OIV-MA-AS323-01A Determination of arsenic in wine by atomic absorption spectrometer

Type IV method

1. Principle

After evaporating ethyl alcohol and reducing the arsenic V in arsenic III, wine arsenic is measured by hydride generation and by atomic absorption spectrometry.

2. Equipment

2.1.   Glass ware:

2.1.1. Graduated flask 50, 100 ml (class A)

2.1.2. Graduated pipettes 1, 5, 10, 25 ml (class A)

2.2.   Water bath at 100°C

2.3.   Filters without ashes

2.4.   Spectrophotometer :

2.4.1. Atomic absorption spectrophotometer

2.4.2. Instrumental parameters

2.4.2.1.  Air-acetylene oxidising flame

2.4.2.2.  Hollow cathode lamp (arsenic)

2.4.2.3.  Wave length: 193.7 nm

2.4.2.4.  Split width: 1.0 nm

2.4.2.5.  Intensity of hollow cathode lamp: 7 mA

2.4.2.6.  Correction of non-specified absorption with a deuterium lamp

2.5.   Accessories:

2.5.1. Hydride absorption cell, placed on an air-acetylene burner.

2.5.2. Vapour generator (liquid gas separator)

2.5.3. Neutral gas (argon)

Figure 1. Hydride generator.

3. Reagents

3.1.   Ultra-pure demineralised water

3.2.   Ultra-pure 65% nitric acid

3.3.   Potassium iodide (KI)

3.4.   10% .Potassium iodide (m/v)

3.5.   Concentrated hydrochloric acid (R)

3.6.   10% Hydrochloric acid (R)

3.7.   Sodium borohydride (NaBH4)

3.8.   Sodium hydroxide (NaOH)

3.9.   0.6% Sodium borohydride (containing sodium hydroxide: 0.5% (m/v))

3.10.         Calcium Chloride CaCl2 (used as a drying agent)

3.11.         1 g/l Arsenic stock solution prepared in the following manner : dissolve 1.5339 g of A in demineralised water, adjust to 1 l.

3.12.         10 mg/l Arsenic solution: place 1 ml of stock solution (3.11.) in a 100 ml flask (2.1.1.) ;add 1 % nitric acid (3.2.) ; fill up to volume with demineralised water (3.1.).

3.13.         100 μg/l Arsenic solution: place 1 ml of 10 mg/l  arsenic solution (3.12.) in a 100 ml flask (2.1.1.) ; fill up to volume with demineralised water (3.1.).

3.14.         Set of callibration standards: 0, 5, 10, 25 μg/l

Successively place 0, 5, 10, 25 ml of 100 µg/l arsenic solution (3.13.) in 4 100 ml flasks (2.1.1.) ; add 10 ml of 10% potassium iodide to each flask (3.4.) and 10 ml of concentrated hydrochloric acid (3.5.) ; leave for 1 hour, fill up to 100 ml with demineralised water.

4. Sample preparation

25 ml of water is evaporated over a 100 °C water bath. This is then brought to 50 ml in the presence of 5 ml of 10% potassium iodide and 5 ml of concentrated hydrochloric acid; leave for 1 hour; filter on an ashless filter.

Make a blank reference sample.

5. Determination

The peristaltic pump sucks in the borohydride solution, the 10% hydrochloric acid solution and the sample solution.

Present the calibration standards in succession (3.14.); take an absorbency reading for 10 seconds; take two readings; the operating software establishes a calibration curve (absorbency according to concentration of arsenic in µg/l).

Then present the samples (4) ; the software establishes the sample’s arsenic concentration in µg/l; deduct the arsenic concentration in the wine in µg/l taking into account that the solution be diluted by 1 / 2 .

6. Quality control

Quality control is assured by placing a control sample of internal quality (*) in a regular manner in 5 samples, or after the set of calibration solutions, or in the middle of a series or at the end the measurement.

Two deviation types are accepted compared to known value.

(*) Samples from the Bureau Communautaire de Référence (Community Bureau of reference): red wine, dry white wine and sweet white wine.

7. Bibliography

• Varian Techtron, 1972. Analytical methods for flame spectroscopy.
• Hobbins B., 1982. Arsenic Determination by Hydride Generation. Varian Instruments at Work.
• Le Houillier R., 1986. Use of Drierite Trap to Extend the Lifetime of Vapor Generation Absorption Cell. Varian Instruments at Work.
• Varian, 1994. Vapor Generation Accessory VGA-77.

OIV-MA-AS323-01B Arsenic

Type IV method

1. Principle

After mineralization, using sulfuric and nitric acids, arsenic V is reduced to arsenic III by means of potassium iodide in hydrochloric acid and the arsenic is transformed into arsenic III hydride (H3As) using sodium borohydride.  The arsenic III hydride formed is carried by nitrogen gas and determined by flameless atomic absorption spectrophotometry at high temperature.

2. Method

2.1.   Apparatus

2.1.1. Kjeldahl flask (borosilicate glass)

2.1.2. Atomic absorption spectrophotometer equipped with arsenic hollow cathode lamp, hydride generator, background corrector and a chart recorder.

The hydride generator includes a reaction flask (which can eventually be put onto a magnetic stirrer) connected by a tube to a nitrogen gas supply (flow rate: 11 L/min) and by a second tube, to a quartz cell which can be brought to a temperature of 900 oC. The reaction flask also has an opening for the introduction of the reagent (borohydride).

2.2.   Reagents

All reagents must be of recognized analytically pure quality, and in particular free of arsenic.  Double distilled water prepared using a borosilicate glass flask or water of similar purity should be used.

2.2.1. Sulfuric acid (₂₀= 1.84 g/mL) arsenic free

2.2.2. Nitric acid (₂₀= 1.38 g/mL) arsenic free

2.2.3. Hydrochloric acid (₂₀= 1.19 g/mL), arsenic free

2.2.4. 10% (m/v) Potassium iodide solution

2.2.5. 2.5% (m/v) Sodium borohydride solution obtained by dissolving 2.5 g of sodium borohydride in 100 mL of 4 % (m/v) of sodium hydroxide solution.  This solution must be prepared at the time of use.

2.2.6. Arsenic reference solution 1 g/L.  Use of a commercial standard arsenic solution is preferred.

Alternatively this solution can be prepared in a 1000 mL volumetric flask, by dissolving 1.320 g of arsenic III trioxide in a minimal volume of 20 % (m/v) sodium hydroxide. The solution is then acidified with hydrochloric acid, diluted 1/2, and made up to 1 liter with water.

2.3.   Procedure

2.3.1.  Mineralization

Place 20 mL of wine in a Kjeldahl flask, boil and reduce the volume by half to eliminate alcohol.  Allow to cool.  Add 5 mL sulfuric acid, and slowly add 5 mL nitric acid and heat. As soon as the liquid turns brown, add just enough nitric acid, dropwise, to lighten the liquid while simmering.  Continue until the color clears and white sulfur trioxide fumes are formed above the solution.

Allow to cool, add 10 mL distilled water, bring back to the boil and simmer until nitrous oxide and sulfur trioxide fumes are no longer produced. Allow to cool and repeat the operation.

Allow to cool and dilute the sulfuric acid residue with a few milliliters of distilled water.Quantitatively transfer the solution into a 40 mL flask, and rinse the flask with water, combine with the diluted residue and make up to the mark with distilled water.

2.3.2. Determination

2.3.2.1. Preparation of the solution

Place 10 mL of the mineralization solution (2.3.1) into the hydride generator reactor flask. Add 10 mL hydrochloric acid, 1.5 mL potassium iodide solution, then switch on the magnetic stirrer and the nitrogen gas (flow rate: 11 L/minute). After 10 sec, add 5 mL of sodium borohydride solution.  The hydride vapor obtained is immediately carried to the measurement cell (at a temperature of 900C) by nitrogen carrier gas, where dissociation of the compound and arsenic atomization occurs.

2.3.2.2. Preparation of standard solutions

From the arsenic reference solution (2.2.6), prepare dilutions having concentrations of 1, 2, 3, 4 and 5 micrograms of arsenic per liter respectively.  Place 10 mL of each of the prepared solutions into the reactor flask of the hydride generator and analyze according to 2.3.2.1.

2.3.2.3. Measurements

Select an absorption wavelength of 193.7 nm. Zero the spectrophotometer using double distilled water and carry out all determinations in duplicate.  Record the absorbance of each sample and standard solution.  Calculate the average absorbance for each of these solutions.

2.4.   Expression of results

2.4.1. Calculation

Plot the curve showing the variation in absorbance as a function of the arsenic concentration in the standard solutions.  The relationship is linear. Note the average absorbance of the sample solutions on the graph and read the arsenic concentration C.

The arsenic concentration in wine, expressed in micrograms per liter is given by: 2 C.

Bibliography

• Jaulmes P. et Hamelle G., Trav. Soc. Pharm. Montpellier, 1967, 27, no 3, 213‑225.
• Jaulmes P., F.V., O.I.V., 1967, no 238
• MEDINA B et SUDRAUD P., F.V., O.I.V., 1983, no 770.

 

OIV-MA-AS323-02A Quantification of total nitrogen according to the Dumas method (Musts and wines)

Type II method

1. Field of application

This method can be applied to the analysis of total nitrogen in musts and wine within the range of 0 to 1000 mg/l.

2. Description of the technique

2.1.  Principle of the Dumas method

The analysis of total nitrogen in an organic matrix can be carried out using the Dumas method (1831). This involves a total combustion of the matrix under oxygen. The gases produced are reduced by copper and then dried, while the CO₂ is trapped. The nitrogen is then quantified using a universal detector.

2.2.  Principle of the analysis (Figure n° 1)

• Injection of the sample and oxygen in the combustion tube at 940°C (1);
• « Flash » Combustion (2);
• The combustion of the gathering ring (3) brings the temperature temporarily up to 1800°C;
• Complementary oxidation and halogen trappings on silver cobalt and granular chromium sesquioxide (4);
• Reduction of nitrogen oxides in N₂ and trapping sulphur components and excess oxygen by copper at 700°C (5);
• Gases in helium include: N₂, CO₂ and H₂O (6);
• Trapping unmeasured elements: H₂O using anhydrone (granular anhydrous magnesium perchlorate) (7) and CO₂ by ascarite (sodium hydroxide on silica) (8);
• Chromatography separation of nitrogen and methane possibly present following very large trial uptake (9);
• Catharometer detection (10);
• Signal gathering and data processing (11).

Figure 1: Diagram of analysis principle

 

3. Reagents and  preparation of reactive solutions

3.1.  Nitrogen (technical quality);

3.2.  Helium (purity 99.99994%);

3.3.  Chromium oxide (chromium sesquioxide me in granules);

3.4.  Cobalt Oxide (silver granule cobalto-cobaltic oxide );

3.5.  Quartz wool;

3.6.  Copper (reduced copper in strings);

3.7.  Ascarite (sodium hydroxide on silica);

3.8.  Anhydrone (granular anhydrous magnesium perchlorate);

3.9.  Oxygen (purity 99.995%);

3.10.        Atropine ;

3.11.        Glumatic-hydric chloride acid;

3.12.        Demineralised water;

3.13.        Tin boat.

4. Apparatus

 

4.1.  Centrifuge with 25 ml pots;

4.2.  Nitrogen analyser;

4.3.  Metallic crucible;

4.4.  Quartz reaction tube (2) ;

4.5.  Precision balance between 0.5 mg and 30 g at  0.3 mg ;

4.6.  Boat carrier;

4.7.  Furnace;

4.8.  Apparatus for folding boats;

4.9.  Sample changer;

4.10.        Computer and printer.

5. Sampling

 

Degas by nitrogen bubbling (3.1) for 5 to 10 mn, sparkling wine. The musts are centrifuged (4.1) for 10 mn at 10°C, at 4200 g.

6. Operating instructions

 

• Open the apparatus programme (4.2 and 4.10) ;
• Put the heating on the apparatus (4.2).
  1.   Principle analytical parameters

• Nitrogen analyser (4.2) under the following conditions:
• gas carrier: helium (3.2);
• metallic crucible (4.3) to be emptied every 80 analyses;
• oxidation tube (4.4), heated to 940° C, containing chromium oxide (3.3) and cobalt oxide(3.4) held back by quartz wool (3.5). The tube and reagent set
• must be changed every 4000 analyses;
• reduction tube (4.4), heated to 700° C, containing copper (3.6) held back by the  quartz wool (3.5). The copper is changed every 450 analyses;
• absorption tube, containing  2/3 of ascarite (3.7) and 1/3 anhydrone (3.8). the ascarite which is taken in block is eliminated and replaced every 200 analyses. The absorbers are completely changed once a year.

The more organic matter to be burned, the more oxygen is needed: the oxygen sampling valve (3.9) is 15 seconds for musts and 5 seconds for wine.

NOTE : The metals are recuperated and sent to a centre for destruction or specialised recycling.

6.2.  Preparation of standard scale

Prepare two samples of atropine (3.10) between 4 to 6 mg. Weigh them (4.5) directly with the boat. The calibration scale goes through 3 points (origin = empty boat).

 

6.3.  Preparation of internal standards

Internal standards are used regularly in the beginning and in the middle of analyses.

Internal checks are carried out using glumatic acid in the form of hydrochloride at 600 mg N/l in demineralised water (3.12).

Molar mass of glumatic acid = 183.59

Molar mass of nitrogen = 14.007

Weigh (4.5) 7.864 g of glumatic acid (3.11) and dilute in demineralised water (3.12) qsp/l, to obtain a 600 mg N/l solution. This solution is diluted by 50% to obtain a 300 mg N/l solution, which is diluted by 50% again to obtain 150 mg/l solution.

6.4.  Preparation of samples:

6.4.1.      In a boat (3.13), weigh (to the nearest 0.01 mg) 20 µl of must or 200 µl of wine with a precision balance (4.5). Repeat this procedure three times per sample;

6.4.2.      Write down the mass

6.4.3.      Place the boats progressively in the boat carrier (4.6) ;

6.4.4.      Place the boats in the furnace (4.7) set at ~ 60° C, until the liquid has completely evaporated (this requires at least one hour) ;

6.4.5.      Fold and crush the boats with an appropriate apparatus (4.8), put them in the changer (4.9) in number order.

7. Expression of results

Results are expressed in  g/l to the fourth decimal.

8. Checking results

Splicing by mass, temperature, and volume.

9. Performance characteristics of the method

Number of laboratories	Average contents	Repeatability	Reproductibility
11	591 mg/l	43 mg/l	43 mg/l

10. Bibliography

 

• Dumas A. (1826) : Annales de chimie, 33,342.
• Buckee G.K. (1994) : Determination of total nitrogen in Barley, Malt and Beer by Kjeldahl procedures and the Dumas combustion method. Collaborative trial. J. Inst. Brew., 100, 57-64.

OIV-MA-AS323-03 Boron (Rapid Colorimetric Method)

Type IV method

 

1. Principle

The alcohol content of the wine is removed by reducing the volume by half by rotary evaporation. The wine is then passed through a column of polyvinylpolypyrrolidone, which retains the coloring agents.  The eluate is collected quantitatively and the boron concentration determined by complexation with azomethine H at pH 5.2 followed by spectroscopic analysis at 420 nm.

2. Apparatus

 

2.1.  Rotary evaporator

2.2.  Spectrophotometer capable of measuring absorbance wavelengths between 300 and 700 nm

2.3.  Cells of 1 cm optical path

Glass column of 1 cm internal diameter and 15 cm in length containing an 8 cm layer of polyvinylpolypyrrolidone.

3. Reagents

 

3.1.  Azomethin H (4‑hydroxy‑5‑(2-hydroxybenzylideneamino)- 2,7‑napthalenedisulfonic acid)

3.2.  Azomethin H solution

Place 1 g of azomethin H and 2 g of ascorbic acid in a 100 mL volumetric flask and add 50 mL double distilled water.  Warm slightly to dissolve and make up to the mark with double distilled water. The reagent is stable for 2 days if kept cold.

3.3.  Buffer solution pH 5.2

Dissolve 3g of EDTA (disodium salt of ethylenediaminetetraacetic acid) in 150 mL of double distilled water. Add 125 mL acetic acid (ρ₂₀ = 1.05 g/mL) and 250 g of ammonium acetate, , and dissolve.  Check the pH with a pH meter and adjust if necessary to pH 5.2.

3.4.  Boron stock standard solution, 100 mg/L

Use of a commercial standard solution is preferable. Alternatively this solution can be prepared by dissolving 0.571 g of boric acid,, dried beforehand at 50 oC until constant weight, in 500 mL double distilled water and made up to 1 liter.

3.5.  Boron standard solution, 1 mg/L

Dilute the stock solution, 100 mg/L (3.4) 1/100 with double distilled water.

Polyvinylpolypyrrolidone or PVPP (see International Enological Codex)

4. Procedure

 

Eliminate alcohol from 50 mL of wine by concentration to half the original volume in a rotary evaporator at 40oC and make up to 50 mL with double distilled water.

Take 5 mL of this solution and pass it through the PVPP column (2.4).  The coloring agents are completely retained.  Collect the eluate and the rinsing waters from the column and place in a 50 mL volumetric flask and make up to the mark with water.

The colorimetric determination is performed in a volume of 5 mL of eluent placed in a 25 mL volumetric flask; dilute to approximately 15 mL with double distilled water and add the following (stirring after each addition):

• 5 mL of azomethin H solution (3.2)
• 4 mL of pH 5.2 buffer solution (3.3)

Make up to 25 mL with double distilled water.

Wait 30 min and determine the absorbance As, at 420 nm.  The zero of the absorbance scale is set using distilled water.

Use a blank consisting of 5 mL of azomethin H solution and 4 mL of pH 5.2 buffer solution in 25 mL of double distilled water.  Wait 30 min and read the absorbance Ab under the same conditions.  The absorbance must be between 0.20 and 0.24; a higher absorbance demonstrates boron contamination in the water or the reagents.

Preparation of the calibration curve

In 25 mL volumetric flasks, place 1 to 10 g of boron, corresponding to 1 to 10 mL of boron standard solution 1 mg/L (3.5) and continue as indicated in 4.0. The calibration graph representing the net absorbance (- ) in relation to the concentration is a straight line passing through the origin.

Where:

• = absorbance of sample

  = absorbance of blank

5. Calculations

The μg of boron contained in 5 mL of eluate, (corresponding to 0.5 mL of wine) obtained from interpolating the net absorbance values of () on the calibration graph is E. The content, B, in milligrams of boron per liter is given by:

Bibliography

• WOLF B., Soil Science and Plant Analysis, 1971, 2(5), 363‑374 et 1974, 5(1), 39‑44.
• CHARLOT C. and BRUN S., F.V., O.I.V., 1983, no771.

OIV-MA-AS323-04A1 Free sulphur dioxide

Type IV method

 

1. Scope

This method is for the determination of free sulphur dioxide in wine and must.

2. Definitions

Free sulphur dioxide is defined as the sulphur dioxide present in the must or wine in the following forms: and , whose equilibrium is dependent on pH and temperature:

represents the molecular sulphur dioxide.

3. Principle

Sulphur dioxide is entrained by a current of air or nitrogen, and is fixed and oxidised by bubbling through a dilute and neutral solution of hydrogen peroxide. The sulphuric acid formed is determined by titration with a standard solution of sodium hydroxide.

The quantity of sulphur dioxide entrained being strongly temperature dependent, the decision was made to work at room temperature (between 18 and 24°C). This temperature, as for that of the currents of air or nitrogen, should be kept constant throughout the determination.

4. Reagents and products
   1.    Pure phosphoric acid at 85% (ρ₂₀ = 1.71 g/mL) (CAS no. 7664-38-2)
   2.    Diluted phosphoric acid (25.5%):

By way of example: Dilute 300 mL of phosphoric acid at 85% (4.1) in 1 L of water for analytical use

4.3.   Indicator reagent:

Methyl red (CAS no. 493-52-7): 100 mg (1 mg)

Methylene blue (CAS no. 7220-79-3) 50 mg (0.5 mg)

Ethanol ( 95%) (CAS no. 64-17-5) 50 mL

Make up to 100 mL with water for analytical use. Respect the proportions for the volumes that differ from 100 mL.

Commercial indicator reagents with the same composition may be used.

4.4.   1 M Sodium hydroxide (3.84%) or in anhydrous form (pellets) (CAS no. 1310-73-2)

4.5.   0.01 M Sodium hydroxide solution:

By way of example: Dilute 10.0 mL of 1 M sodium hydroxide (4.4) in 1 L of water for analytical use.

If necessary, check the titre of the solution regularly (correction factor to be applied) and keep it away from atmospheric CO₂.

4.6.   Hydrogen peroxide solution in 3 volumes (= 9.1 g/L = 0.27 mol/L :), prepared or commercial (e.g. 30% : mixture with CAS no. 7722-84-1)

Note: A solution of 30% by mass corresponds to a titre of 110 volumes (ρ₂₀ 1,11 g/mL), implying the volume of oxygen ideally released per litre of under standard conditions of temperature and pressure, while a solution of 3% by mass (ρ₂₀ 1 g/mL) corresponds to a titre of 10 volumes (0.89 mol/L). The preparation thus depends on the commercial solution used, considering that in any case the volume used in the method will be in excess.

5. Apparatus

 

The apparatus to be used should conform to the diagram below, especially with regard to the condenser.

The gas supply tube to bubbler B ends in a small sphere of 1 cm in diameter with 20 holes of 0.2 mm in diameter around its largest horizontal circumference. Alternatively, this tube may end in a sintered glass plate that produces a large number of very small bubbles and thus ensures good contact between the liquid and gaseous phases.

The gas flow through the apparatus should be approximately 40 L/h. The bottle situated on the right of the apparatus is intended to restrict the pressure reduction produced by the water pump to 20-30 cm water. In order to regulate the pressure reduction to achieve the proper flow rate, it is preferable to install a flow meter with a semi-capillary tube between the bubbler and the bottle.

Flask A should be kept at a temperature of between 18°C and 24°C throughout aspiration. Each flask should consequently be temperature-controlled (e.g. using a thermostatic bath) if the room temperature of the laboratory is not within these limits or if 85% phosphoric acid is used, which can significantly increase the temperature in the flask during addition.

Figure 1- The dimensions are indicated in milimetres. The internal diameters of the 4 concentric tubes that make up the condenser are 45,34, 27 and 10 mm

6. Procedure

Air- or nitrogen-rinsing the apparatus before each new determination (e.g. for 5 minutes) is recommended. If a blank test is carried out, the colour of the indicator in the neutralised hydrogen peroxide solution at the exit of the gas-supply tube should not change.

Connect the water from the condenser.

Control the laboratory temperature or stabilise the bath in advance (at between 18 °C and 24 °C).

In bubbler B of the entrainment apparatus, introduce 2-3 mL hydrogen peroxide solution (4.6) and 2 drops of indicator reagent (4.3), and neutralise with the 0.01 M sodium hydroxide solution (4.5); a neutral pH = green colour.

Note: For large sample series, it is also possible to prepare an already neutralised solution before introducing it into the flask. Adapt the concentrations and volumes accordingly, bearing in mind that the oxidative power of the solution must be maintained (reduced shelf life).

Adapt this bubbler to the apparatus.

Transfer 50 mL of sample to the 250-mL flask A and attach it to the apparatus.

Introduce 15 mL of diluted phosphoric acid (4.2) into bulb C.

Note: If the expected concentration of free sulphur dioxide is higher than 50 mg/L, it is necessary to use phosphoric acid at 85% (4.1). However, ensure that the temperature in flask A does not increase during addition.

Open the tap to add the acid to the sample while simultaneously starting the gas flow and setting the timer to 15 minutes. The entrained free sulphur dioxide is oxidised into sulphuric acid.

After 15 minutes, take bubbler B out and rinse the gas supply tube in water (via the socket).

Titrate the acid formed by the 0.01 M sodium hydroxide solution (4.5) up to the green bend.

The number of millilitres used is expressed by n.

7. Calculation and expression of results

The free sulphur dioxide is expressed in milligrams per litre (mg/L), in whole numbers.

Calculation: Free sulphur dioxide in milligrams per litre: 6.4 n

8. Bibliography

Paul, F., Mitt. Klosterneuburg, Rebe u. Wein, 1958, ser. A, 821

Collaborative study

Method validation for the determination of free sulphur dioxide

1. Scope of application

An international collaborative study, in accordance with Resolution OIV-OENO 6-2000, for the validation of updates to the methods for the determination of free sulphur dioxide and total sulphur dioxide (OIV-MA-AS323-04A), based on the decision of the OIV “Methods of Analysis” Sub-Commission, April 2018.

2. Standard references

• Update (draft) to the OIV-MA-AS323-04A methods,
• ISO 5725,
• Resolution OIV-OENO 6-2000.

3. Protocol

A total of 20 samples were prepared using homogeneous volumes of 10 wines from various wine regions in France and Portugal. Each sample was made up twice (the second as a blind duplicate), according to the double-blind principle.

The samples were prepared between 18 and 20 June 2018, then shipped without delay to the participating laboratories.

Sample no.	Blind duplicate no.	Nature of sample
A	1-14	Dry white wine
B	2-16	Dry white wine
C	3-19	Dry rosé wine
D	4-12	Dry rosé wine
E	5-20	Dry red wine
F	6-18	Dry red wine
G	7-11	Dry red wine
H	8-15	White liqueur wine
I	9-17	Red liqueur wine
J	10-13	Red liqueur wine

The analyses were carried out simultaneously by all participating laboratories between 16 and 20 July 2018. Samples were kept in refrigerated cabinets by all laboratories between the date of reception and the date of analysis, according to the protocols sent.

The following laboratories provided their results:

Laboratory	City	Country
Estación de Viticultura e Enoloxía de Galicia	Leiro (Ourense)	Spain
Laboratorio arbitral agroalimentario	Madrid	Spain
ASAE	Lisbon	Portugal
SCL Montpellier	Montpellier Cdex 5	France
HBLA und BA für Wein- und Obstbau	Klosterneuburg	Austria
Laboratorio de Salud Pública	Madrid	Spain
Laboratorio Agroambiental de Zaragoza	Zaragoza	Spain
Laboratoire SCL Bordeaux	Pessac Cedex - CS 98080	France
Unione Italiana Vini Servizi	Verona	Italy
Laboratorio Agroalimentario de Valencia	Burjassot (Valencia)	Spain
Agroscope	Nyon	Switzerland
Laboratoires Dubernet	Montredon des Corbières	France
Laboratoire Dioenos Rhône	Orange	France
Laboratoire Natoli	Saint Clément de Rivière	France

NB: The order of laboratories in the table does not correspond with the order in the following tables, in order to preserve the anonymity of results.

4. Free sulphur dioxide

4.1.   Free data

Free SO2 (mg/L)	A		B		C		D		E		F		G		H		I		J
Sample	1	14	2	16	3	19	4	12	5	20	6	18	7	11	8	15	9	17	10	13
Labo 3			31	36	18	18	21	23	20	18	6	6	20	17	5	6
Labo 5			37	35	21	24	24	25	20	20	8	7	20	20	3	4
Labo 6	4	1	38	33	21	20	20	26	19	20	7	6	21	19	7	8	1	3	1	1
Labo 7	1	1	37	40	20	22	24	26	20	22	9	8	20	23	8	8	2	1	1	1
Labo 8			31	32	18	19	23	22	22	20	6	7	19	20	5	3	1	1
Labo 9			35	34	23	19	25	24	21	24			17	17
Labo 10	2	1	35	34	20	21	24	24	22	21	9	8	21	20	7	7	2	2	1	1
Labo 11	0	0	33	30	17	11	22	16	16	21	6	4	15	19	6	3	1	1	0	0
Labo 15			15	19	15	13	18	20	8	16	6	5	8	15	5	5
Labo 17	0	0	37	38	24	26	28	28	26	23	8	8	24	22	7	7	1	2	0	0
Labo 18	0	4	33	31	21	11	23	27	15	19	6	4	9	20	3	4	1	1	0	0
Labo 20	0	0	32	32	20	19	21	21	29	21	8	8	20	18	12	4	1	1	0	0
Labo 21	2	1	33	38	19	15	25	22	19	21	6	6	19	20	8	7	2	1	0	0

Results left blank were rendered non-quantifiable (< limit of quantification).

	Result removed by the COCHRAN test at 5%
	Result removed by the GRUBBS test at 5%

4.2.   Free SO₂ results

Free SO2 (mg/L)	A	B	C	D	E	F	G	H	I	J
No. of laboratories selected	7	9	11	10	10	12	11	11	9	8
No. of repetitions	2	2	2	2	2	2	2	2	2	2
Min.	0	31.5	14	19	17	5	17	3.5	1	0
Max.	2.5	38.5	25	28	24.5	8.5	23	8	2	1
Mean	0.9	34.2	19.8	23.4	20.6	6.8	19.6	5.7	1.4	0.4
Standard deviation	0.98	2.67	2.91	2.46	2.04	1.31	1.77	1.72	0.42	0.52
Repeatability variance	0.79	1.67	2.59	1.20	2.60	0.58	2.23	0.82	0.39	0.00
Inter-laboratory standard deviation	0.98	2.67	2.91	2.46	2.04	1.31	1.77	1.72	0.42	0.52
Reproducibility variance	1.35	7.97	9.76	6.64	5.46	2.00	4.25	3.38	0.37	0.27
Repeatability standard deviation	0.89	1.29	1.61	1.10	1.61	0.76	1.49	0.90	0.62	0.00
r limit	2.48	3.61	4.51	3.07	4.51	2.14	4.18	2.53	1.75	0.00
Repeatability %CV (k=2)	191	8	16	9	16	23	15	32	90	0
Reproducibility standard deviation	1.16	2.82	3.12	2.58	2.34	1.41	2.06	1.84	0.61	0.52
R limit	3.25	7.90	8.75	7.22	6.54	3.96	5.78	5.15	1.70	1.45
Reproducibility %CV (k=2)	250	16	32	22	23	42	21	64	87	276
Horwitz PRSDR (%)	16.18	9.40	10.21	9.95	10.15	12.00	10.22	12.30	15.23	18.55
Horwitz sR	0.15	3.22	2.02	2.33	2.09	0.81	2.00	0.70	0.21	0.07
Horwitz R	0.42	9.10	5.71	6.59	5.91	2.29	5.67	1.99	0.60	0.20
Horwitz Ratio	7.64	0.87	1.53	1.10	1.11	1.73	1.02	2.58	2.84	7.37

Figure 1: Modelling of the repeatability coefficient of variation, %CV(r) (k=2), as a function of the concentration, C:

Figure 2: Modelling of the inter-laboratory reproducibility coefficient of variation, %CV(R) (k=2), as a function of concentration, C:

5. Total sulphur dioxide
   1.    Total SO₂ data

Total SO2 (mg/L)	A		B		C		D		E		F		G		H		I		J
Sample	1	14	2	16	3	19	4	12	5	20	6	18	7	11	8	15	9	17	10	13
Labo 3			128	127	72	73	128	131	61	59	28	28	57	56	102	102	47	45
Labo 5			122	121	68	71	112	114	42	53	22	22	51	42	102	101	35	34
Labo 6		1	128	131	72	72	126	131	53	54	22	20	42	49	98	99	31	34	3	1
Labo 7	3	3	131	131	70	74	130	131	54	59	26	23	46	48	106	101	37	40	1	1
Labo 8	2	1	125	127	72	72	129	128	58	57	22	23	46	45	97	99	42	39	1	1
Labo 9			120	128	77	75	132	108	71	59	21	25	44	47	110	99	38	48
Labo 10	2	2	130	130	74	76	130	130	61	61	28	32	55	56	103	104	43	44	3	4
Labo 11	4	3	119	125	71	74	118	118	39	40	18	21	45	41	89	94	26	38	2	2
Labo 14	3	3	129	128	72	72	127	129	58	58	32	29	50	49	102	101	42	41	3	4
Labo 15			134	136	76	78	134	136	60	58	39	27	52	61	110	106	51	50
Labo 17	3	3	134	132	82	76	136	133	59	50	24	23	46	44	107	105	35	38	0	0
Labo 18	5	3	130	129	78	73	133	133	62	59	29	32	58	52	105	105	50	48	2	2
Labo 20	1	1	128	131	72	74	130	130	58	56	26	28	48	45	98	93	41	43	0	0
Labo 21		0	124	125	69	72	124	126	45	51	19	20	42	42	97	97	35	34	0	1

Results left blank were rendered non-quantifiable (< limit of quantification).

	Result removed by the COCHRAN test at 5%
	Result removed by the GRUBBS test at 5%

5.2.   Total SO₂ results

Total SO2 (mg/L)	A	B	C	D	E	F	G	H	I	J
No. of laboratories selected	7	12	13	13	8	13	10	13	12	9
No. of repetitions	2	2	2	2	2	2	2	2	2	2
Min.	1	121.5	69.5	113	53.5	19.5	42	91.5	32.5	0
Max.	3.5	135	77	135	61	30.5	56.5	108	50.5	3.5
Mean	2.4	128.8	73.0	128.0	58.3	24.7	47.6	100.9	40.8	1.5
Standard deviation	0.93	3.63	2.20	6.24	2.42	4.04	4.89	4.61	5.80	1.35
Repeatability variance	0.14	1.46	3.27	2.35	1.44	3.04	2.30	3.96	2.21	0.17
Inter-laboratory standard deviation	0.93	3.63	2.20	6.24	2.42	4.04	4.89	4.61	5.80	1.35
Reproducibility variance	0.94	13.93	6.49	40.11	6.57	17.84	25.03	23.28	34.72	1.90
Repeatability standard deviation	0.38	1.21	1.81	1.53	1.20	1.74	1.52	1.99	1.49	0.41
r limit	1.1	3.4	5.1	4.3	3.4	4.9	4.2	5.6	4.2	1.1
Repeatability %CV (k=2)	31	2	5	2	4	14	6	4	7	54
Reproducibility standard deviation	0.97	3.73	2.55	6.33	2.56	4.22	5.00	4.82	5.89	1.38
R limit	2.7	10.5	7.1	17.7	7.2	11.8	14.0	13.5	16.5	3.9
Reproducibility %CV (k=2)	80	6	7	10	9	34	21	10	29	184
Horwitz PRSDR (%)	14.00	7.70	8.39	7.71	8.68	9.87	8.95	7.99	9.16	15.05
Horwitz sR	0.34	9.92	6.13	9.86	5.06	2.44	4.26	8.06	3.73	0.23
Horwitz R	0.96	28.05	17.33	27.90	14.31	6.91	12.04	22.80	10.56	0.64
Horwitz Ratio	2.82	0.37	0.41	0.64	0.50	1.71	1.16	0.59	1.56	6.04

Figure 3: Modelling of the repeatability coefficient of variation, %CV(r) (k=2), as a function of concentration, C:

Figure 4: Modelling of the inter-laboratory reproducibility coefficient of variation, %CVR (k=2), as a function of concentration, C:

OIV-MA-AS323-04A2 Total sulphur dioxide

Type II method

 

1. Scope

This method is for the determination of total sulphur dioxide in wine and must.

2. Definitions

Total sulphur dioxide is defined as the sum of all of the different forms of sulphur dioxide present in the wine in free form or bound to the wine’s constituents.

3. Principle

Sulphur dioxide is aspirated by a current of air or nitrogen, and is captured and oxidised by bubbling through a dilute and neutral solution of hydrogen peroxide. The sulphuric acid formed is determined by titration with a standard solution of sodium hydroxide.

The total sulphur dioxide is extracted from the wine by aspiration at high temperature (around 100 °C).

4. Reagents and products
   1.   Pure phosphoric acid at 85% (ρ₂₀ = 1.71 g/mL) (CAS no. 7664-38-2)
   2.   Indicator reagent:

Methyl red (CAS no. 493-52-7) 100 mg (1 mg)

Methylene blue (CAS no. 7220-79-3): 50 mg (0.5 mg)

Ethanol (≥ 95%) (CAS no. 64-17-5): 50 mL

Make up to 100 mL with water for analytical use. Respect the proportions for the volumes that differ from 100 mL.

Commercial indicator reagents with the same composition may be used.

4.3.  1 M Sodium hydroxide (3.84%) or in anhydrous form (pellets) (CAS no. 1310-73-2)

4.4.  0.01 M Sodium hydroxide solution:

By way of example: Dilute 10.0 mL of 1 M sodium hydroxide (4.4) in 1 L of water for analytical use.

If necessary, check the titre of the solution regularly (correction factor to be applied) and keep it away from atmospheric CO₂.

4.5.  Hydrogen peroxide solution in 3 volumes (= 9.1 g/L = 0.27 mol/L H₂O₂), prepared or commercial (e.g. 30% : mixture with CAS no. 7722-84-1)

Note: A solution of 30% by mass corresponds to a titre of 110 volumes (ρ₂₀ 1,11 g/mL), implying the volume of oxygen ideally released per litre of under standard conditions of temperature and pressure, while a solution of 3% by mass (ρ₂₀ 1 g/mL) corresponds to a titre of 10 volumes (0.89 mol/L). The preparation thus depends on the commercial solution used, considering that in any case the volume used in the method will be in excess.

5. Apparatus

The apparatus to be used should conform to the diagram below, especially with regard to the condenser.

The gas supply tube to bubbler B ends in a small sphere of 1 cm in diameter with 20 holes of 0.2 mm in diameter around its largest horizontal circumference. Alternatively, this tube may end in a sintered glass plate that produces a large number of very small bubbles and thus ensures good contact between the liquid and gaseous phases.

The gas flow through the apparatus should be approximately 40 L/h. The bottle situated on the right of the apparatus is intended to restrict the pressure reduction produced by the water pump to 20-30 cm water. In order to regulate the pressure reduction to achieve the proper flow rate, it is preferable to install a flow meter with a semi-capillary tube between the bubbler and the bottle. For the determination of total sulphur dioxide, using a burner (with a 4-5 cm high flame or infrared) allowing for boiling point to be reached very quickly is preferable. Do not place a wire gauze under flask A, but rather a deflector with a 2-4 cm orifice. The pyrogenation of non-volatile matter in the wine on the flask walls is thus avoided.

Use a 250-mL flask for a 50 mL sample and a 100-150 mL flask for a 20 mL sample.

Figure 1 : The dimensions are indicated in millimetres. The internal diameters of the 4 concentric tubes that make up the condenser are 45, 34, 27 and 10 mm

6. Procedure

Air- or nitrogen-rinsing the apparatus before each new determination (e.g. for 5 minutes) is recommended. If a blank test is carried out, the colour of the indicator in the neutralised hydrogen peroxide solution at the exit of the gas-supply tube should not change.

Connect the water from the condenser.

In bubbler B of the entrainment apparatus, introduce 2-3 mL hydrogen peroxide solution (4.5) and 2 drops of indicator reagent (4.2), and neutralise with the 0.01 M sodium hydroxide solution (4.4); a neutral pH = green colour.

Note: For large sample series, it is also possible to prepare an already neutralised solution before introducing it into the flask. Adapt the concentrations and volumes accordingly, bearing in mind that the oxidative power of the solution must be maintained (reduced shelf life).

Adapt this bubbler to the apparatus.

Transfer 50 mL of sample to flask A if the presumed total SO₂ content in the sample is <50 mg/L, and 20 mL of sample if the presumed total SO₂ content is ≥ 50 mg/L and attach it to the apparatus.

Introduce 15 mL of phosphoric acid (4.1) into bulb C if the presumed total SO₂ content of the sample is <50 mg/L and 5 mL phosphoric acid (4.1) if the presumed total SO₂ content of the sample is  50 mg/L.

Open the tap to add the acid to the sample and activate the heat source, while simultaneously starting the gas flow and setting the timer to 15 minutes. Maintain at boiling point for the duration of the gas flow. The entrained total sulphur dioxide is oxidised into sulphuric acid.

After 15 minutes, turn off the heat source, take bubbler B out, and rinse the gas supply tube (via the socket) with water.

Titrate the acid formed by the 0.01 M sodium hydroxide solution (4.4) up to the green bend.

The number of millilitres used is expressed by n.

7. Calculations and expression of results

The total sulphur dioxide is expressed in milligrams per litre (mg/L), in whole numbers.

Calculations:

Samples low in sulphur dioxide (50 mL sampling):  6.4 n

Other samples (20 mL sampling): 16 n

 

8. Precision

8.1.  Repeatability (r)

Content < 50 mg/L (50 mL sampling), r = 1 mg/L

Content  50 mg/L (20 mL sampling), r = 6 mg/L

8.2.  Reproducibility (R)

Content < 50 mg/L (50 mL sampling), R = 9 mg/L

Content  50 mg/L (20 mL sampling), R = 15 mg/L

 

9. Bibliography

• Paul, F., Mitt. Klosterneuburg, Rebe u. Wein, 1958, ser. A, 821.

Collaborative study

1. Scope of application

An international collaborative study, in accordance with Resolution OIV-OENO 6-2000, for the validation of updates to the methods for the determination of free sulphur dioxide and total sulphur dioxide (OIV-MA-AS323-04A), based on the decision of the OIV “Methods of Analysis” Sub-Commission, April 2018.

2. Standard references

• Update (draft) to the OIV-MA-AS323-04A methods,
• ISO 5725,
• Resolution OIV-OENO 6-2000.

3. Protocol

A total of 20 samples were prepared using homogeneous volumes of 10 wines from various wine regions in France and Portugal. Each sample was made up twice (the second as a blind duplicate), according to the double-blind principle.

The samples were prepared between 18 and 20 June 2018, then shipped without delay to the participating laboratories.

Sample no.	Blind duplicate no.	Nature of sample
A	1-14	Dry white wine
B	2-16	Dry white wine
C	3-19	Dry rosé wine
D	4-12	Dry rosé wine
E	5-20	Dry red wine
F	6-18	Dry red wine
G	7-11	Dry red wine
H	8-15	White liqueur wine
I	9-17	Red liqueur wine
J	10-13	Red liqueur wine

The analyses were carried out simultaneously by all participating laboratories between 16 and 20 July 2018. Samples were kept in refrigerated cabinets by all laboratories between the date of reception and the date of analysis, according to the protocols sent.

The following laboratories provided their results:

Laboratory	City	Country
Estación de Viticultura e Enoloxía de Galicia	Leiro (Ourense)	Spain
Laboratorio arbitral agroalimentario	Madrid	Spain
ASAE	Lisbon	Portugal
SCL Montpellier	Montpellier Cdex 5	France
HBLA und BA für Wein- und Obstbau	Klosterneuburg	Austria
Laboratorio de Salud Pública	Madrid	Spain
Laboratorio Agroambiental de Zaragoza	Zaragoza	Spain
Laboratoire SCL Bordeaux	Pessac Cedex - CS 98080	France
Unione Italiana Vini Servizi	Verona	Italy
Laboratorio Agroalimentario de Valencia	Burjassot (Valencia)	Spain
Agroscope	Nyon	Switzerland
Laboratoires Dubernet	Montredon des Corbières	France
Laboratoire Dioenos Rhône	Orange	France
Laboratoire Natoli	Saint Clément de Rivière	France

NB: The order of laboratories in the table does not correspond with the order in the following tables, in order to preserve the anonymity of results.

4. Free sulphur dioxide

 

4.1.  Free SO₂ data

Free SO2 (mg/L)	A		B		C		D		E		F		G		H		I		J
Sample	1	14	2	16	3	19	4	12	5	20	6	18	7	11	8	15	9	17	10	13
Labo 3			31	36	18	18	21	23	20	18	6	6	20	17	5	6
Labo 5			37	35	21	24	24	25	20	20	8	7	20	20	3	4
Labo 6	4	1	38	33	21	20	20	26	19	20	7	6	21	19	7	8	1	3	1	1
Labo 7	1	1	37	40	20	22	24	26	20	22	9	8	20	23	8	8	2	1	1	1
Labo 8			31	32	18	19	23	22	22	20	6	7	19	20	5	3	1	1
Labo 9			35	34	23	19	25	24	21	24			17	17
Labo 10	2	1	35	34	20	21	24	24	22	21	9	8	21	20	7	7	2	2	1	1
Labo 11	0	0	33	30	17	11	22	16	16	21	6	4	15	19	6	3	1	1	0	0
Labo 15			15	19	15	13	18	20	8	16	6	5	8	15	5	5
Labo 17	0	0	37	38	24	26	28	28	26	23	8	8	24	22	7	7	1	2	0	0
Labo 18	0	4	33	31	21	11	23	27	15	19	6	4	9	20	3	4	1	1	0	0
Labo 20	0	0	32	32	20	19	21	21	29	21	8	8	20	18	12	4	1	1	0	0
Labo 21	2	1	33	38	19	15	25	22	19	21	6	6	19	20	8	7	2	1	0	0

Results left blank were rendered non-quantifiable (< limit of quantification).

	Result removed by the COCHRAN test at 5%
	Result removed by the GRUBBS test at 5%

4.2.  Free SO₂ results

Free SO2 (mg/L)	A	B	C	D	E	F	G	H	I	J
No. of laboratories selected	7	9	11	10	10	12	11	11	9	8
No. of repetitions	2	2	2	2	2	2	2	2	2	2
Min.	0	31.5	14	19	17	5	17	3.5	1	0
Max.	2.5	38.5	25	28	24.5	8.5	23	8	2	1
Mean	0.9	34.2	19.8	23.4	20.6	6.8	19.6	5.7	1.4	0.4
Standard deviation	0.98	2.67	2.91	2.46	2.04	1.31	1.77	1.72	0.42	0.52
Repeatability variance	0.79	1.67	2.59	1.20	2.60	0.58	2.23	0.82	0.39	0.00
Inter-laboratory standard deviation	0.98	2.67	2.91	2.46	2.04	1.31	1.77	1.72	0.42	0.52
Reproducibility variance	1.35	7.97	9.76	6.64	5.46	2.00	4.25	3.38	0.37	0.27
Repeatability standard deviation	0.89	1.29	1.61	1.10	1.61	0.76	1.49	0.90	0.62	0.00
r limit	2.48	3.61	4.51	3.07	4.51	2.14	4.18	2.53	1.75	0.00
Repeatability %CV (k=2)	191	8	16	9	16	23	15	32	90	0
Reproducibility standard deviation	1.16	2.82	3.12	2.58	2.34	1.41	2.06	1.84	0.61	0.52
R limit	3.25	7.90	8.75	7.22	6.54	3.96	5.78	5.15	1.70	1.45
Reproducibility %CV (k=2)	250	16	32	22	23	42	21	64	87	276
Horwitz PRSDR (%)	16.18	9.40	10.21	9.95	10.15	12.00	10.22	12.30	15.23	18.55
Horwitz sR	0.15	3.22	2.02	2.33	2.09	0.81	2.00	0.70	0.21	0.07
Horwitz R	0.42	9.10	5.71	6.59	5.91	2.29	5.67	1.99	0.60	0.20
Horwitz Ratio	7.64	0.87	1.53	1.10	1.11	1.73	1.02	2.58	2.84	7.37

Figure 1: Modelling of the repeatability coefficient of variation, %CV(r) (k=2), as a function of the concentration, C:

Figure 2: Modelling of the inter-laboratory reproducibility coefficient of variation, %CV(R) (k=2), as a function of concentration, C:

5. Total sulphur dioxide

5.1.  Total SO₂ data

Total SO2 (mg/L)	A		B		C		D		E		F		G		H		I		J
Sample	1	14	2	16	3	19	4	12	5	20	6	18	7	11	8	15	9	17	10	13
Labo 3			128	127	72