL was also adopted as this slightly improved resolution of peaks. The samples were matched to the starting chromatography conditions by making up samples to volume with 2% formic and 0.01% TFA. Both of these measures improved the retention time variability. A typical HPLC-DAD chromatogram is shown in Figure A-1 with some of the major peaks identified. The trace arising largely from benzoic acids, flavan-3-ols and other phenol- ring containing compounds can be seen when A280 being the largest. 104
AWRI: Identification Of The Major Drivers Of ‘Phenolic’ Taste In White Wines 105 Table A-1 : Resolving power of phenolic species in chardonnay and Riesling hard pressings wine (2009) using standardized integration parameters 1 Column details Conditions used Synergi Polar RP 2 (Phenomenex) 4.6 x 250 mm; 4µm Synergi Hydro RP 2 (Phenomenex) 2.1 x 150 mm; 4µm UDC Cholesterol (Cogent) 4.6 x 250 mm; 4µm Spherisorb Phenyl (Waters) 4.6 x 250 mm; 5µm Ascentis Phenyl (Supelco Sigma) 2.1 x 150 mm; 3µm Gemini C6-Phenyl (Phenomenex) 2.1 x 150 mm; 3µm XBridge (Waters) 2.1 x 150 mm; 3.5µm Kintex PFP 3 (Phenomenex) 2.1 x 150 mm; 3µm Start: 1% ACN in water/1% formic acid/ 0.1% MeOH; ramp to 80% ACN /2% formic acid/10% MeOH from 5’ to 50’; hold for 5’ return over 5’; 1 mL/min Start: 1% MeCN in water/1% formic acid; ramp to 80% MeCN /2% formic acid from 5’ to 50’; hold for 5’ return over 5’; 1 mL/min Start: 1% ACN in water/1% formic acid/ 0.1% MeOH; ramp to 80% ACN /2% formic acid/10% MeOH from 5’ to 50’; hold for 5’ return over 5’; 1 mL/min Start: 1% MeOH in water/1% formic acid; ramp to 80% MeOH /2% formic acid from 5’ to 50’; hold for 5’ return over 5’; 0.2 mL/min; 45°C 2% formic acid constant; ramp to 40% MeOH from 0’ to 50’; ramp to 100% for 5’ return over 2’; 0.3 mL/min; 45°C 2% formic acid constant; ramp to 40% MeOH from 0’ to 50’; ramp to 100% for 5’ return over 2’; 0.2 mL/min; 45°C 2% formic acid constant; ramp to 45% MeOH from 0’ to 50’; ramp to 90% for 5’ return over 2’; 0.3 mL/min; 45°C 2% formic acid constant; ramp to 40% MeOH from 0’ to 50’; ramp to 100% for 5’ return over 2’; 0.2 mL/min; 50°C No. peaks 280 nm No. peaks A320 A280 67 6 No. peaks 320 nm 39 49 7 41 49 8 41 41 2 12 38 76 9 51 108 12 86 82 9 65 106 13 75 1 standardized integration parameters: slope sensitivity = 1; peak width = 0.2 min; area reject = 50 mAublns; height reject = 5 mAu 2 RP: reversed phase 3 PFP: pentafluorophenyl The cinnamic acids and GRP-analogues are represented where the A320 trace is the biggest and flavonols and their glyco-conjugates when A370 is biggest. In the example shown, the acids caftaric, coutaric, fertaric and ferulic (red A320 trace) are the most dominant and quercetin-3-glucuronide is the major flavonol glyco-conjugate. Note all peaks elute within 70 minutes. Coupling two column together in series can give an even better separation was possible if two columns were connected. It was necessary to subsequently reduce the flow rate to 0.16 mL/min to
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Identification of the major drivers of 'phenolic' taste in ... - GWRDC

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