SO2-Втора част

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SO2-Втора част

Мнениеот vinoirakia » Нед Май 16, 2010 7:04 am

Технолог/ако беше енолог, разговора нямаше да се проведе/ към техник винар:
- На резервоар 45 трябва да вдигнем свободния с 20
- Това какво означава-пита техник-винара
- Една кофа киселина-е отговора
- Малка или голяма
- Каквато намериш- отговаря технолога

8. SO2 and Temperature
As temperature increases, free SO2 increases and bound SO2 decreases. (SO2 bound to acetaldehyde remains constant.) This is because increased temperatures cause partial dissociation of the bound SO2 form, resulting in increased free SO2 and hence increased molecular SO2 concentrations.

For example, a wine containing 68 mg of free SO2 at 0°C (30°F) will contain 85 mg at 15°C (57°F) and 100 mg at 30°C (84°F) [Peynaud, 1984]. Sudraud [1977] showed 64 mg/l of free SO2 at 16°C increased to 120 at 48°C and to 200 at 80°C, as BSO2 was released.

Sometimes, wines with high molecular SO2 levels are served cold to hide the sulphurous aroma they would exhibit with their high SO2 content.

9. Sensory Threshold
It is the molecular SO2 form which is responsible for the sensory threshold. Hence, the sensory threshold of SO2 depends on the pH and temperature. There exists considerable variation in threshold within the population. Nevertheless, the sensory threshold is generally considered to be around 2 mg/l molecular SO2.

10. SO2 Loss
SO2 can be lost from wine under a number of circumstances. Molecular SO2 is volatile and some is lost from both juice and wine through vaporisation to the air, especially if the juice/wine is agitated. This loss is higher in wines stored in barrels. However, the quantity lost in this way is usually negligible.
SO2 will be lost during alcoholic fermentation. This is partially through vaporisation with escaping carbon dioxide from the fermentation. At the end of fermentation it is common for a wine to possess zero to just a few milligrams per litre of total SO2, however significant deviations from this norm can be found.
Losses additionally occur through the oxidative protection of SO2. This is largely due to SO2 reacting with hydrogen peroxide to form sulphuric acid. Interactions of SO2 with quinones to form monosulphonates may also result in SO2 loss [Lu Valle, 1952].
SO2 is also lost in bottled wine [Ough, 1985]. Mьller-Spдth [1982] found that the total SO2 had dropped by 20-30% after 5 years at 12°C in two bottled white wines. The rate of total SO2 loss appears to be 2-3 times faster in reds than in whites [Ough, 1985]. Peynaud notes that SO2 losses in bottle are a few mg/l per year [Peynaud, 1984, p.271]. The causes for SO2 loss in bottle are numerous. SO2 vapour may be lost through the cork, but this is not substantial under normal temperatures of storage. Oxidation of the SO2 with oxygen in the bottle will certainly occur, but this reaction is very slow. Oxidation of the SO2 by formerly oxidised phenols will lead to the production of sulphate and a loss in total SO2. SO2 loss may also occur due to rearrangements in the reaction processes within the wine after long time periods, favouring redox/equilibrium reactions rather than kinetic reaction rates.

Given this information, it should be kept in mind that the total SO2 is not the same as the amount of SO2 that has been added to the must/wine, since some of the added SO2 will be oxidised irreversibly to sulphate and some lost through volatisation.

Whenever losses occur, the equilibrium between free and bound SO2 will re-establish, resulting in a small decrease in bound SO2.

11. SO2 and Oxidation
11.1. In must
Without the presence of SO2 in musts, juice undergoes enzymatic oxidation. The enzymatic oxidation of phenolic compounds governs oxidation over and above chemical oxidation reactions because of their much faster reaction speed. The oxidase responsible is polyphenol oxidase (PPO), and in the case of Botrytis infected fruit, laccase [Dubernet and Ribйreau-Gayon, 1973 and 1974].

PPO is also known as tyrosinase, catecholoxidase, catecholase, phenolase, phenoloxidase, and o-diphenoloxidase. Its activity depreciates with time and is usually completely inactive following fermentation. However, it is primarily responsible for oxidation in juice. SO2 is usually added to musts to inhibit the activity of (or destroy) oxidase enzymes and subsequently prevent oxidation. (For exceptions to this practise, see Section 12, "Hyperoxidation".)

The rate of oxygen uptake in juice is determined by the temperature, enzyme activity, phenolic concentrations, the substances "consumed" by the enzyme, and the competition between different substances for binding [White and Ough, 1973]. PPO activities vary widely even within fruits of the same variety [Traverso-Rueda and Singleton, 1973; Hooper et al., 1985].

However, in the absence of SO2, oxygen uptake is generally rapid. When first coming into contact with air, an SO2-free juice can exceed uptake of 2 mg oxygen/l/min. Uptake of dissolved oxygen (from a saturated juice state) can be complete in a white grape must within 4-20 minutes [Dubernet and Ribйreau-Gayon, 1974]. In an apple juice, also saturated with dissolved oxygen, consumption was complete within 1 hour (at pH 3.45) and most uptake took place within the first 10 minutes. The process was slower in orange juice at pH 3.57 with an initial rapid uptake of around 2 mg/l oxygen within the first hour, followed by around 0.4 mg/l uptake per hour thereafter. Uptake in lemon juice at pH 2.35 progressed at around 0.7 mg/l oxygen per hour. [Lьthi, 1953, 1954, 1960; Biedermann, 1956].

Upon the addition of SO2 to a must, oxygen consumption will tail off over a period of time (typically 1-6 minutes for SO2 additions of 10-100 mg/l) until it completely stops and the oxygen concentration remains constant [Dubernet and Ribйreau-Gayon, 1974]. This behaviour is shown in Figure 8.

Based on the fact that the effectiveness of the SO2 is delayed, and that such enzymatic oxidation is rapid, it is important to ensure that SO2 is added as soon as possible to prevent oxidation.

The addition of 25-75 mg/l SO2 to clarified juices has been shown to inhibit PPO activity by 75 and 97%, respectively [Dubernet and Ribйreau-Gayon, 1973; Amano et al., 1979]. A rough representation of these results is presented in Figure 9.

Due to the binding of SO2 to fruit particulates, the required doses may in practise be 75-100 mg/l for unclarified must, and 30-50 mg/l for clarified juices.

Laccase (also known as p-phenoloxidase) is another important oxidative enzyme. It is found mostly in fruit infected with Botrytis cinerea and it causes rapid oxidation [Peynaud, 1984]. Less data exists on the inactivation of laccase by SO2. However, it appears more difficult to inactivate than PPO enzymes, and it consumes oxygen over longer periods of time than PPO enzymes. One study found that even with a free SO2 level of 150 mg/l, only a 20% reduction in activity occurred [Dubernet and Ribйreau-Gayon, 1973]. In general, laccase activity exists in fermented wines made from Botrytis infected fruit. Ascorbic acid is often used as an antioxidant in such situations.

11.2. In wine
The excessive oxidation of wines causes increased browning, increased production of stale-smelling aldehydes (for example, the oxidation of ethanol to acetaldehyde [Kielhofer and Wьrdig, 1960]), and ultimately leads to a loss of fruit/varietal character. Limiting oxidation (to a certain extent) is normal practise in most wines and exceptions to this are limited (e.g. Sherry, Madeira).

11.2.1. Mechanisms
In wine, oxidative enzymes no longer exist and the primary oxidative impact is through chemical oxidation.

The precise mechanisms by which SO2 protects wine from oxidation are not fully understood, nor widely agreed upon. Nevertheless, SO2 is deemed to possess antioxidative activity because it is preferentially oxidised over other compounds which, when oxidised, would lead to undesirable aromatic/flavour changes.

The direct reaction of SO2 with oxygen appears to be insignificant in wine. Oxygen reacts with polyphenols before it can be removed by SO2. Instead, the main antioxidant role of SO2 appears to be its reaction with hydrogen peroxide (H2O2) [Danilewicz, 2003]. The most reactive polyphenol grouping in grape (and apple) wine are the catechols (in this explanation catechols are used as a simple example to show the reaction of the most reactive wine polyphenol grouping). The following equation represents their reaction with O2 in wine:

1,2-benzenediol (catechol), through Fe(III) catalysis, reacts with O2 to form 1,2-benzoquinone and hydrogen peroxide [Wildenradt and Singleton, 1974].

Essentially, transition metals oxidise catechol (1,2-benzenediol) and these reduced metals are then reoxidised by oxygen. Indeed, work by Poulton [1970] suggests that an intermediate is formed in the presence of oxygen which then reacts rapidly with SO2 since, under model wine conditions, a half time of approximately 30 days was required for SO2 to consume the oxygen in a saturated solution. In a white wine, the half time was 1.2 days.

Hydrogen peroxide is a strong oxidising agent and will oxidise other wine compounds, potentially leading to an undesirable aroma. For example, ethanol may be oxidised to form acetaldehyde, which possesses a stale aroma:

H2O2 + ethanol ===> acetaldehyde + 2H2O

However, when SO2 is present, the bisulphite ion (HSO3-) is deemed to undergo nucleophilic attack by hydrogen peroxide (H2O2) with displacement of water and the formation of a peroxymonosulphite ion:

HSO3- + H2O2 ===> HOOSO2- + H2O

Acid catalyzed rearrangement subsequently results in the formation of a sulphate dianion:

HOOSO2- + H+ ===> SO42- + 2H+

Thus, SO2 removes the strong oxidising agent H2O2, preventing it from oxidising other wine compounds which would potentially lead to undesirable oxidised aromas. (This action is, however, competitive and formation of acetaldehyde has been shown to occur even in model wine solutions with high (177 mg/l) concentrations of SO2 [Wildenradt and Singleton, 1974].)

From the equations above it can be seen that, for every mole of oxygen uptaken, one mole of SO2 and one mole of a quinone will form.

Quinones are highly reactive with bisulphite and the reaction of 1,2-benzoquinone with bisulphite proceeds as follows:

However, some of the quinone will not react with bisulphite due to the fact that they condense with other compounds. If all the quinone reacted with bisulphite then a further mole of SO2 would be taken up, resulting in the uptake of two moles of SO2 for every mole of oxygen. However, since not all quinones will react in this way, the overall SO2 loss for every mole of oxygen should be between one and two moles of SO2. The antioxidant activity of bisulphite is primarily restricted to its reaction with hydrogen peroxide. The direct reaction of SO2 with oxygen seems to be a chain process, prevented by the reaction of scavanging phenols with SO2 [Danilewicz, 2003].

Wines made from botrytised fruit present an exception to the oxidation of wine being solely chemically driven. In this case, the enzyme laccase may be involved in the oxidation (see Section 11.1 for more).
Since SO2 does not prevent oxidation directly - it simply prevents undesirable oxidation reactions from taking place - measures to protect wine from oxidation should still be practised even when SO2 is used.

11.2.2. Reaction
Under ideal conditions, 2 moles of SO2 would be required to remove 1 mole of oxygen (molecular weight of 32 vs 64) in a direct reaction. However, as noted in the above section, the overall SO2 loss for each mole of oxygen should be between one and two moles of SO2, depending on whether quinones react with bisulphite (see Section 11.2.1). This seems unlikely since condensation with numerous compounds (including polyphenols) has been shown to occur. Thus, in practise, it appears more likely that 1 mole of SO2 will be lost for every 1 mole of oxygen consumed. It should, however, be stressed that the direct reaction of oxygen with SO2 is unlikely to occur to any significant level. Therefore this reaction ratio is assumed as purely a general and theoretical estimate of O2 consumption by SO2.

11.2.3. Oxygen uptake
Given oxygen exposure, the oxygen uptake of wine is around 1-2 mg/l/day. This is a generalisation however, since different wines have different rates of O2 consumption, and this appears to change with increased O2 exposure (Rossi and Singleton 1966, Perscheid and Zьrn 1977).

It takes several days for O2 saturated wine (8-8.6 mg/l O2) to be consumed by SO2 (in a synthetic medium). At room temperature, dissolved O2 usually drops to undetectable levels after about a week. SO2 depletion
Over time, free SO2 decreases in wine. Average estimates indicate that SO2 depletion may be around 5 mg/l per month in wines stored in large tanks in cool cellars with small headspaces. Wines stored in warm cellars with large headspaces often lose 10-20 mg/l per month, or more. [Eisenman, 2001]. (SO2 in bottle exhibits a depletion of no more than a few milligrams per year.) Because of this decrease, SO2 levels must be continually maintained.

SO2 depletion increases with an increase in temperature, headspace, and oxygen exposed surface area to volume ratio. Since it is dependant on many variables, SO2 depletion varies from set-up to set-up and wine to wine. Safe assumptions can be made based on the past experience witnessed with each set up. For this, free SO2 levels must have been measured to determine the level of decrease over a given time and situation. Saturation level
The saturation level of dissolved oxygen in juice/wine depends on temperature (it increases with a decrease in temperature) and the alcohol content of the wine (it increases with an increase in alcoholic content). At 20°C (68°F) 8 mg/l (6 ml/l) is the saturation level, whereas at 0°C (32°F) it is 11 mg/l (8 ml/l). [Peynaud, 1984, p.248] Thus, the oxygen saturation range in wine is generally 7-11 mg/l (5-8 ml/l). [Supported by Rankine, 1995, p.187-188; Jackisch, 1985, p.115]. This level cannot be surpassed unless the temperature or pressure changes.
The oxygen content of commercially extracted orange juice is reported as 2.5-4.7 ml/l [Pulley and von Loesecke, 1939], of laboratory extracted orange juice as 2.7-5 ml/l [Loeffler, 1940], and of hand-ream extracted as 5 ml/l [Kefford et al., 1950]. Though Eisenman [2001] claims oxygen saturated juice contains about 10 mg/l of oxygen.

Note that the figures quoted outside of parentheses below are in milligrams per litre and those inside parentheses are in millilitres per litre. Racking
Gentle racking often causes an oxygen uptake of 1-3 mg/l (0.8-2.3 ml/l), whereas those with more turbulence and air exposure might absorb 3-8 mg/l (2.3-6 ml/l) during each racking. [3-4 ml/l in Peynaud, 1984, p.249; 5-6 ml/l in Jackisch, 1985, p.117; 4 mg/l from Kelly and Wollan, 2003]. Barrels
Oxygen uptake by wines in barrels is highly variable. Some quote 3-7 mg/l per year (2-5 ml/l/yr). Others find 20-27 mg/l/yr (15-20 ml/l/yr) [Mountonet et al., 1998]. The highest diffusion rate has been estimated at 26.4 ml/l/yr, and oxygen exposure due to topping and ullage has been estimated at 5 ml/l/yr [Kelly and Wollan, 2003]. This increases with less close-grained wood and smaller cask sizes - in tuns of 5cm thickness it was considered to be practically nil [Peynaud, 1984, p.248].

However, when considering that barrels are often opened for testing/tasting, oxygen absorption may be around 40-53 mg/l per year (30-40 ml/l/yr) [Jackisch, 1985, p.117]. Peynaud notes that absorption through surface exposure is about 20-27 mg/l per year (15-20 ml/l/yr), whether at the bung-on-top position with regular toppings or the bung-on-side position [Peynaud, 1984, p.248]. A partially filled container of wine with a surface area of 100 cm2 will absorb oxygen at 2 mg/l per hour (1.5 ml/l/hr). [Peynaud, 1984, p.248]

(The conversions from oxygen's volumetric measures to oxygen's by weight measures are calculated at 1 atm and 20°C (68°F). Under these conditions, 1 ml/l of oxygen weighs 1.33 mg/l. At 0°C (32°F) and 1 atm it's 1.43 mg/l, a difference of only 7% which is considered reasonably comparable.)

11.2.4. pH alteration due to sulphate formation
Since wine is an acid solution, H+ ions are present and the sulphate (2SO42-) forms sulphuric acid (HSO4-). (See reaction equations below.) The formation of sulphuric acid lowers pH. This can result in a harsher tasting wine. However, the production of sulphuric acid is small (0.82 g/l titratable acidity as tartaric acid when 350 mg/l SO2 is used). In wines with botrytised fruit, and non-botrytised sweet wines with high SO2 concentrations, a considerable amount of sulphate can be formed (0.5 g/l as tartaric). For wines stored in barrels over long periods, this can result in reduced wine quality.

SO2 + H2O ===> HSO3- + H+
2HSO3- + O2 ===> 2SO4>= + 2H+

11.2.5. Overview
Given that SO2 does not react directly with O2, all measures to protect wines from O2 should be taken to avoid O2 exposure when the desire is to minimise wine oxidation. The presence of SO2 does not guarantee the avoidance of oxidation entirely.

12. Hyperoxidation
12.1. Hyperoxidation theory
Members of "the brown juice club" do not add SO2 to white wines before fermentation. The intention is to allow rapid polyphenol oxidase (PPO) enzymatic oxidation of the many phenolic compounds in the juice which would later be chemically oxidised in the wine. The brown quinone polymers formed are adsorbed to solids and precipitate during or soon after fermentation. The process of adding oxygen to musts to achieve this result is called "hyperoxidation" or "hyperoxygenation".

Wines made in this way are claimed to be more stable with regard to later SO2 additions and less susceptible to oxidation later in their life. The technique enhances colour stability [Mьller-Spдth, 1977]. It may have a desirable impact on aroma (Chardonnay) [Mьller-Spдth, 1988; Fabre, 1998; Cheynier et al., 1989], however it can reduce the aromatic intensity of varieties (for e.g., Sauvignon Blanc) [Dubourdieu and Lavigne, 1990]. Indeed, some (e.g. the UC Davis team) believe this technique reduces varietal character and is disadvantageous for making fruit-driven wine styles in particular. For example, the volatile sulphur compounds (4-MMP, 4-MMPOH and 3-MH) contribute significantly to the characteristic varietal aroma of Sauvignon Blanc (smelling of box tree, citrus, and grapefruit/passion fruit, respectively) [Denis Dubourdieu, 2004 noted in Zoecklein [2005]]. They are easily oxidised. Therefore, to retain varietal character it is important to protect Sauvignon Blanc juice from oxidation.

The success of hyperoxidation depends on the amount of oxygen required to fully oxidise the flavonoid phenols in the juice, the pH and temperature, and the fraction of phenols which will oxidise under PPO activity. Inconsistent results have been found with the use of this technique because of these variables. Firstly, the PPO activity of juices varies widely (even within juices of the same variety) [Traverso-Rueda and Singleton, 1973; Hooper et al., 1985]. This means that some juices will require more exposure to oxygen than others. For example, Perscheid and Zurn [1977] found that 40 oxygen saturations were required before any significant decrease in the rate of PPO activity was observed. Whilst Amano et al. [1979] saw significant decreases as soon as the juice was exposed to oxidative treatment.

The premise behind this technique is that the phenols oxidised by PPO enzymes are the same as those which will later be oxidised and browned in the wine. This may not be the case and, in fact, many flavonoides are not oxidised fully through PPO activity [Singleton, 1987]. Additionally, the lack of SO2 in must can contribute to the occurrance of a slow or stuck fermentation [Zoecklein, 2001, under "Sulfur Dioxide in the Fermenter"]. The consumption of significant amounts of oxygen in the must can potentially lead to an insufficient oxygen content for healthy yeast growth.

12.2. Practical aspects of hyperoxidation
To successfully manage this technique, PPO activity should be maximised. Aside from avoiding SO2, this also means the juice should not be fined or clarified in any way. The juice should be sparged with pure oxygen (or else compressed air) whilst mixing the juice significantly. Around 20-30 mg/l oxygen (15-23 ml/l oxygen) or 95-140 mg/l air (70-105 ml/l air) is required. Repeating the sparging procedure is recommended. The juice should then be separated (clarified) from the precipitated oxidised phenols. SO2 may be added after clarification, but is usually avoided altogether.

13. Accounting for SO2 Binding: Practical Examples
13.1. Approximations for SO2 binding
Having made an SO2 addition, winemakers should re-test the free SO2 concentration some days after the addition has been made to assure that the free SO2 level in a wine has been attained. Of course, it is preferable to make a single addition which will, having accounted for the SO2 binding which will occur, arrive reasonably close to the desired level. Using the approximate rules of SO2 binding outlined in Section 5.5 above, the following examples detail how winemakers might account for such binding.

13.2. Accounting for binding lost to bisulphite addition products
Using the rule that 50% of any SO2 addition becomes bound whilst the total SO2 content of the wine is under 50 mg/l, and that 10% of any addition becomes bound thereafter, the following example illustrates the additions a winemaker might make.

35 mg/l of SO2 is added to a white must at crush. Following fermentation, the wine has a pH of 3.1 and it is (safely assumed or) assessed that the free SO2 content is negligible and all SO2 is bound. It is desired to take the molecular SO2 level to 0.6 mg/l. 12 mg/l free SO2 is required for 0.6 mg/l molecular at pH 3.1 (see Figures 6 and 7 or Table 2 above). If all SO2 added became free, 12 mg/l would be added to obtain this level. However, it has been assumed (from the above rule) that 50% of the SO2 addition will become bound. Thus, for 12 mg/l to remain after binding, 24 mg/l (12*2 or 12*100/50) SO2 must be added.

Some time later when the wine is bulk ageing, the total amount of SO2 that has been added to the wine is larger than 50 mg/l. The pH remains 3.1 and the free SO2 has depleted to 10 mg/l.
Again, 12 mg/l free SO2 is required for 0.6 mg/l molecular at pH 3.1 (see Figures 5 and 7 or Table 2 above). Since 10 mg/l is already present, 2 mg/l (12-10) free SO2 is therefore the required addition assuming no binding occurs.

If, instead, it is assumed that binding still occurs at the lower rate of 10%, then 10% of all SO2 added at this stage becomes bound. 2.2 mg/l (2 / 90% which is also 2 / (90/100)) SO2 is required to be added to the wine to obtain the 12 mg/l free SO2 level for 0.6 mg/l molecular SO2.

13.3. Accounting for Oxygen Binding: Examples
The bottling of 5 litres of wine is conducted with some splashing. It was assumed that the wine would become almost saturated with oxygen after such a racking and 12 ml of headspace would remain in the bottle once corked. Two calculations are required: (a) to account for the SO2 required to bind with the oxygen uptake during the bottling operation, and (b) to account for the SO2 required to bind with the airspace in the bottle. It is assumed that SO2 binding with wine compounds is negligible in this case (which is likely by the time bottling is due).

(a) 7 mg/l of oxygen is assumed to be dissolved into the wine following the racking procedure. A maximum of 14 mg/l of free SO2 (7*2) is required. 70 mg might be added to the bulk 5 litres (14*5), or alternatively, 10.5 mg to each 750 ml bottle (14*0.75).
(b) Each bottle contains 12 ml of airspace. Using the fact that air is 21% oxygen, the oxygen content in the headspace is 2.5 ml (12*0.21). 2.5 ml weighs 3.3 mg (2.5 mg * 1.33 mg/ml). Therefore 6.6 mg of SO2 is required (3.3*2) to bind with the oxygen in the headspace in each bottle.

If the SO2 is added to each bottle and not to the bulk, the total amount of SO2 in each bottle should be 17.1 mg (10.5+6.6).

13.4. Accounting for Oxygen and Binding: Combined Example
Combining the above sections, a typical SO2 addition accounting for both bound SO2 (due to wine components) and oxygen binding (using up the oxygen the wine is exposed to during, for example, a racking) may be calculated.

A wine is to be racked gently. The current free SO2 level is assumed to be zero, and the total SO2 level is under 50 mg/l which means that approximately 50% of all added SO2 will become bound. The oxygen uptake due to racking is assumed to be 3 mg/l. The pH is 3.1 and the aim is to obtain 0.8 mg/l molecular SO2.
Accounting for the racking oxygen uptake, 6 mg/l SO2 is required (3*2). For 0.8 mg/l molecular at pH 3.1, 16 mg/l is required. Thus, a total of 22 mg/l is required assuming no wine component binding. Yet 50% of the SO2 amount added will become bound, so 44 mg/l is required (100/50*22).

14. Testing for SO2 (Ripper and AO methods)
Due to its widespread and historic use in the wine and the food industries in general a number of analytical methods exist for measuring SO2. The most common methods are probably the Ripper method [Ripper, 1892] and the aeration oxidation (AO) method.

14.1. Ripper method
The Ripper method for SO2 uses an iodine standard to titrate the SO2 in a sample. Free SO2 is determined directly while total SO2 can be ascertained by treating the sample with sodium hydroxide before the titration to release bound SO2.

The free and total SO2 analysis used in the Ripper test is based on the redox reaction:

SO2 + 2H2O + I2 --> H2SO4 + 2HI

A starch indicator is added to the wine sample and it is acidified with H2SO4. The sample is then rapidly titrated with an iodine solution. The completion of the reaction is noted when excess iodine is complexed. This is determined by a blue-black colour end point in the presence of a previously added starch indicator.

A simple and cheap way to conduct a Ripper test is to use Chemetrics "Titrets" kits ( (See Figure 10.)

The Ripper method is, however, slightly inaccurate. The method suffers from the fact that the iodine reacts with oxidisable substances in wine (e.g. phenols, ascorbic acid), resulting in increased consumption of iodine and subsequently a false-high SO2 estimation. The correct assessment of free SO2 using this method is susceptible to the fact that bound SO2 interferes with the measurement. The reduction of the free SO2 during both the Ripper and AO methods results in a low bisulphite level and consequently, some bisulphite is released from the bound SO2 to re-attain equilibrium. In red wines, the release of SO2 from bound SO2-anthocyanin can significantly result in a false high measurement of free SO2. How rapidly the carbonyl bound SO2 compounds dissociate to release free SO2 depends on the dissociation rates for SO2 binding with those respective compounds (see section 5.3. above). Acetaldehyde bound SO2, for example, will be slow to release. However, pyruvate (and possibly alpha-keto-glutarate) will dissociate faster. Wines high in pyruvate will therefore result in an increased release of free SO2 from the bound form, yielding a false-high measurement of the free SO2 content of the wine. This is another reason why such titrations should be conducted rapidly.

It can also be particularly difficult using this method with reds, since the dark colour of red wines makes it difficult to identify the end point of the titration. Use of an oxidation-reduction electrode will not solve this problem since the end point is dependent on the actual blue starch end point.

Additionally, the potential volatilisation of SO2 during titration, and the reduction of the iodine titrant by non-sulphite compounds such as phenols or pigments, can effect the result significantly. Other interferences include botrytis and ascorbic acid (results are false-high due to the competitive oxidation of ascorbic acid and SO2 by the iodine titrant).

Despite the inaccuracies, the Ripper method remains the most common method used for free SO2 determination in winemaking (including commercial winery labs) due to its speed and simplicity.

14.2. AO method
The AO method, also called the Rankine method after Rankine [1962] or the Tanner method after Tanner [1963], is a modification of the Monier-Williams method. It involves the acidification of a sample, followed by the distillation of SO2 (with nitrogen sweeping gas or air aspiration) out of the sample into a peroxide solution. The SO2 and peroxide react to form H2SO4 according to the following equation:

H2O2 + SO2 --> SO3 + H2O --> H2SO4

The acid formed is then titrated with NaOH to an end point, and the volume of NaOH required used to calculate the SO2 level. This method avoids the iodine-phenol binding which occurs in the Ripper method. However, it is not without its problems. The acidification of the solution causes a shift in the equilibrium between bisulphite and anthocyanins, and the SO2 bound anthocyanins (red wine pigments). This causes a freeing of some anthocyanin bound SO2, resulting in an increased free SO2 concentration and a false-high free SO2 measurement. An inefficient condenser (used in the distillation) accompanied by high volatile acidity will also result in a false-high measurement. Additionally, the flow rate of the aspiration is important in AO testing. An excessively high aspiration rate may prevent sufficient time to allow the H2O2-SO2 reaction to complete. A 15-20 minute aspiration time with a flow rate of 1-1.5 L/min is recommended [Buechsenstein and Ough, 1978].
Despite these potential inaccuracies, the AO method gives reproducible results and has been noted as having an accuracy with just a 2.5-5% error [Buechsenstein and Ough, 1978].

14.3. Countering inaccuracies
To counteract the inaccuracies due to these testing techniques, winemakers sometimes dilute the sample with distilled water. This helps determine the end-point but lowers accuracy. Additionally, dilution may further affect the free-total SO2 equilibrium.
The use of hydrogen peroxide (H2O2) may also be employed to obtain better free SO2 evaluations. In this case, a wine sample is titrated in the usual way. Then a second sample is treated with excess H2O2 to remove the free SO2, and is subsequently titrated. The free SO2 in the wine is then assumed to be the value obtained in the first titration minus the value obtained in the second titration. The argument here is that the H2O2 removes all the SO2 present such that, when the sample is then titrated, the iodine is reacting with the phenols in the sample. Thus, the value obtained when solely accounting for phenol-binders is subtracted from that when they are included, leaving the true SO2 value. It is possible that the addition of the H2O2 in this method upsets the equilibrium between free and total SO2 leading to a false reading. Nevertheless, numerous winemakers have found this method compares favourably with values obtained from the AO method [e.g. Eisenmann, 2004].
The use of a high intensity light to illuminate the sample (especially with reds), and the use of calibrated control solutions may also help.

Use of a baseline value might also improve the accuracy of testing results. This is achieved by testing for free SO2 immediately after fermentation (before any SO2 addition has been made), when the free SO2 content in the wine is expected to be zero. The testing result obtained at this time is then used to correct all later test result values. Of course, this method will fail to give accurate results when the SO2 content in newly fermented wine is not zero.

14.4. Applying value adjustments
Value adjustments ("fudge factors") are usually applied to the readings to account for their inaccuracies. Such value adjustments are based on previous values attained from more reliable sources. For example, value adjustments made to values obtained from the Ripper method might be corrected based on values made by historical comparison using the alternative aeration oxidation (AO) method.

The true SO2 content obtained using the Ripper method is often considered to be an overestimate of the true value by around 10-20 mg/l (some quote 10 for whites and 20 for reds, whilst others do not adjust for whites at all). Breeden [2002a] noted that the Ripper method commonly measured 15-20 mg/l false high for red wines and 8 mg/l high for Chardonnay wines when compared with measured values using the AO method. Additionally, he claimed differences of up to 30 mg/l were possible [Breeden, 2002b]. He suggested a correction factor (subtraction from Ripper result value) of 20 mg/l be used on reds [Breeden, 2003]. Results obtained by Eisenman [2004] comparing Ripper to AO show a similar disparity. He used a correction factor of 15-20 mg/l for medium-heavy reds, respectively. The largest difference between AO and standard Ripper that he had encountered was 19 mg/l (with a dark, tannic Syrah). Breeden also compared results obtained from Titrets and regular Ripper. Testing showed that, on average, Titrets overestimated regular Ripper values by an average of 6 mg/l (differences ranged 3-8.5) for Chardonnay (and one Pinot Noir) wines [data analysed from Breeden, 1999]. Such differences may be deemed due to operator error only.

This data suggests that the difference between values obtained from AO and Ripper methods varies from zero to 20 or 30 mg/l, depending on the wine style. For less phenolic wines, the difference may be typically zero to 10 mg/l, whilst for more heavily extracted wines the difference may be 15-20 mg/l. These factors may be used when correcting values obtained with the Ripper method. Of course, such correction factors assume that the AO method gives completely accurate measurement of SO2 in wine, which is not precisely the case.

14.5. Minimising operator error
With any analytical test method there is the risk of operator error entering into the determination. Aside from the usual issues of correct reagant concentrations, it should be borne in mind that SO2 is a volatile substance. Samples of wine used for SO2 analysis should be tested as soon as possible after taking them from the bulk wine. In cases where this is not possible, air contact with the sample should be minimised. Samples should be taken from a homogeneous source. If the wine sample for testing is solely sourced from near the headspace of the storage vessel or, alternatively near/amongst the lees, the SO2 concentration is likely to be slightly lower than elsewhere. Samples should be made with this in mind, and should be taken to be representative of the vessel as a whole.

15. Removing Free SO2
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