1. Best Practice Measurement of Total Sulfur Dioxide
Dr Eric Wilkes
Australian Wine Research Institute

2. What is Sulfur Dioxide
Sulfur dioxide (SO2) is an important component of the wine making process providing both antimicrobial and antioxidant properties.
It is not only used in wine but in a vast range of foods and beverages.
It is one of the most commonly analysed components in wine production.
It is also one of the most commonly regulated wine components.

3. SO2 Testing in the Real world
The accurate and reproducible measurement of SO2 in wine is of prime importance to ensure the free and efficient movement of wine between markets.
Unfortunately, the quality of measurement of total SO2 in different markets can vary significantly.
This is clearly demonstrated by the APEC WRF Ring test programs where laboratories from a range of APEC economies test the same wine and compare their performance to an industry wide proficiency program.

4. Total Sulfur Dioxide 2016 Ring Test Results These results are not good
White
Red
APEC
IWAG
Mean
115.66
125
67.03 63
Standard Deviation
21.99 6
11.87 5
Total Sulfur Dioxide
These results are not good

5. Pathways into wine
SO2 can be added to wine at many points during its production.
Added to harvest bins as the salt potassium meta bisulfite (KMBS).
Added as KMBS or as a liquid during grape crushing
SO2 is produced by wine yeast as an intermediate in their sulfate assimilation metabolic pathway during fermentation.
Added as KMBS or as a liquid to the finished wine during maturation or immediately before packaging.
SO2 performs its functions in a sacrificial manner, and the measured levels decrease over time.

6. Forms of Sulfur Dioxide in wine
In wine, SO2 exists in a range of forms in a dynamic equilibrium with water and other wine components.
It should be noted that all the different forms in which SO2 can be added, liquid, gas or as salt, equilibrate to the same forms in solution.
That is, it is essentially impossible in wine to differentiate if the SO2 present has been naturally generated by yeast, added in the form of liquid SO2 or as the salt KMBS.
The forms that it is found in wine are dependant on its interaction with other wine components.

7. Forms of Sulfur Dioxide in wine
SO2 interacts with water to form the following three chemical species.
At wine pH between 92% and 99% of the SO2 is present in the bisulfite form.

8. Forms of Sulfur Dioxide in wine
The bisulfite is also involved in dynamic chemical equilibria with other wine components.
Principle among these is acetaldehyde, however bisulfite will also bind weakly to other carbonyls, phenolics, colour compounds and sugars found in wine.
SO2 bound strongly to wine components in this way is generally considered to be unavailable for antimicrobial and antioxidant activities and is referred to as the bound SO2. The unbound component is referred to as the free SO2.
Free SO2 + Bound SO2 = Total SO2

9. Forms of Sulfur Dioxide in wine
The balance between the different forms of SO2 is highly dynamic and interdependent on a range of wine components.
It is also continually changing over time.
For this reason from a governance point of view it is most practical to only refer the total SO2.
Regulatory limits for SO2 are usually phrased in term of total SO2 with units of mg/L.
free
bound
total
SO HSO SO32-
SO2 bound to acetaldehyde, phenolics, colour, sugars
SO2 bound to phenolics, colour, sugars
SO2 bound to acetaldehyde

10. Methods for Measuring Sulfur Dioxide
There are 3 generally accepted methods for the determination of total SO2.
Very few economies specify the actual method required for the determination.
1. Aeration / Oxidation
Also know as Rankine, Monier/Williams and some other variations on the theme.
It is the current OIV referenced method.
Requires specialist glassware and approximately 20 minute per analysis.
It is, however, very robust and suffers from little interference from other wine components.

11. Methods for Measuring Sulfur Dioxide
2. Automated Flow methods
Provide very good correlation with aeration / oxidation
Relatively expensive to implement using specialist equipment
Mostly only suited to high throughput laboratories specialising in wine chemistry.
3. Iodic Titration
Relatively simple and cheap to implement
Very quick and can be automated
Suffers from significant interferences from other wine components, usually giving artificially high results.
Cannot be used on wines containing the antioxidants ascorbic or erythorbic acid, without appropriate compensation.

12. Sample Handling and Management
SO2 is a relatively volatile substance and readily oxidises.
As such a range of specific precautions are needed during its analysis.
Samples should not be opened until immediately before analysis to avoid loss of SO2 through volatility or oxidation.
If a sample must be decanted, it should be done very carefully to avoid any aeration into a glass container which can be sealed with no air space under the closure.
If decanted samples must be stored they should be refrigerated. Freezing samples is not recommended.
Refrigerated samples should be allowed to equilibrate to 20C in sealed vessels before analysis.
During transfer and pipetting of samples great care should be taken to avoid aeration of the sample.

13. Best Practice Quality Assurance
Despite the existence of a robust reference method in the form of AO, the volatile nature of SO2 mean a high quality, wine specific quality assurance program is required to ensure comparable results.
This would include;
Known total SO2 wine control samples run at least every 10th sample,
Regular duplicate samples,
Standard checks against 100 mg/L control samples,
Checks against 1g/L acetic acid solutions for VA interference.

14. COnclusions
SO2 is an important component of the wine making process providing both antimicrobial and antioxidant properties.
In wine, it exists in a range of forms in a dynamic equilibrium dependant on other wine components and changes over time.
It is often only practical to refer to the total SO2 present when it is deemed necessary to define limits.
It is essentially impossible to analytically determine the form in which the SO2 was originally added to the wine.
There is a significant need for agreement on best practice methodologies for the measurement of total SO2.
Aeration / Oxidation (AO) is most easily implemented in non-specialist laboratories and is free of significant interferences.
Independent of the method used, the volatile nature of SO2 and its sensitivity to oxidation mandate that careful sample handling and rigorous quality assurance procedures using matrix matched control samples are required to achieve accurate and precise results.

15. Ackowledgements
I wish to acknowledge Wine Australia who funded the work which has contributed to this paper and the staff of the commercial services group of the AWRI who provided the feedback during its preparation.
I would also like to thank Paul Huckaba and Gregory Hodson and members of the FIVS STE for their input and contributions.