Warning: most methods are probably greatly overestimating free SO2 in reds wines!

Recent development of protocols for free SO₂ determination using gas detection tubes has highlighted significant problems with the accuracy of traditional methods. The acidulation of wine in both Ripper and AO testing releases SO₂ that is bound to anthocyanins and measures it as free. This information was reported as early at 1975 by Bourroughs¹, however, corrective steps required several spectrophotometric measurements using solutions not commonly found in wine labs, and therefore, corrections are almost never made. Unfortunately, the amount of overestimation is significant (up to 66%) but depends on the concentration of each form of anthocyanins in the wine, so cannot be directly correlated to values routinely measured in the winery lab.

Experiments with gas detection tubes began with the goal of measuring free SO₂ without interference from the wine matrix (as if found in Ripper) but also without complex and expensive glassware (as is the case with AO). Pegram et al (2013)² developed a method using gas detection tubes to register sulfur in the gas phase. To move SO₂ from the wine to the tube, wine was acidulated to convert SO₂ into the gas phase, then alka-seltzer tablets were used to create a current that delivered SO₂ to the tubes. However, the alka-seltzer tabs also buffered the acidulated solution, leading to a limit in the range of the test. To solve this problem, Coelho et al (2015) retained the use of the gas detection tubes but, instead of acidulation and airflow, they loaded wine into a closed syringe with known headspace, allowed molecular SO₂ in the wine to equilibrate in the headspace, then depressed the plunger to move headspace air through the syringe³. This methodology measures molecular (gaseous) SO₂ directly, from which free SO₂ can be calculated. 

The headspace gas detection tube method correlated well with AO measurements for white and Rosé wines (R² = 0.97) but the correlation with red wines was poor (R² = 0.72), with headspace methods 2-3 times lower than those reported with AO. After investigating sources of systemic error, the researchers found that in traditional methods the acidulation of wine led to release of SO₂ from anthocyanins while in the HS-GDT method this SO₂ remained bound. They confirmed this was the cause of the difference in free SO₂ values by measuring anthocyanins directly, calculating SO₂ binding to various forms of anthocyanins present at that pH, then correlating back to the difference in free SO₂ between AO and HS-GST. They found a very strong correlation (R² = 0.94)³. Other non-perturbing methods also show this decrease in free SO₂ measures.

Further investigation by Howe et al (2018)⁴ has shown that anthocyanin-bisulfite complexes do not appear to have any antimicrobial properties⁴. This group used sweet wines inoculated with EC1118 yeast to determine effective antimicrobial levels of SO₂ using both AO and HS-GDT methods. They tested both white wine and white wine with added anthocyanin (to mimic red wine, but still compare to the white). In white wines, molecular SO₂ values needed to prevent EC1118 growth were similar using either SO₂ determination method, indicating no change is needed in our practice. In the pseudo-red wine however, a 2-log reduction was found with molecular SO₂ of 0.5-2.0 mg/L when measured by HS-GDT while values greater than 2.0 mg/L when measured by AO were still not sufficient to affect cell counts. (In this study, HS-GDT measures of molecular SO₂ were only 37% those found for AO³.) This means that if SO₂ additions are made based on AO test results in red wines, it is unlikely molecular targets are being met and wine is left susceptible to microbial spoilage.

Unfortunately there is not an easy answer to this problem. The HS-GDT methodology is still very new and has yet to be optimized for the commercial winery. The gas detection tubes used in this method were manufactured for the mining industry, not for wine, and only one of three brands tested by the initial researchers worked^2,3, and even these were discolored by the ethanol found in wine. Several sources of error have been identified, including the timing of syringe depression and leaks in the tubing attachments⁵. The manufacturer’s markings must be converted to linear scale, and calculations must be done based on temperature, pH and ethanol to determine free SO₂⁶. To aid in these calculations, researchers at Cornell have developed a downloadable Excel file (HSGDT calculator). Variation in readings is still large enough for the developers to recommend 5 replicate readings per wine, with each tube costing $7.50 and accommodating 2-3 readings each⁵. So far, this method is not automated, though work done by Jenkins et al (2020)⁷ converts the concept of headspace SO₂ detection to an automatable form. If you would like to try this method in your own winery, Dlubac and Sacks (n.d.)⁵ provide sourcing information and step by step instructions for setup. Make sure you validate the method (by comparing values with white wines) with a known reliable reference method to ensure you are getting reliable numbers.

References

(1) Burroughs, L. F. DETERMINING FREE SULFUR DIOXIDE IN RED WINE. Am. J. Enol. Viticult. 1975, 26 (1), 5.

(2) Pegram, Z.; Kwasniewski, M. T.; Sacks, G. L. Simplified Method for Free SO ₂ Measurement Using Gas Detection Tubes. Am. J. Enol. Vitic. 2013, 64 (3), 405–410.  

(3) Coelho, J. M.; Howe, P. A.; Sacks, G. L. A Headspace Gas Detection Tube Method to Measure SO2 in Wine without Disrupting SO2 Equilibria. American Journal of Enology and Viticulture 2015, 66 (3), 257–265.  .

(4) Howe, P. A.; Worobo, R.; Sacks, G. L. Conventional Measurements of Sulfur Dioxide (SO2) in Red Wine Overestimate SO2 Antimicrobial Activity. Am J Enol Vitic. 2018, 69 (3), 210–220.  

(5) Dlubac, G.; Sacks, G. L. Measuring Sulfur Dioxide (SO2) in Wine Using a Headspace Gas Detection Tube Method.

(6) Sacks, G. L.; Howe, P. A. “Free” Doesn’t Always Mean Free: Rethinking SO2 Measurements in the Winery. 6.

(7) Jenkins, T. W.; Howe, P. A.; Sacks, G. L.; Waterhouse, A. L. Determination of Molecular and “Truly” Free Sulfur Dioxide in Wine: A Comparison of Headspace and Conventional Methods. Am J Enol Vitic. 2020, 71 (3), 222–230.