Free & Total Sulfur Dioxide Measurement SPECTROPHOTOMETRIC VS. RIPPER AND A/O METHODS G. Anderson, Unitech Scientific LLC

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1 Free & Total Sulfur Dioxide Measurement SPECTROPHOTOMETRIC VS. RIPPER AND A/O METHODS G. Anderson, Unitech Scientific LLC ABSTRACT: Unitech Scientific LLC has introduced UniFLEX TM Free Sulfite and Total Sulfite reagent kits for spectrophotometric measurement of sulfites in wine samples. Recently, sulfite concentrations in a variety of wine samples was measured in an independant winery using UniFLEX kits compared to traditional wet chemistry and instrument detection methods. UniFLEX results correlate well with traditional methods; e.g. Free Sulfite: UniFLEX agrees (95% Confidence) with Ripper within 6ppm; with A/O within 5 ppm. Total Sulfite: UniFLEX agrees with Ripper results within 11ppm; with A/O within 9 ppm. METHODS The UniFLEX Free Sulfite (SO 2 -Fr) reagent is based on the reaction of bisulfite (SO3-2 ) with fucsin compounds and formaldehyde to produce a magenta chromophor measured photometrically at 545nm; SO 2 -Fr reagents are linear from ppm. The UniFLEX Total Sulfite (SO 2 -T) reagent is based on the dissociation of bound sulfites at neutral ph and the reaction of bisulfite (SO3-2 ) with Ellman's reagent to produce a colored reaction product measured photometrically at 420nm; SO 2 -T reagents are linear from ppm. In these SO 2 -Fr and SO 2 -T tests, sample blanking corrects for the absorbance from polyphenols and wine pigments. The Net ABS was calculated for samples and standards; sample ppm concentrations were derived by comparison to Net ABS of the 5-level standard curve. Traditional free sulfite concentration in white wines was measured manually to a visual end-point using modified Ripper reagents and, in red wines, using Aeriation Oxidation (A/O). Total sulfites were measured using modified Ripper. The Modified Ripper method uses the more stable Potassium Iodate solution to create Iodine in situ with Potassium Iodide. Pre-bottling wine samples were received from the cellar and tested without pretreatment by the traditional method, then immediately tested using the ChemWell automated wine analyzer and UniFLEX reagents. RESULTS Free SO 2 : All UniFLEX F-SO 2 results were within 7 ppm of traditional free SO 2 results. Average agreement varied by wine variety and method: Chardonnay results were within 1.5 ppm of Ripper, red wine varieties (Pinot Noir, Mourvedre, and Syrah) averaged 5

2 ppm higher than Aeration Oxidation results. Note that, for red wines, both UniFLEX and Ripper results average 5-7 ppm higher than A/O results. The Ripper method has been found to be less accurate than other A/O for testing free SO 2. Ripper accuracy is ± 5 ppm;. All red wine free SO 2 analyses are by the Aeration Oxidation method (A/O). The A/O method accuracy is ± 2 ppm. A/O results are typically 5 to 7 ppm lower than Ripper results; one might expect A/O results to also be approximately 7 ppm lower than the UniFLEX photometric method. The A/O method requires all wine samples be tested below 68 ºF; free sulfites recovery increase as the temperature of the wine increases. Free SO 2 Results: Traditional VS. UniFLEX Ripper A O Average (delta) 3 5 Standard Deviation % Confidence Minimum 0 1 Maximum 7 7 CF Total Variance 24% 6% Total SO 2 : While individual wine samples exhibited a significant method difference (i.e. Delta) - likely due to the presence of interferrences discussed below - total sulfite concentrations on average were fairly close between methods. Chardonnay and Pinot Noir UniFLEX T-SO 2 results were, on average, within 2 ppm of Ripper results; all individual UniFLEX results were within 10 ppm of Ripper results. A greater T-SO 2 method Delta was found with Syrah, as was the case with F-SO 2 : UniFLEX averaged 11ppm higher than Ripper results and all Syrah UniFLEX results were with 15 ppm of Ripper. UniFLEX T-SO 2 Chardonnay results are consistent from day to day; replicates for Fz-check wines were very close within the run and from day to day. Most UniFLEX T-SO 2 results are within the error of ± 5 ppm expected in the Modified Ripper method.

3 Total SO 2 Results: Traditional VS. UniFLEX Ripper A O Winery: #1 Without Syrah #2 Average (delta) Standard Deviation % Confidence Minimum Maximum CF Total Variance 9% 6% 2% Recovery of Added T-SO 2 : Sulfite added to a Syrah wine sample was measured by both UniFLEX and Ripper Total SO 2 methods. Expected values were obtained; the error was within 3%. SAMPLE Total SO 2 by Ripper Total SO 2 by UniFLEX SY CA ppm 52 ppm SY CA ppm spike 116 ppm 128 ppm Expected ppm 132 ppm 119 ppm (% error) (-3%) (-2.5%) DISCUSSION Free SO 2 by Aeration Oxidation is considered the standard method in the wine industry. It consists of three sample preparation steps: Sample is acidified to force HSO 3 - to molecular form Gas stream or vacuum strips molecular SO 2 from the sample to a peroxide trap SO 2 is oxidized by peroxide to sulfuric acid in the trap and the detection step: Sulfuric acid is titrated with NaOH to determine SO 2 concentration. Page 3

4 Potential Sources of Analytical Error Sulfites chemistry in wine complicates its accurate measurement. This is especially true of the Ripper, but aeration oxidation (AO) analysis (a method alleged free of interference) are also affected by details of Sulfite binding in wine matrices. HSO 3 - readily binds (i.e. forms adducts) with a variety of compounds in wine or juice: Acetaldehyde, Anthocyanins, Sugars, Pyruvate, Alpha-ketoglutarate. Sulfite Adducts vary in stability: Anthocyanin adduct is not stable it will hydrolyze readily with acid addition or to reestablish equilibrium if free SO 2 is consumed; Sugars, pyruvate & alpha-keto-glutarate are moderately stable, but will hydrolyze with acid to re-establish equilibrium. 1. Fr-S02 and T-SO2 Errors RedOx Interferrence: Another source of error that can lead to overestimation of free SO 2 is the presence of other oxidizable compounds in the wine which react with the iodine titrant, leading to overestimation of the SO 2 level. In some white wines, there are appreciable levels of ascorbic acid which react readily with the titrant. In red wines, the presence of phenolic compounds can also lead to overestimation. A suggested approach to estimate the error in a modified Ripper method is to titrate duplicate samples, one having been pretreated with a drop of 3% hydrogen peroxide, the other untreated. The difference between these values is the non-sulfite substances reacting with the titrant, since peroxide primarily removes only the SO 2. This 'peroxide blanking' approach is incorporated into the UniFLEX SO 2 -F method, thereby greatly reducing the potential for RedOx interferrence. Visual Endpoint: In the Ripper titration, standard iodine (or iodate with iodide) is measured into an acidified wine sample. The iodine reacts with free SO 2 ; when all SO 2 has all been titrated, the residual free iodine produces a dark blue starch-iodine complex signals the endpoint. This original Ripper method is fast but its accuracy and precision are notably poor, primarily because it relies on the appearance of a dark end point, which is difficult to see (especially in red wines) and subject to errors of interpretation. Some modifications to the Ripper method improve the reliability of detecting the endpoint. The Hanna Instruments titrators like the HI (also sold by Unitech Scientific LLC) use the ORP electrode in an automated device. This electrode measures the oxidation status of the solution that occurs as free iodine appears (eliminating the need for visual detection.) Numerous reports describe potential error and interferences in the Ripper method. Standardization: The traditional Ripper method depends on freshly-made Iodine solution; Iodine is unstable and traditional Ripper needs to be calibrated daily. The Modified Ripper method uses the more stable Potassium Iodate solution to create Iodine in situ with Page 4

5 Potassium Iodide. The Ripper Iodine method is more accurate, but Modified Ripper is more frequently used in wineries due to this improved convenience. Metabisulfite solutions, typically used as standards and process checks in wine sulfite testing, tend to be unstable - they are typically prepared fresh prior to an assay. UniFLEX SO 2 Reagents include a single standard; multi-level standard sets are also available from Unitech Scientific for use in automated SO 2 testing. These standards are stable, thus improving analytical quality and assay convenience. 2. Fr- SO 2 Measurement Acidification: Both Ripper and A/O Fr- SO 2 start with an acidification step. Many sulfite adducts are acid labile and, as a result, some bound SO 2 is slowly released during these analyses, forming more free SO 2. This leads to a systematic overestimation of free SO 2. For this reason, "Free SO 2 " can better be characterized as "free plus some dissociated bound SO 2 ". The amount of dissociated bound SO 2 depends on the types of adducts present in the wines and the speed of the analysis ( The overestimation due to the acidification step can be minimized by performing the Ripper titration quickly (within 2 minutes). The stability of sulfite adducts is critical to free SO 2 analysis, as most analytical approaches acidify the sample to drive the bisulfite to the H 2 SO 3 molecular form. It should be noted that the acid lability of SO 2 Adducts is taken into account by the short incubation time a low reaction temperature of UniFLEX F-SO 2 manual and automated procedures. CONCLUSIONS UniFLEX reagents are highly sensitive, and require a much smaller sample volume than traditional detection. As a result, the accuracy of the Ripper and A/O analyses is more likely to compromised by pigment and other matrix interferrence. UniFLEX Sulfite reactions are fast and can be performed in batch mode using either spectrophotometers or automated analyzers; the reagents and SO 2 standards provided are stable and easy to use. The UniFLEX F-SO 2 manual and automated test procedures take into account the above-mentioned time-temperature dependancy in Fr-SO 2 testing; UniFLEX Fr-SO 2 results were reproducible and the bias compared to Aeration Oxidation results was constant; the evaluating Winery concluded that UniFLEX F-SO 2 testing can replace their traditional F-SO 2 methods. Page 5

6 The UniFLEX T-SO 2 photometric method is very simple and easy to use. It is a quick batch test, thereby improving laboratory efficiency over the 10 minute sequential testing required by the Ripper method. UniFLEX T-SO 2 and Ripper Total SO 2 methods are comparable. By converting from traditional to photometric SO 2 analyses, both lab work-flow and reduce labor costs are improved. UniFLEX Free SO 2 and Total SO 2 reagents are excellent replacements for Ripper and Aeriation/Oxidation in wine samples. RAW DATA UniFLEX Fr-SO 2 vs Ripper (Chardonnays) Sample Variety Free SO 2 Ripper Free SO 2 UniFLEX Delta Fz-Check CH B10 CH B46 CH B47 CH B64 CH B65 CH TK 226 CH TK 227 CH TK 228 CH TK 229 CH TK 238 CH TK 231 CH TK 233 CH Average: Page 6

7 UniFLEX Fr-SO2 vs A/O Sample Variety AO ppm UniFLEX. ppm Delta Tank 048 CH Tank 049 CH CA1051 SY CA1052 SY CA1050 SY CJ1097 SY CJ1098 SY CJ1095 GN CJ1096 MV CW1056 PN CW1084 PN CW1085 PN CW1065 PN CW1079 PN Average: Page 7

8 UniFLEX T-SO2 vs Ripper Sample Variety Ripper ppm UniFLEX. ppm Delta Fz-Check CH CW1004 CH CW1003 CH CW1006 CH Fz-Check CH Fz-Check CH Fz-Check CH B10 CH B46 CH B64 CH TK 227 CH TK 228 CH TK 238 CH TK 231 CH TK 232 CH EL1006 CH EL1007 CH EL1008 CH EL1009 CH CJ1039 CH CJ1032 CH CJ1011 CH Fz-Check CH Fz-Check CH Fz-Check CH Fz-Check CH CH-Average: CW1076 PN CW1083 PN CW1063 PN CW1077 PN CW1087 PN B23 PN B26 PN B27 PN B28 PN B30 PN B31 PN B32 PN CW1088 PN CJ1082 PN CJ1084 PN CJ1091 PN CJ1092 PN CW1050 PN CW1051 PN CW1052 PN CW1053 PN CW1055 PN CW1059 PN CW1072 PN CW1086 PN CW1070 PN CW1075 PN CJ1080 PN CJ1081 PN CJ1083 PN CJ1085 PN CJ1086 PN Page 8

9 B51 PN B52 PN B53 PN B54 PN B55 PN B56 PN B57 PN B60 PN B61 PN B62 PN BF1003 PN PN-Average: CA850 SY CA1050 SY CA1051 SY CA1052 SY CA951 SY CA952 SY CA953 SY CA954 SY B63 SY SY-Average: Overall Average Page 9