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	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-AS323-04B Sulfur dioxide

Type IV method

 

1. Definitions

Free sulfur dioxide is defined as the sulfur dioxide present in the must or wine in the following forms: H₂SO₃, HSO₃, whose equilibrium as a function of pH and temperature is:

H₂SO₃ represents molecular sulfur dioxide.

Total sulfur dioxide is defined as the total of all the various forms of sulfur dioxide present in the wine, either in the free state or combined with their constituents.

 

2. Free and Total Sulfur Dioxide

2.1.  Principle

Free sulfur dioxide is determined by direct titration with iodine. The combined sulfur dioxide is subsequently determined by iodometric titration after alkaline hydrolysis. When added to the free sulfur dioxide, it gives the total sulfur dioxide.

2.2.  Rapid Method

2.2.1.      Reagents

2.2.1.1.                        EDTA: ethylenediaminetetraacetic acid, di-sodium salt

2.2.1.2.                        4 M Sodium hydroxide solution (160 g/L).

2.2.1.3.                        Dilute sulfuric acid: 10% sulfuric acid (ρ20 = 1.84 g/mL) diluted 10% (v/v).

2.2.1.4.                        Starch solution, 5 g/L.

Mix 5 g starch with approx. 500 mL water.  Bring to a boil stirring continuously and keep boiling for 10 minutes.  Add 200 g of sodium chloride. Cool and make to 1 liter.

2.2.1.5.                        0.025 M Iodine solution

2.2.2.      Free sulfur dioxide

Place in a 500 mL conical flask place:

• 50 mL of wine
• 5 mL starch solution
• 30 mg EDTA
• 3 mL H₂SO₄

Immediately titrate with 0.025 M iodine, until the blue color persists clearly for 10 to 15 seconds.  Let n mL be the volume of iodine used.

2.2.3.      Combined sulfur dioxide

Add 8 mL of 4 M sodium hydroxide solution, shake the mixture once and allow to stand for 5 minutes.  Add, with vigorous stirring and in one operation, the contents of a small beaker in which 10 mL of sulfuric acid have been placed.  Titrate immediately with the 0.025 M iodine solution; let n' be the volume used.

Add 20 mL of sodium hydroxide solution, shake once and allow to stand for 5 minutes.  Dilute with 200 mL of ice-cold water.

Add, while stirring vigorously and in one operation, the contents of a test tube in which 30 mL sulfuric acid has previously been placed.  Titrate the free sulfur dioxide immediately with the 0.025 M iodine, and let n" be the volume of iodine used.

2.2.4.      Expression of the results

2.2.4.1.                        Calculation

Free sulfur dioxide in milligrams per liter is given by:

32·n

Total sulfur dioxide in milligrams per liter is given by:

32 (n + n' + n")

Remarks:

1. For red wines with low SO₂ concentrations, the 0.025 M iodine may be diluted (for example: 0.01 M).  In this case, replace the coefficient 32 by 12.8 in the above formula.

2. For red wines, it is useful to illuminate the wine from below with a beam of yellow light from an ordinary electric light bulb shining through a solution of potassium chromate or from a sodium vapor lamp.  The determination should be carried out in a dark room and the transparency of the wine observed: it becomes opaque when the starch endpoint is reached.

3. If the quantity of sulfur dioxide found is close to or exceeds the legal limit, the total sulfur dioxide should be determined with the reference method.

4. If the determination of free sulfur dioxide is specifically required, carry out a determination on a sample kept under anaerobic conditions for two days at 20 °C before analysis.  Carry out the determination at 20 °C.

5. Because certain substances are oxidized by iodine in an acid medium, the quantity of iodine used in this way must be assessed for more accurate determinations.  To achieve this, combine the free sulfur dioxide in an excess of ethanal or propanal before beginning the titration with iodine. 

Place 50 mL of wine into a 300 mL conical flask, add 5 mL of 7 g/L ethanol solution or 5 mL of a 10 g/L propanal solution.

Stopper the flask and allow to stand for at least 30 minutes.  Add 3 mL of sulfuric acid and sufficient iodine, 0.025 M, to cause the starch to change color.  Let n''' mL be the volume of iodine used. This must be subtracted from n (free sulfur dioxide), and from n + n' + n'' (total sulfur dioxide).

n''' is generally small, from 0.2 to 0.3 mL of 0.025 M iodine.  If ascorbic acid has been added to the wine, n''' will be much higher and it is possible, at least approximately, to measure the amount of this substance from the value of n''' given that 1 mL of 0.025 M iodine will oxidize 4.4 mg ascorbic acid.  By determining n''', it is possible to detect quite easily the presence of residual ascorbic acid in amounts greater than 20 mg/L, in wines to which it has been added.

Bibliography

 

 

Rapid method:

• RIPPER M., J. Prakt. Chem., 1892, 46, 428.
• JAULMES, P., DIEUZEIDE J.-C., Ann. Fals. Fraudes, 1954, 46, 9; Bull. O.I.V., 1953, 26, n° 274, 52.
• KIELHOFER E., AUMANN H., Mitt. Klosterneuburg, Rebe u. Wein, 1957, 7, 289.
• JAULMES P., HAMELLE Mme G., Ann. Fals. Exp. Chim., 1961, 54, 338

OIV-MA-AS323-04C Sulfur dioxide

Type IV method

 

1. Definitions

Free sulfur dioxide is defined as the sulfur dioxide present in the must or wine in the following forms: H₂SO₃, HSO₃, whose equilibrium as a function of pH and temperature is:

H₂SO₃ represents molecular sulfur dioxide.

Total sulfur dioxide is defined as the total of all the various forms of sulfur dioxide present in the wine, either in the free state or combined with their constituents.

2. Molecular Sulfur Dioxide

2.1.  Principle of the Method

The percentage of molecular sulfur dioxide, H₂SO₃, in free sulfur dioxide, is calculated as a function of pH, alcoholic strength and temperature.

For a given temperature and the alcoholic strength:

where

L = [] + []

pk_M = pk_T 

I = ionic strength

A & B = Coefficients which vary according to temperature and alcoholic strength.

= Thermodynamic dissociation constant; the value of pk_T is given in Table 1 for various alcoholic strengths and temperatures.

= Mixed dissociation constant

Taking a mean value 0.038 for the ionic strength I, Table 2 gives the values of pk_M for various temperatures and alcoholic strengths.

The molecular sulfur dioxide content calculated by the relationship given in (1) is presented in Table 3 for various values of pH, temperature and alcoholic strength.

2.2.  Calculations

Knowing the pH of wine and its alcoholic strength, the percentage of molecular sulfur dioxide is given in Table 3 for a temperature t °C.  Let this be X %.

The amount of molecular sulfur dioxide in mg/L is given by:X · C

C = the free sulfur dioxide in mg/L

 

Table I

Values of the thermodynamic constant pk_T

Alcohol % by volume	Temperature oC
	20	25	30	35	40
0	1.798	2.000	2.219	2.334	2.493
5	1.897	2.098	2.299	2.397	2.527
10	1.997	2.198	2.394	2.488	2.606
15	2.099	2.301	2.503	2.607	2.728
20	2.203	2.406	2.628	2.754	2.895

 

Table II

Values of the Mixed Dissociation Constant pk_M (I= 0.038)

Alcohol % by volume	Temperature °C
	20	25	30	35	40
0	1.723	1.925	2.143	2.257	2.416
5	1.819	2.020	2.220	2.317	2.446
10	1.916	2.116	2.311	2.405	2.522
15	2.014	2.216	2.417	2.520	2.640
20	2.114	2.317	2.538	2.663	2.803

 

Table III

Molecular Sulfur Dioxide as a Percentage of Free Sulfur Dioxide (I=0.038)

	pH		T = 20 oC Alcohol % by volume
					0		10		15		20
	2.8		7.73		9.46		11.55		14.07		17.09
	2.9		6.24		7.66		9.40		11.51		14.07
	3.0		5.02		6.18		7.61		9.36		11.51
	3.1		4.03		4.98		6.14		7.58		9.36
	3.2		3.22		3.99		4.94		6.12		7.58
	3.3		2.58		3.20		3.98		4.92		6.12
	3.4		2.06		2.56		3.18		3.95		4.92
	3.5		1.64		2.04		2.54		3.16		3.95
	3.6		1.31		1.63		2.03		2.53		3.16
	3.7		1.04		1.30		1.62		2.02		2.53
	3.8		0.83		1.03		1.29		1.61		2.02
T = 25 oC
	2.8		11.47		14.23		17.15		20.67		24.75
	2.9		9.58		11.65		14.12		17.15		22.71
	3.0		7.76		9.48		11.55		14.12		17.18
	3.1		6.27		7.68		9.40		11.55		14.15
	3.2		5.04		6.20		7.61		9.40		11.58
	3.3		4.05		4.99		6.14		7.61		9.42
	3.4		3.24		4.00		4.94		6.14		7.63
	3.5		2.60		3.20		3.97		4.94		6.16
	3.6		2.07		2.56		3.18		3.97		4.55
	3.7		1.65		2.05		2.54		3.18		3.98
	3.8		1.32		1.63		2.03		2.54		3.18
T = 30 oC
	2.8		18.05		20.83		24.49		29.28		35.36
	2.9		14.89		17.28		20.48		24.75		30.29
	3.0		12.20		14.23		16.98		20.71		25.66
	3.1		9.94		11.65		13.98		17.18		21.52
	3.2		8.06		9.48		11.44		14.15		17.88
	3.3		6.51		7.68		9.30		11.58		14.75
	3.4		5.24		6.20		7.53		9.42		12.08
	3.5		4.21		4.99		6.08		7.63		9.84
	3.6		3.37		4.00		4.89		6.16		7.98
	3.7		2.69		3.21		3.92		4.95		6.44
	3.8		2.16		2.56		3.14		3.98		5.19

 

Table III (continued)

Molecular Sulfur Dioxide as a Percentage of Free Sulfur Dioxide (I=0.038)

pH	T=35 oC Alcohol % by volume
	0	5	10	15	20
2.8	22.27	24.75	28.71	34.42	42.18
2.9	18.53	20.71	24.24	29.42	36.69
3.0	15.31	17.18	20.26	24.88	31.52
3.1	12.55	14.15	16.79	20.83	26.77
3.2	10.24	11.58	13. 82	17.28	22.51
3.3	8.31	9.42	11.30	14.23	18.74
3.4	6.71	7.63	9.19	11.65	15.49
3.5	5.44	6.16	7.44	9.48	12.71
3.6	4.34	4.95	6.00	7.68	10.36
3.7	3.48	3.98	4.88	6.20	8.41
3.8	2.78	3.18	3.87	4.99	6.80
T = 40 oC
2.8	29.23	30.68	34.52	40.89	50.14
2.9	24.70	26.01	29.52	35.47	44.74
3.0	20.67	21.83	24.96	30.39	38.85
3.1	17.15	18.16	20.90	25.75	33.54
3.2	14.12	14.98	17.35	21.60	28.62
3.3	11.55	12.28	14.29	17.96	24.15
3.4	9.40	10.00	11.70	14.81	20.19
3.5	7.61	8.11	9.52	12.13	16.73
3.6	6.14	6.56	7.71	9.88	13.77
3.7	4.94	5.28	6.22	8.01	11.25
3.8	3.97	4.24	5.01	6.47	9.15

Bibliography

Molecular sulfur dioxide:

• BEECH F.W. & TOMAS Mme S., Bull. O.I.V., 1985, 58, 564-581.
• USSEGLIO-TOMASSET L. & BOSIA P.D., F.V., O.I.V., 1984, n° 784.

OIV-MA-AS323-06 Determination of mercury in wine by vapour generation and atomic spectroglurimeter

Type IV method

1. Field of application

This method applies to the analysis of mercury in wines with a concentration range between 0 to 10 ug/l.

2. Description of technique

2.1.   Principle of the method

2.1.1. Mineralisation of wine takes place in an acid environment: heating under reflux; mineralisation is achieved with a potassium permanganate.

2.1.2. Reduction of non-consumed permanganate by hydroxylamine hydrochlorate

2.1.3. Reduction in mercury II (metal mercury by stannous chloride (II).

2.1.4. Mercury pick up by an argon current at ambient temperature

2.1.5. Dosage of mercury in monoatomic vapour state by atomic flourescence spectometre with wavelength of 254 nm. Mercury atoms are excited by a mercury vapour lamp; the atoms thus excited emit a radiation called flourescent which allows the quantification of mercury present using a photonics detector to obtain good linearity while eliminating memory effects.

2.2.   Principle of analysis (figure 1)

The peristaltic pump absorbs the stannous chloride solution, the blank solution (demineralised water containing 1% nitric acid) and the sample of mineralised wine.

The mercury metal is taken up in a gas-liquid separator by a current of argon. After going through a drying tube, the mercury is detected by florescence. Then, the gaseous current goes through a permanganate potassium solution in order to capture the mercury.

 

3. Reagents and preparation of reactive solutions

3.1.   Ultra-pure demineralised water

3.2.   Ultra-pure 65% nitric acid

3.3.   White: demineralised water (3.1) containing 1% of nitric acid (3.2)

3.4.   Nitric acid solution 5.6 M (3.1):

Put 400 ml of nitric acid (3.2) into a 1000 ml flask; fill with demineralised water (3.1).

3.5.   Sulphuric acid (d= 1.84)

3.6.   Sulphuric acid solution 9M:

Put 200 ml of demineralised water (3.1), 50 g of potassium permanganate (3.7) into a 1000 ml flask; fill with demineralised water (3.1).

3.7.   Potassium permanganate KMnO4

3.8.   5% Potassium permanganate solution:

Dissolve 50 g of potassium permanganate (3.7) with demineralised water (3.1), in a 1000 ml flask. Fill with demineralised water (3.1).

3.9.   Hydroxylamine hydrogen chloride NH2OH, HCl

3.10.         Reducing solution:

Weigh 12g of hydroxylamine hydrogen chloride (3.9) and dissolve in 100 ml of demineralised water (3.1).

3.11.         Stannous chloride (SnCl2, 2 H2O)

3.12.         Concentrated hydrochloric acid

3.13.         Stannous chloride solution:

Weigh 40 g of stannous chloride (3.11) and dissolve in 50 ml of hydrochloric acid (3.12). Fill with 200 ml of demineralised water (3.1).

Mercury standard solution at 1g/l prepared by dissolving 1708 g of Hg (NO₃). H₂O in an aqueous nitric acid solution at 12% prepared from metal mercury.

Reference mercury solution at 10 mg/l :

Place 1 ml of mercury standard solution (3.14) in a 100 ml volumetric flask, add 5 ml of nitric acid, fill will demineralised water (3.1)

Mercury solution at 50 mg/l:
Place 1 ml of 10 mg/l (3.15) solution in a 200 ml flask. Add 2 ml of nitric acid (3.2). Fill with demineralised water (3.1).

4. Apparatus

4.1.   Glass ware

4.1.1. Volumetric flasks 100, 200, and 1000 ml (class A)

4.1.2. Volumetric pipette 0.5,1.0, 2.0, 5, 10 and 20 ml (class A)

4.1.3. Precautionary action: Before using, the glass ware must be washed with 10% nitric acid, leave in contact 24 hours, then rinse with demineralised water.

4.2.   Mineralisation apparatus (figure 2)

4.3.   Temperature controlled heating mantle

4.4.   Squeeze pump

4.5.   Cold vapour generator

4.5.1. Liquid gas separator

4.6.   Desiccant (Hygroscopic membrane) covered by an air current (supplied from a compressor) and placed before the detector

4.7.   Spectrofluorimeter

4.7.1. Mercury vapour lamp regulated to 254 nm wave length

4.7.2. Atomic fluorescence specific detector

4.8.   Computer

4.8.1. Software which regulates the parameters of the vapour generator and the atomic fluorescence detector and enables calibration and usage of the results.

4.8.2. Printer which stores results

4.9.   Neutral gas bottle (argon)

5. Preparation of calibration solutions and samples

5.1.   Set of calibration solutions: 0; 0.25; 0.5; and 1.0 ug/L

Introduce : 0; 0.5; and 1.0 and 2.0 ml of the mercury solution to 50 ug/l (3.16.) in 4 100 ml flasks; add 1 % nitric acid (3.2.); fill with demineralised water (3.1.).

5.2.   Preparation of samples (figure 2)

Wine is mineralised in a glass pyrex apparatus made up of three parts joined by spherical honing: a 250 ml balloon, a vapour recuperation chamber, a refrigerant.

Using a pipette put 20 ml of wine in a 250 ml reaction flask; assemble the mineralisation apparatus.

Add 5 ml of sulphuric acid (3.6.) and 10 ml of nitric acid (3.4.) slowly; leave overnight.

Heat slowly under reflux until the nitrous vapours disappear ; leave to cool. Recover the condensed vapours in the reaction flask. Rinse the recipient with demineralised water. Pour the contents of the reaction flask into a 100 ml volumetric flask. Add potassium permanganate  solution (3.8.) until the colour remains. Solubilize the precipitate (MnO₂) with a reducing solution (3.10.). Fill with demineralised water (3.1.).

Carry out a blank test on demineralised water.

6. Operating procedure

6.1.   Analytical measurement

Turn on the fluorimeter; the apparatus is stable after 15 minutes. The squeeze pump absorbs the white (3.3), the stannous lead II (3.13) and the sample calibrations (5.1) or (5.2.) Verify that bubbling occurs in the liquid gas separator. Present the calibration samples successively (5.1); set off the vapour generator program. The computer software establishes a calibration curve (percentage of fluorescence according to concentration of mercury ug/l). Then present the samples (5.2).

6.2.   Automatic checks

A blank analysis and a calibration are analysed every five tests to correct any possible spectrofluorimeter derivitives.

7. Expression of results

Results are provided by the computer software and expressed in ug/l. Deduct the mercury concentration in wine in ug/l keeping into account 1/5 dilution.

8. Checking results

Quality control is carried out by placing reference material in which the mercury content is known, following the set of calibrations and every 5 samples. Following the analytical series, the reference material is red wine, dry white wine or sweet white wine.

The check card is set for each reference material used. The check limits are set at: +/- 2S_R  intra (2S_R  intra : reproducibility spread-type)

The uncertainly calculation, carried out on check cards, resulted in a red wine reference of: 3.4 +/- 0.8 ug/l and for reference dry white wine : 2.8+/-0.9 ug/l.

9. Bibliography

• CAYROL M., BRUN S., 1975. Dosage du mercure dans les vins. Feuillet Vert de l’O.I.V. n°371.
• REVUELTA D., GOMEZ R., BARDON A., 1976. Dosage du mercure dans le vin par la méthode des vapeurs froides et spectrométrie d’absorption atomique. Feuillet Vert de l’O.I.V. n°494.
• CACHO J., CASTELLS J.E., 1989. Determination of mercury in wine by flameless atomic absorption spectrophotometry. Atomic Spectroscopy, vol. 10, n°3.
• STOCKWELL P.B., CORNS W.T., 1993. The role of atomic fluorescence spectrometry in the automatic environmental monitoring of trace element analysis. Journal of Automatic Chemistry, vol. 15, n°3, p 79-84.
• SANJUAN J., COSSA D., 1993. Dosage automatique du mercure total dans les organismes marins par fluorescence atomique. IFREMER, Rapport d’activité.
• AFNOR, 1997. Dosage du mercure total dans les eaux par spectrométrie de fluorescence atomique. XPT 90-113-2.
• GAYE J., MEDINA B., 1998. Dosage du mercure dans le vin par analyse en flux continu et spectrofluorimétrie. Feuillet Vert de l’O.I.V. n°1070.

OIV-MA-AS323-07 Multielemental analysis using ICP-MS

Type II method

 

1. Scope of application

This method can be applied to the analysis of the elements present in wines within the range indicated and featured in the following list:

• Aluminium between 0.25 and 5.0 mg/l
• Boron between 10 and 40 mg/l
• Bromine between 0.20 and 2.5 mg/l
• Cadmium between 0.001 and 0.040 mg/l
• Cobalt between 0.002 and 0.050 mg/l
• Copper between 0.10 and 2.0 mg/l
• Strontium between 0.30 and 1.0 mg/l
• Iron between 0.80 and 5.0 mg/l
• Lithium between 0.010 and 0.050 mg/l
• Magnesium between 50 and 300 mg/l
• Manganese between 0.50 and 1.5 mg/l
• Nickel between 0.010 and 0.20 mg/l
• Lead between 0.010 and 0.20 mg/l
• Rubidium between 0.50 and 1.2 mg/l
• Sodium between 5 and 30 mg/l
• Vanadium between 0.003 and 0.20 mg/l
• Zinc between 0.30 and 1.0 mg/l

This technique can also be used to analyze other elements.

The sample sometimes requires mineralization. This is the case, for example, of wines with more than 100 g/L of sugar where it can be necessary to realise mineralization of the sample before. In this case, it is recommended to perform a digestion with nitric acid in a microwave.

The technique can also be applied to musts, after mineralization.

 

2. Basis

Multielemental quantitative determination using Inductively Coupled Plasma Mass Spectometry or ICP-MS.

Injection and nebulization of the sample in high-frequency plasma. The plasma causes the desolvation, atomization and ionization of the elements in the sample. The ions are extracted using a vacuum system fitted with ionic lenses. The ions are separated according to the mass-to-charge ratio in a mass spectrometer, for example, a quadrupole. Detection and quantification of ions using an electron multiplier system.

3. Reagents and solutions

3.1.   Ultrapure, demineralized water with resistivity ( 18 MΩ), in accordance with ISO 3696.

3.2.   Certified solution(s) (for example, 100 mg/l) containing the metals to be analyzed. Multielemental or monoelemental solutions can be used.

3.3.   Indium and/or rhodium solution as an internal standard (normally 1 g/l).

3.4.   Nitric acid 60% (metal impurities 0.1 μg/l).

3.5.   Argon, minimum purity of 99.999%.

3.6.   Nitrogen (maximum impurity content: H₂O 3 mg/l, O₂ 2 mg/l and

CnHm 0.5 mg/l).

Solution concentration and internal standards are given by way of reference.

Preparation of standard solutions:

Acid concentration in the standards and in the final dilution of the wine samples must be the same and must not exceed 5%. The following is an example.

3.7.   Stock solution (5mg/l).

Place 0.5ml of solution (3.2) in a 10 ml (4.5) tube and add 0.1 ml of nitric acid (3.4). Level off to 10 ml with demineralized water (3.1) and homogenize.

Shelf life: 1 month.

3.8.   Internal standard solution (1 mg/l).

Using micropipettes (4.4), place 50 µl of indium or rhodium solution (3.3) and 0.5 ml of nitric acid (3.4) in a 50 ml tube (4.6). Level off to 50 ml with demineralized water (3.1) and homogenize.

Shelf life: 1 month.

3.9.   Standard solutions of the calibration curve.

Adapt the range of the series of standards according to the dilution on the  sample or the equipment used.

Use 1000 μl and 100 µl pipettes (4.4).

Shelf life of standard solutions: 1 day

These standard solutions can also be prepared gravimetrically. Add internal standard in the same concentration as for the samples.

3.10.         Internal control wine of known concentrations (MRC, MRE, MRI, etc.).

4. Material and equipment

4.1.   Inductively coupled mass spectrometer with/without collision/reaction cell.

4.2.   Computer with data processing software and printer.

4.3.   Autosampler (optional).

4.4.   1000 μl and 100 µl micropipettes

4.5.   10 ml plastic, graduated test tubes with bung or glass volumetric flasks.

4.6.   50 ml plastic, graduated test tubes with bung or glass volumetric flasks.

 

All volumetric material (micropipettes and test tubes) must be duly calibrated.

Note: material that will come into contact with the sample, such as, for example, tubes and tips, must remain for at least 24 hours in a nitric acid solution (3.4) at a concentration of 10% and must subsequently be rinsed several times in water (3.1).

5. Sample preparation

Samples of sparkling wine must be degasified. This can be done through nitrogen bubbling (3.6) for 10 minutes or by using an ultrasound bath.

Remove the bung carefully to ensure that the wine is not contaminated. Wash the bottle neck in an acid solution (2% H). Wine samples are taken directly from the bottle.

Use a micropipette (4.4) to insert 0.5 ml of wine, 0.1 to 0.5 ml of nitric acid (3.4) and 100 μl of internal standard solution (3.8) into a 10 ml tube (3.5).Level off with water (3.1) and homogenize.

For certain elements a higher dilution may be necessary owing to their high natural content in the sample.

Br has high ionization potential and its ionization in plasma may be incomplete because of the presence of high concentrations of other elements in wines with low ionization potential. This may result in the incorrect quantification of Br and therefore a 1/50 dilution is recommended to avoid this effect (in the event of another dilution being used, confirm the results by checking recovery after an addition).

When the standards are prepared gravimetrically, the final dilution of the sample must also be obtained gravimetrically.

6. Procedure

Switch on the device (pump working and plasma on).

Clean the system for 20 minutes using 2% nitric acid (3.4).

Check that the device is functioning correctly.

Analyze a blank and the series of standard solutions in increasing concentrations, then a standard solution (e.g. no. 2 of series 3.9) to check for correct calibration and finally the blank to ensure that there is no memory effect. Read the samples in duplicate. For the internal control, use a wine of known concentrations (3.10) to confirm that the results are coherent.

Element	m/z*
Aluminium	27
Boron	11
Bromine	79
Cadmium	114
Cobalt	59
Copper	63
Strontium	88
Iron	56/57
Lithium	7
Magnesium	24
Manganese	55
Nickel	60
Lead	average of 206, 207 and 208
Rubidium	85
Sodium	23
Vanadium	51
Zinc	64

*The above table is given by way of example. Other isotopes may be required, depending on the equipment.

In the event of using equipment with no collision/reaction cell, correction equations may be necessary for some elements.

7. Results

The software can calculate the results directly.

Element concentrations are reported in mg/l to two decimal points.

Obtain, by interpolating in the calibration curve, the concentration of the elements in the diluted samples. Use the following equation to calculate the concentration of the elements in the sample:

Where:

C =  Concentration of the element in the sample

= Concentration of the elements in the diluted sample

  = Final volume of the measurement solution, in ml

= Aliquot volume of wine, in ml.

8. Quality control

Ensure traceability by using certified standards.

In each analytical series, use a CRM (Certified Reference Material) as an internal control of wine or a wine used as reference material from an interlaboratory test campaign.

It is recommended that control graphs be created from the results of the quality control analysis.

Participation in interlaboratory test campaigns.

9. Precision

The results of the statistical parameters of the collaborative trial are shown in Appendix A.

9.1.   Repeatability (r)

The difference between two independent results, obtained using the same method, in the same sample, in the same laboratory, by the same operator, using the same equipment in a short time interval. r results are given in Tables 1 to 17 of Appendix A

9.2.   Reproducibility (R)

The difference between two results, obtained using the same method, in the same sample, in a different laboratory, by a different operator and with different equipment. R results are given in Tables 1 to 17 of the Appendix A.

Table 1 represents the % of the relative standard deviation of Repeatability and Reproducibility (RSD_r% et RSD_R%) of the method. (*) C = Concentration

Element	Concentration	RSDr %	RSDR %
Aluminium	0,25 – 5,0 mg/l	4	10
Boron	10 - 40 mg/l	3,8	6,3
Bromine	0,20– 1,0 mg/l	4,1	16,3
	≥ 1,0 – 2,5 mg/l	2.1	8,0
Cadmium	0,001 – 0,020 mg/l	0,06 C*+0,18	10
	≥ 0,020 – 0,040 mg/l	1,5	10
Cobalt	0,002 – 0,050 mg/l	3,2	13,2
Copper	0,10 – 0,50 mg/l	3,8	11,4
	≥ 0,50 – 2,0 mg/l	2,0	11,4
Strontium	0,30 – 1,0 mg/l	2,5	7,5
Iron	0,80– 1,0 mg/l	4,2	15,7
	≥ 1,0-5,0 mg/l	4,2	7,8
Lithium	0,010 – 0,050 mg/l	7	12
Magnesium	50 - 300 mg/l	2	6
Manganese	0,50-1,5 mg/l	3	7
Nickel	0,010 – 0,20 mg/l	5	8
Lead	0,010 – 0,050 mg/l	8	7
	≥ 0,050 – 0,20 mg/l	2	7
Rubidium	0,50 – 1,2 mg/l	3	6
Sodium	5 - 10 mg/l	2	10
	≥ 10 - 30 mg/l	0,3 C*-2,5	10
Vanadium	0,003 – 0,010 mg/l	8	10
	≥ 0,010 – 0,20 mg/l	3	10
Zinc	0,30 – 1,0 mg/l	5	12

Table 1: relative standard deviation of Repeatability and Reproducibility

10. Bibliography

• ISO 5725:1994, Precision of test methods-Determination of repeatability and reproducibility for a Standard test method by interlaboratory test.
• ISO 17294:2004.
• ALMEIDA M. R, VASCONCELOS T, BARBASTE M. y MEDINA B. (2002), Anal. Bioanal Chem., 374, 314-322.
• CASTIÑEIRA et al. (2001), Frenesius J. Anal. Chem., 370, 553-558.
• DEL MAR CASTIÑEIRA GOMEZ et al. (2004), J. Agric Food Chem., 52, 2962-2974.
• MARISA C., ALMEIDA M. et VASCONCELOS T. (2003), J. Agric. Food Chem., 51, 3012-3023.
• MARISA et al., (2003), J. Agric Food Chem., 51, 4788-4798.
• PÉREZ-JORDAN M. Y., SOLDEVILLA J., SALVADOR A., PASTOR A y de la GURDIA M. (1998), J. Anat. At. Spectrom., 13, 33-39.
• PEREZ-TRUJILLO J.-P., BARBASTE M. y MEDINA B. (2003), Anal. Lett., 36(3), 679-697.
• TAYLOR et al. (2003), J. Agric Food Chem., 51, 856-860.
• THIEL et al. (2004), Anal. Bioanal. Chem, 378, 1630-1636.

Appendix A: Results of the collaborative trials

The method has been checked with two collaborative trials, by evaluating precision in accordance with ISO 5725.The trueness of the method has been obtained through recovery studies.

1st Collaborative Trial

8 samples (A, B, C, D, E, F, MH1 and MH2) were used from the following origins:

• Three samples of red wine, with and without addition.

• Three samples of white wine, with and without addition.
• Two samples of synthetic hydroalcoholic mixture, prepared with ethanol and water.

Hydroalcoholic sample MH1 presented problems of instability during the trial and the results have not been taken into account.

	MH2	A	B	C	D	E	F
Metal (mg/l)	Hydroalcoholic mixture	RW2	RW3	WW2	WW3	Natural red wine	Natural white wine
Aluminium	5	0.5	2	2	1	No addition	No addition
Cadmium	0.001	0.005	0.02	0.05	0.01	No addition	No addition
Strontium	0.300	No addition	No addition	No addition	No addition	No addition	No addition
Lithium	0.020	0.01	0.02	0.04	0.01	No addition	No addition
Magnesium	50	100	200	50	25	No addition	No addition
Manganese	0.500	0.5	1	1	0.5	No addition	No addition
Nickel	0.070	0.025	0.2	0.1	0.1	No addition	No addition
Lead	0.010	0.05	0.1	0.15	0.05	No addition	No addition
Rubidium	1.0	No addition	No addition	No addition	No addition	No addition	No addition
Sodium	20	10	10	20	5	No addition	No addition
Vanadium	0.010	0.05	0.2	0.1	0.1	No addition	No addition
Zinc	0.500	0.1	1	0.5	0.5	No addition	No addition

2nd Collaborative Trial

Sixteen samples (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P) from the following origins were used:

 Four samples of red wine, with and without addition.

 Four samples of Port wine, with and without addition.

 Six samples of white wine, with and without addition.

 Two samples of champagne.

Amounts added to the samples

Samples	Code	Addition	B	Co	Cu	Fe
			mg/l	μg/l	mg/l	Mg/l
White wine	F-N	No addition	0.0	0.0	0.0	0.0
	C-I	Addition 1	5.0	5.0	5.0	1.0
	A-O	Addition 2	10.0	10.0	1.0	2.0
Liqueur wine	B-K	No addition	0.0	0.0	0.0	0.0
	E-L	Addition 3	15.0	20.0	1.5	3.0
Red wine	D-M	No addition	0.0	0.0	0.0	0.0
	H-J	Addition 4	20.0	50.0	2.0	5.0
Sparkling wine	G-P	No addition	0.0	0.0	0.0	0.0

Precision parameters (Tables 1 to 17)

The values of Horrat_r and Horrat_R have been obtained by using the Horwitz equation taking into account Thompson’s modification for the concentration below 120 µg/L.

Table 1: Aluminium (mg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	10	0,68	0,020	0,06	2,9	11	0,26	0,077	0,22	11	17	0,66
B	11	9	2,1	0,043	0,12	2,0	9,4	0,22	0,21	0,61	10	14	0,71
C	11	9	2,1	0,032	0,09	1,5	9,5	0,16	0,21	0,59	10	14	0,69
D	11	10	1,2	0,041	0,12	3,4	10	0,34	0,10	0,29	8,3	16	0,56
E	11	10	0,34	0,014	0,04	4,1	12	0,34	0,029	0,08	8,5	19	0,46
F	11	10	0,27	0,006	0,02	2,2	13	0,17	0,028	0,08	10	20	0,52
MH2	11	8	5,2	0,26	0,73	5,0	8,2	0,60	0,56	1,6	11	13	0,86

Table 2: Boron (mg/l)

SAMPLE:	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A-O	8	6	18	0,77	2,2	4,3	6,8	0,62	0,94	2,69	5,2	10	0,50
B-K	8	4	4,5	0,27	0,76	6,0	8,4	0,72	0,40	1,14	8,9	13	0,70
C-I	8	4	13	0,31	0,89	2,4	7,2	0,33	0,33	0,94	2,5	11	0,24
D-M	8	7	11	0,26	0,74	2,4	7.4	0,31	1,1	3,11	10	11	0,90
E-L	8	5	21	0,47	1,3	2,2	6.7	0,33	0,85	2,43	4,0	10	0,40
F-N	8	5	8,3	0,43	1,2	5,2	7.7	0,68	0,47	1,34	5,7	12	0,48
G-P	7	4	3,1	0,094	0,27	3,0	8.9	0,34	0,18	0,51	5,8	14	0,43
H-J	8	5	31	1,0	3,0	3,2	6.3	0,54	1,6	4,43	5,2	9,6	0,52

Table 3: Bromine (mg/l)

SAMPLE:	LAB. No.	Accepted	Vref	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A-O	6	2	1,21	0,028	0,08	2,3	10,3	0,22	0,041	0,12	3,4	15,6	0,22
B-K	5	2	0,19	0,006	0,02	2,9	13,6	0,21	0,0043	0,012	2,3	20,5	0,11
C-I	6	3	0,81	0,017	0,05	2,1	10,9	0,19	0,062	0,18	7,7	16,5	0,47
D-M	6	4	0,38	0,017	0,05	4,5	12,2	0,37	0,066	0,19	17,4	18,5	0,94
E-L	6	3	1,72	0,030	0,09	1,7	9,7	0,17	0,22	0,62	12,8	14,8	0,86
F-N	6	3	0,22	0,014	0,04	6,4	13,3	0,48	0,046	0,13	20,9	20,1	1
H-J	6	2	2,30	0,061	0,17	2.7	9,3	0,28	0,092	0,26	4	14.1	0.28

Table 4: Cadmium (μg/l)

SAMPLE:	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	12	11	6	0,2	0,6	3,3	15	0,22	1	3	17	22	0,77
B	12	11	16	0,4	1	2,5	15	0,17	2	6	13	22	0,59
C	12	9	40	0,4	1	1,0	15	0,07	3	8	7,5	22	0,34
D	12	10	10	0,3	0,8	3,0	15	0,20	0,9	3	9,0	22	0,41
E	8	7	0,3	0,20	0,6	67	15	4,47	0,20	0,67	67	22	3,05
F	8	6	0,3	0,04	0,1	13	15	0,87	0,20	0,45	67	22	3,05
MH2	9	5	0,9	0,08	0,2	8,9	15	0,59	0,10	0,29	11	22	0,50

Table 5: Cobalt (μg/l)

SAMPLE:	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A-O	10	6	22	0,5	1	2,3	15	0,15	2	6	9,1	22	0,41
B-K	10	6	8	0,3	0,9	3,8	15	0,25	1	4	13	22	0,59
C-I	10	8	19	0,4	1	2,1	15	0,14	3	7	16	22	0,73
D-M	10	3	3	0,07	0,2	2,3	15	0,15	0,1	0,3	3,3	22	0,15
E-L	10	8	27	1	3	3,7	15	0,25	3	9	11	22	0,50
F-N	10	7	12	0,5	2	4,2	15	0,28	1	4	8,3	22	0,38
G-P	9	5	2	0,2	0,5	10	15	0,67	0,3	0,8	15	22	0,68
H-J	10	6	49	0,5	1	2,3	15	0,15	6	18	12	22	0,55

Table 6: Copper (mg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A-O	10	8	1,1	0,013	0,040	1,2	10	0,12	0,11	0,32	10	16	0,63
B-K	10	8	0,21	0,006	0,020	2,9	13	0,22	0,021	0,060	10	20	0,50
C-I	10	7	0,74	0,009	0,030	1,2	10	0,12	0,046	0,13	6,2	17	0,36
D-M	10	8	0,14	0,007	0,020	5,0	14	0,36	0,015	0,043	11	22	0,50
E-L	10	9	1,7	0,061	0,17	3,6	7,8	0,5	0,16	0,46	9,0	15	0,60
F-N	10	7	0,16	0,006	0,020	3,8	14	0,27	0,029	0,083	18	21	0,86
G-P	9	4	0,042	0,004	0,010	9,5	15	0,63	0,006	0,017	14	22	0,64
H-J	10	7	2,1	0,018	0,050	0,86	9,5	0,09	0,24	0,69	11	14	0,79

Table 7:  Strontium (μg/l)

SAMPLE	LAB. Nº	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R		RSDR (%)	HorwitzR RSDR (%)	HorratR
A	12	11	1091	33	93	3,0	10	0,30	78	222		7,2	16	0,45
B	12	8	1139	66	188	5,8	10	0,58	69	195		6,1	16	0,38
C	12	9	328	6	18	1,8	13	0,14	19	54	5,8		19	0,31
D	12	10	313	7	20	2,2	13	0,17	22	61		7,0	19	0,37
E	12	10	1176	28	80	2,4	10	0,24	86	243		7,3	16	0,46
F	12	10	293	3	9	1,0	13	0,08	22	62		7,5	19	0,39
MH2	12	9	352	7	19	2,0	12	0,17	24	69		6,8	19	0,36

Table 8: Iron (mg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A-O	10	6	3,2	0,017	0,05	0,53	8,9	0,06	0,23	0,66	7,2	13	0,55
B-K	10	6	1,5	0,085	0,24	5,7	9,9	0,58	0,11	0,31	7,3	15	0,49
C-I	10	5	2,1	0,036	0,10	1,7	9,4	0,18	0,18	0,51	8,6	14	0,61
D-M	10	5	3,1	0,033	0,094	1,1	8,9	0,12	0,29	0,83	9,4	14	0,67
E-L	10	5	4,3	0,120	0,34	2,8	8,5	0,33	0,29	0,83	6,7	13	0,52
F-N	10	6	1,1	0,051	0,15	4,6	10	0,46	0,16	0,46	15	16	0,94
G-P	9	6	0,83	0,024	0,07	2,9	11	0,26	0,14	0,40	17	16	1,06
H-J	10	7	7,8	0,180	0,52	2,3	7,8	0,29	1,2	3,52	15	12	1,25

Table 9:  Lithium (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	10	34	2	5	5,9	15	0,39	4	11	11	22	0,50
B	11	11	42	3	8	7,1	15	0,47	4	12	10	22	0,45
C	11	11	47	1	4	2,1	15	0,14	5	13	9,8	22	0,45
D	11	11	18	1	4	5,6	15	0,37	2	7	14	22	0,64
E	11	11	25	1	3	4,0	15	0,27	3	9	12	22	0,55
F	11	9	9	0,3	1	3,8	15	0,25	0,6	2	7,2	22	0,33
MH2	11	7	22	1	3	4,6	15	0,31	1	3	5,3	22	0,24

Table 10: Magnesium (mg/l)

SAMPLE:	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	10	7	182	2,9	8,1	1,6	4,3	0,37	9,3	26	5,1	7,3	0,70
B	10	6	280	3,9	11	1,4	4,5	0,31	6,0	17	2,1	6,9	0,30
C	10	7	104	2,4	6,9	2,3	5,3	0,43	6,8	19,25	6,5	8,0	0,81
D	10	6	85	1,4	4,0	1,7	5,4	0,31	2,2	6,1	2,6	8,2	0,32
E	10	7	94	2,2	6,2	2,3	5,3	0,43	5,5	16	5,9	8,1	0,73
F	10	7	65	0,95	2,7	1,5	5,6	0,27	3,8	11	5,9	8,5	0,69
MH2	10	7	51	0,90	2,5	1,8	5,8	0,31	2,4	6,9	4,7	8,9	0,53

Table 11: Manganese (mg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	10	1,3	0,014	0,040	1,1	10	0,11	0,13	0,37	10	15	0,67
B	11	9	1,8	0,14	0,40	7,8	9,7	0,80	0,20	0,56	11	15	0,73
C	11	8	1,5	0,028	0,080	1,9	9,9	0,19	0,084	0,24	5,6	15	0,37
D	11	8	1,0	0,035	0,10	3,5	11	0,32	0,049	0,14	4,9	16	0,31
E	11	9	0,84	0,019	0,050	2,3	11	0,21	0,057	0,16	6,8	16	0,43
F	11	9	0,59	0,015	0,040	2,5	11	0,23	0,031	0,090	5,3	17	0,31
MH2	11	8	0,52	0,029	0,080	5,6	12	0,47	0,037	0,10	7,1	18	0,39

_{Table 12: Nickel (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	10	40	2	6	5,0	15	0,33	5	13,90	13	22	0,59
B	12	10	194	7	20	3,6	14	0,26	17	48,96	8,8	21	0,42
C	12	8	148	4	10	2,7	14	0,19	5	15,12	3,4	21	0,16
D	12	8	157	4	12	2,6	14	0,19	8	23,10	5,1	21	0,24
E	11	8	15	0,6	2	4,0	15	0,27	1	3,33	6,7	22	0,30
F	12	9	66	1	4	1,5	15	0,10	4	10,58	6,1	22	0,28
MH2	11	7	71	5	14	7,0	15	0,47	4	11,41	5,6	22	0,25

Table 13: Lead (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	12	9	59	1	4	1,7	15	0,11	3	9	5,1	22	0,23
B	12	10	109	2	6	1,8	15	0,12	8	23	7,3	22	0,33
C	12	9	136	3	9	2,2	14	0,16	13	37	9,6	22	0,44
D	12	9	119	2	6	1,7	15	0,11	5	13	4,2	22	0,19
E	12	10	13	1	3	7,7	15	0,51	1	4	7,7	22	0,35
F	12	9	92	1	4	1,1	15	0,07	4	11	4,4	22	0,20
MH2	12	10	13	1	3	7,7	15	0,51	1	3	7,7	22	0,35

Table 14: Rubidium (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	6	717	14	41	2,0	11	0,18	13	36	1,8	17	0,11
B	11	7	799	25	70	3,1	11	0,28	30	86	3,8	17	0,22
C	11	8	677	10	27	1,5	11	0,14	34	96	5,0	17	0,29
D	11	7	612	18	51	2,9	11	0,26	18	50	2,9	17	0,17
E	11	9	741	19	53	2,6	11	0,24	66	187	8,9	17	0,52
F	11	9	617	10	28	1,6	11	0,15	43	123	7,0	17	0,41
MH2	11	7	1128	10	28	0,89	10	0,09	64	181	5,7	16	0,36

Table 15: Sodium (mg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	10	9	19	0,59	1,7	3,1	6,8	0,46	2,2	5,7	12	10	1,20
B	10	9	20	1,3	3,6	6,5	6,7	0,97	2,2	6,3	11	10	1,10
C	10	7	28	0,33	0,93	1,2	6,4	0,19	1,9	5,4	6,8	9,7	0,70
D	10	8	11	0,24	0,68	2,2	7,4	0,30	1,1	3,0	10	11	0,91
E	10	8	9,8	0,19	0,53	1,9	7,5	0,25	0,89	2,5	9,1	11	0,83
F	10	8	6,1	0,093	0,26	1,5	8,1	0,19	0,74	2,1	12	12	1,00
MH2	10	8	24	1,8	5,0	7,5	6,6	1,14	2,6	7,2	11	9,9	1,11

Table 16: Vanadium (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	12	11	46	1	3	2,2	15	0,15	5	13	11	22	0,50
B	12	11	167	5	15	3,0	14	0,21	19	54	11	21	0,52
C	12	11	93	3	8	3,2	15	0,21	12	33	13	22	0,59
D	12	9	96	3	8	3,1	15	0,21	8	22	8,3	22	0,38
E	10	7	3	0,2	0,7	6,7	15	0,45	0,3	0,9	10	22	0,45
F	10	8	3	0,2	0,6	6,7	15	0,45	0,2	0,7	6,7	22	0,30
MH2	12	9	11	0,3	1	2,7	15	0,18	0,9	3	8,2	22	0,37

Table 17: Zinc (μg/l)

SAMPLE	LAB. No.	Accepted	Vréf	Sr	r	RSD r (%)	Horwitz  RSDr  (%)	Horratr	SR	R	RSDR (%)	HorwitzR RSDR (%)	HorratR
A	11	8	405	22	61	5,4	12	0,45	45	128	11	18	0,61
B	11	9	1327	49	138	3,7	10	0,37	152	429	11	15	0,73
C	11	9	990	14	41	1,4	11	0,13	86	243	8,7	16	0,54
D	11	9	1002	28	79	2,8	11	0,25	110	310	11	16	0,69
E	11	9	328	13	37	4,0	13	0,31	79	224	24	19	1,26
F	11	9	539	15	42	2,8	12	0,23	61	172	11	18	0,61
MH2	11	8	604	72	204	12	11	1,09	89	251	15	17	0,88