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.
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.)
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.
Note: this protocol is not appropriate for elemental S analyses in wine; there are unidentified interferences that will give false positives in wines.
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.)
Add four times the weight of the sample of PEG (e.g,. if using 1 gram of sample add 4 grams of PEG).
Place jar in heated water bath for 5 minutes, swirling periodically to mix sample with PEG.
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.
Swirl contents of jar until well mixed.
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.
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.
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.
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.
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:
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.
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:
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