Evaluation of a fully automated method for the measurement of glycerol in wine

Significance of the topic

The concentration of glycerol in wine is an important chemical parameter reflecting yeast metabolism during alcoholic fermentation and contributing to wine mouthfeel, perceived viscosity and sweetness. Typical concentrations are ~5 g/L but can reach 15–20 g/L depending on fermentation conditions and sulfur dioxide levels. Reliable, high-throughput, and automatable glycerol assays support quality control in wineries, fermentation monitoring, regulatory testing and research into fermentation management.

Aims and study overview

This application note evaluated a fully automated enzymatic photometric method for glycerol measurement implemented on the Thermo Scientific Arena 20XT discrete analyzer. Goals were to demonstrate analytical performance (accuracy, repeatability, linear range) and to compare results to those from a WineScan FT120 (FOSS) and an accredited enzymatic reference method used by ALKO, Inc. The method was also positioned for use on Thermo Scientific Gallery and Gallery Plus discrete analyzers.

Materials and samples

• Total samples: 53 commercial wines (17 white, 3 rosé, 33 red).
• Controls: two levels of water-based glycerol controls prepared from a glycerol standard (AnalaR, BDH).
• No sample pretreatment was applied prior to analysis.

Methodology

The assay is an enzymatic end-point photometric method based on sequential enzymatic reactions involving glycerokinase (GK), ADP-dependent hexokinase (ADP-HK) and glucose-6-phosphate dehydrogenase (G6P-DH). The formation of NADH (measured at 340 nm) is stoichiometrically linked to glycerol concentration. Key procedural parameters on the Arena 20XT: a 5 µL sample is incubated with 100 µL of buffer (containing D-glucose and enzymes) at 37 °C for 120 s followed by a blank reading; then 25 µL of reagent containing NAD+ and ATP is added and incubated 420 s before absorbance measurement at 340 nm. Calibration is performed as a two-point linear calibration (two replicates per calibrator). The stated measuring range is 0.07–30.00 g/L.

Used instrumentation

• Arena 20XT discrete analyzer (Thermo Fisher Scientific, Vantaa, Finland) used for measurements in this study.
• WineScan FT120 analyzer (FOSS, Hillerød, Denmark) used for method comparison.
• Method adapted for Thermo Scientific Gallery and Gallery Plus discrete analyzers.

Calibration and analytical setup

Calibration was performed with two-point calibration (two replicates each). The example calibration data indicate stable linear response across the working range, enabling direct calculation of glycerol concentration from absorbance. The system reagent glycerol kit and a glycerol standard were used as calibrators.

Key results and discussion

• Correlation with reference methods: comparison of Arena results to WineScan FT120 produced a correlation coefficient of 0.964. Comparison to ALKO, Inc. accredited enzymatic method for five proficiency test samples yielded a correlation coefficient reported as 1.000.
• Proficiency test samples: Arena results fell within stated tolerances for five diverse wines (examples: white 5.22 g/L vs tolerance 5.4 ± 0.8 g/L; red 6.82 g/L vs 7.1 ± 1.1 g/L; sparkling 5.61 g/L vs 6.0 ± 0.9 g/L; aromatized 8.33 g/L vs 8.7 ± 1.3 g/L).
• Repeatability: within-run standard deviation (SD) observed for water-based standard and wine samples in the 4.25–10.39 g/L range was between 0.036 and 0.099 g/L, indicating good precision at both low and moderate concentrations.
• Suitability across matrices: analysis of white, rosé and red wines without pretreatment demonstrates robustness of the enzymatic assay against typical wine matrix effects within the tested range.

Benefits and practical applications

• High-throughput and automation: small sample volume (5 µL) and the discrete analyzer workflow allow multiple automated tests on the same sample, increasing laboratory throughput and reducing manual handling.
• Accuracy and precision: results comparable to established analyzers and accredited enzymatic methods support use in QA/QC, proficiency testing and regulatory contexts.
• Wide linear range (0.07–30.00 g/L): suitable for typical and elevated glycerol levels found in wines.
• No sample pretreatment required: simplifies routine implementation in winery and central laboratories.

Future trends and potential uses

• Integration with laboratory information management systems (LIMS) to streamline data flow and regulatory reporting.
• Multiplexed discrete analyzer panels combining glycerol with other fermentation and quality markers (e.g., ethanol, sugars, organic acids) for comprehensive fermentation profiling.
• Adoption for in-line or at-line monitoring in production with further miniaturization and faster incubation chemistries.
• Expansion of accredited methods and standardized calibrators to support inter-laboratory comparability and proficiency testing.
• Application of similar enzymatic photometric approaches to research on yeast metabolism, strain selection and process optimization to control glycerol formation intentionally.

Conclusion

The Thermo Scientific glycerol reagent kit combined with the Arena 20XT discrete analyzer provides an automated, accurate and repeatable enzymatic method for glycerol determination in wines. The method demonstrates strong agreement with both a commonly used FTIR-based WineScan analyzer and an accredited enzymatic reference method, delivers good precision across relevant concentration ranges, and requires no sample pretreatment. These characteristics make it suitable for routine laboratory use in quality control, proficiency testing and fermentation monitoring.

References

1. Ribéreau-Gayon P., Glories Y., Maujean A., Dubourdieu D. Handbook of Enology, Volume 2: The Chemistry of Wine Stabilization and Treatments. John Wiley & Sons, New York, 2000.
2. O’Kennedy K. Glycerol: The Myth. New World Winemaker, 2010.
3. Patterson T. Many Roads to Mouthfeel. Wines & Vines, article.
4. Organisation Internationale de la Vigne et du Vin (OIV). Compendium of International Methods of Wine and Must Analysis, 2009.