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

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.
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