**2.3 Chemiluminometry**

360 Macro to Nano Spectroscopy

epicatechin, (+)-catechin and quercetin. Interesting, gallic acid, as modelling solution by

137 wine samples (73 red, 54 white and 10 rosé wines) were purchased from wineries and directly analysed. Most of the red wine samples were from grape varieties Blue Frankish, Merlot, Cabernet Sauvignon, Pinot Noir, Refošk and Barbera. Cviček was used in the study as a typical Slovenian mixture of red and white wines, whereas from among the white wines, most samples were from grape varieties Welsh Riesling, Chardonnay, Traminer,

The HPLC system Waters 600E was composed of isocratic pump W600, autosampler and Waters 996 photodiode array detector. The HPLC column Synergi Hydro RP 150 × 4.6 mm, 4 µm (Phenomenex, Torrance, California, USA) was used at 35 °C. Wavelengths of detection: (+)-catechin and (-)-epicatechin 210 nm, quercetin 253 nm, gallic acid 278 nm and *trans*resveratrol 303 nm. Gallic acid, (+)-catechin hydrate, (-)-epicatechin, *trans*-resveratrol and quercetin dihydrate were purchased from Sigma-Aldrich (St. Louis, USA). All reagents and

The experimental conditions were: mobile phase A: 0.1% H3PO4; mobile phase B: MeOH; gradient elution: 0 min 90% A, 10% B; 15 min 78% A, 22% B; 25 min 50% A, 50% B; 34 min 34% A, 66% B; 35 min 90% A, 10% B for reconditioning of the system (8min); flow rate: 1.0 mL/min; injection volume: 50 μL; MeOH and ortophosphoric acid were HPLC-grade (Fluka, St. Gallen, Suisse) and were filtered and degassed before their use. The wine samples

Stock solutions of standards were diluted in the mobile phase to obtain working standard solutions. Concentrations of the analytes were calculated from chromatogram peak areas on the basis of calibration curves. The method linearity was assessed by means of linear regression of the mass of analyte injected vs. its peak area. The repeatability was expressed

Typical standard deviations for determinations of the sum of phenolic antioxidants determined using HPLC/UV-vis were 0.015 mmol/L for red wines, 0.004 mmol/L for rosé

Spectrophotometric determination of total antioxidant potential (TAPSP) was performed according to the Singleton-Rossi procedure (Singelton Rossi, 1965). TAPSP of an antioxidant sample was estimated by measuring its reducing capacity with the Folin-Ciocalteu reagent using a spectrophotometer. The Folin-Ciocalteu reagent is a mixture of phosphowolframic acid (H3PW12O40) and phosphomolybdenic acid (H3PMo12O40), the absorbance of which was measured after the reaction at 765 nm using a Cary 1E spectrophotometer (Varian, California, USA). The Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). It contains sodium tungstate, sodium molybdate,

spectrophotometric determination of TAPSP, shows the lowest contribution to TAP.

Rhine Riesling, Yellow Muscat and Sauvignon. Vintages ranged from 1997 to 2006.

standards were prepared using Milli Q deionized water (Millipore, Bedford, USA).

were diluted ten times with the respective mobile phases described above.

as standard deviation (SD) of three separate determinations.

and 0.050 mmol/L for white wines.

**2.2 Spectrophotometry** 

**2. Experimental** 

**2.1 HPLC- UV/VIS method** 

The Abel®-21 M2 antioxidant test kit (Knight Scientific Limited, Plymouth, UK) was used for chemiluminometric determination of total antioxidant potential (TAPCL). Superoxide, generated in a tube containing Pholasin® with and without a sample of wine with unknown antioxidant potential leads to appearance of chemiluminescence, which was measured using a micro plate luminometer model Lucy (Anthos Labtec Instruments, Wals, Austria). Pholasin® is a bioluminescent photo protein of *Pholas dactylus*, which is a marine, rockboring, bivalve mollusc. Antioxidant test kit used contains assay buffer (pH 7.2).

The Antioxidant Test procedure for Superoxide is provided by the supplier of the test kit (Hipler and Knight, 2001). The amount of sample was optimised to obtain not more than 90% and typically 50% signal inhibition. This signal was then corrected for sample dilution: 10 µL of sample was used, however, red wines and rosé wines were first diluted with water (1:10) while white wines were not.

The results are calculated as TAPCL, expressed as % signal inhibition. Typical measurement uncertainty was 0.024 mmol/L for red wines, 0.016 mmol/L for rosé and 0.002 mmol/L for white wines.
