Analyzing Elemental Sulfur Residues on Grapes

Overview of Method       Equipment       Reagents       Apparatus       Sample Prep       Sample Analysis       Calculations      Figures of Merit   Data Interpretation       References       Video

Elemental Sulfur Residue Analysis Instructions, v1.3

Misha T. Kwasniewski (kwasniewskim@missouri.edu), University of Missouri, and Gavin L. Sacks (gls9@cornell.edu), Cornell University

This instruction sheet is based on Misha T. Kwasniewski, Rachel B. Allison, Wayne F. Wilcox, Gavin L. Sacks, “Convenient, inexpensive quantification of elemental sulfur by simultaneous in situ reduction and colorimetric detection,” Analytica Chimica Acta, Volume 703, Issue 1, 3 October 2011, pages 52-57.

A video version of the protocol is available below, and at http://www.youtube.com/watch?v=yH83vDX8ORQ.

Overview of Method

This method quantifies elemental sulfur in liquid or solid samples.  The sample is first dispersed in polyethylene glycol (PEG-400), diluted, and then buffered and dearated by addition of Alka-Seltzer.   The elemental sulfur is then converted to hydrogen sulfide (H₂S) by addition of a chemical reducing agent (DTT).  Two more Alka-Seltzer tablets are then added to generate CO₂ and sparge the H₂S into disposable sulfide detection tubes.

*Take care to minimize risk of the jar bursting from pressure generated from Alka-Seltzer addition (i.e., wear lab safety goggles and make sure there is no blockage of escaping CO₂.)

Equipment

• 120 mL flask with hole drilled in top (see below)
• Short length of plastic tubing (see below)
• Volumetric pipette (see below)
• Balance — able to measure 1-100 grams with 0.1 g resolution
• Blender, either immersion or standard
• Hot water bath, must be able to remain above 80°C for five minutes
• Ruler, metric scale with millimeters​

Reagents

• Distilled water
• Alka-Seltzer tablets, 3 tablets per test
• PEG 400 (Polyethylene Glycol).  250 ml is sufficient for 50-100 assays.
  • CAS No. 25322-68-3
• Dithiothreitol (DTT), 1 g sufficient for 50 assays
  • Store in freezer when not in use.
  • Can also be prepared and used as a dilution in ethanol, prepared weekly. 
  • CAS No. 3483-12-3
• 4M Gastec Hydrogen Sulfide (H₂S) detection tubes
  • Available for $70-$80 for 10 tubes through online retailers
  • Unreacted portion of a tube can be used for future tests on the same day by marking portion already reacted, although the detection range will be diminished by the amount of tube already reacted.
  • Other brands and ranges of H₂S detection tubes are also usable but will have different responses and require separate calibration curves

Apparatus

A -120 ml jar, with hole drilled through the top

B – 5 ml glass pipette, attached to jar and detection tube with plastic tubing. Aquarium tubing works well.

C – H₂S detection tube, will darken with exposure

Note: Both ends of the sulfide detection tube must be opened prior to attaching it to the apparatus. Pliers work well.

Once assembled, check the integrity of the apparatus by adding an Alka-Seltzer tablet to water and submerging the apparatus to just below the top of the gas detection tube. No bubbles should be visible. Zip ties or hose clamps may be needed depending on the fit of the tubing.

 

Sample Preparation

• Fresh or frozen samples of juice or grapes can be used.  If using frozen samples it may be easier to add an equal weight of water to ensure easy blending (e.g., add 120g water for 120g grape sample), but remember to make a correction for this dilution in your final calculations. 

• Try to minimize sample handling prior to blending to ensure residue is not rubbed off.  You might leave the sulfur on your hands rather than on grapes.

• For fruit samples, blend until completely homogenized.  There should be no pieces of skin or stem clearly distinguishable; it should be the consistency of a smoothie. Juice and wine can be used without preparation.

Note: this protocol is not appropriate for elemental S analyses in wine; there are unidentified interferences that will give false positives in wines.

Sample Analysis

1. Weigh out 1-5 grams of macerate or juice sample into the 120ml jar.  Five grams allows for the detection of lower concentrations of sulfur, but will compromise the ability to measure higher concentrations of sulfur above the range of the detection tube. (One gram of sample will be appropriate for most situations.)

2. Add four times the weight of the sample of PEG (e.g,. if using 1 gram of sample add 4 grams of PEG).

3. Place jar in heated water bath for 5 minutes, swirling periodically to mix sample with PEG.

4. Add distilled water to jar to bring total volume to 80ml. If sample shows signs of foaming, a crushed Gas-X tablet or silicone oil can be added at this step.

5. Swirl contents of jar until well mixed.

6. Add first Alka-Seltzer tablet and quickly attach apparatus top, allow to continue until fizzing stops, about 5 minutes. This step is to remove dissolved oxygen; after this point care should be taken to minimize oxygen getting inside the jar.

7. Add approximately 20 mg DTT (about the size of a BB pellet) to the jar, and quickly re-attach jar to the apparatus top. Allow to react for 5 minutes.

8. Add second Alka-seltzer to jar and quickly re-attach jar to apparatus.  Wait until fizzing stops, about five minutes. Wait until fizzing stops, about five minutes. At this point you should start to see the bottom of the detection tube change color from white to brown/grey if elemental sulfur is present.

9. Add third Alka-Seltzer tablet and allow to fizz until it stops. After fizzing has stopped measure the length of detection tube reacted to the nearest 0.5 mm.

Calculations

Recovery spikes of wettable sulfur should be used at regular intervals to evaluate the accuracy of the method. Recommended recovery spike concentrations are 2 and 10 µg/g. The stock solution can be 0.4 mg/mL wettable sulfur in PEG 400, as described above. Recovery should be in the range of 80-110%.

Poor recovery may be due to:

• Impure standards
• Degraded reagents or detection tubes
• Leaks in apparatus
• Inappropriate calibration curves, e.g., because detection tubes are a different model

Figures of Merit

Limit of detection with 1g sample = 0.32 µg/g

Quantification Range with 1 g sample = 1.2-22.5 µg/g

Recovery from grapes and juice as compared to water: 85-95%

Precision: <10% RSD for 2-10 µg/g

Known Interferences: Other species capable of liberating H₂S or low molecular weight mercaptans under reducing conditions, for example disulfides or trisulfides. 

            Note 1: H₂S and low molecular weight mercaptans present in the initial sample will be removed by the first Alka-Seltzer tablet.

            Note 2: Sulfite and sulfate do not cause interferences.

 Note 3: This protocol has not been validated for wine, only for grapes and juice.

Interpretation of Data

Currently, we recommend a limit of 1 µg/g elemental S for skin fermented red grapes, and 10 µg/g for white grapes, although these values are subject to further revision.  Some authors have reported that concentrations as low as 1µg/g may cause increased H₂S production (Wenzel et al. 1980), but other studies suggest concentrations below 3µg/g do not increase H₂S production (Thomas et al. 1993). In white wine production, much of the sulfur residue (>90%) can be lost during pressing and clarification of the must (Wenzel et al. 1980), so higher residues on fruit are tolerable.

Providing precise recommendations for elemental S limits is challenging for several reasons:

• Different yeast strains can convert elemental S to H₂S with different efficiency.
• Yeast can produce H₂S through means other than elemental S reduction, e.g., as a byproduct of amino acid biosynthesis, which is dependent on fermentation conditions and must nutrient availability.
• Elemental S may be lost prior to fermentation during processing, e.g., during pressing and settling of a white wine must.
• Finally, most current recommendations are based on avoiding excess formation of H₂S during fermentation. It is not clear whether there can be other long-term effects of elemental S on wine quality, and whether these should be considered separately.

References

Brenner, M.W. and J.L. Owades (1954). Stable colloidal sulfur solutions. Science 119(3104):911.

Kwasniewski, M.T., R.B. Allison, W.F. Wilcox and G.L. Sacks (2011). Convenient, inexpensive quantification of elemental sulfur by simultaneous in situ reduction and colorimetric detection. Analytica Chimica Acta 703:52-57.

Thomas, C.S., R.B. Boulton, M.W. Silacci and W.D. Gubler (1993). The effect of elemental sulfur, yeast-strain, and fermentation medium on hydrogen-sulfide production during fermentation. American Journal of Enology and Viticulture 44(2):211-216.

Wenzel, K. H.H. Dittrich, H.P. Seyffardt and J. Bohnert (1980). Sulphur residues on grapes and in must and their effect on H2S formation. Wein-Wissenschaft (6):414-420.

Revised October 7, 2013