RESOLUTION OENO 18/2003

ANALYTICAL AND CONTROL TECHNIQUES (Oenological Codex)

Chemical Section

The GENERAL ASSEMBLY,

Considering Article 5, paragraph 4 of the International Convention of the Unification of

Methods of Analysis and Appraisal of Wine of 13 October 1954,

Upon the proposal of the Sub-commission of the Methods of Analysis and Appraisal of Wine,

DECIDES:

To replace and complete Chapter II of the International Oenological Codex by the following
analytical and control techniques:

CHAPTER II: ANALYTICAL AND CONTROL TECHNIQUES

ANALYSES COMMON TO ALL MONOGRAPHIES

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 RESOLUTION OENO 18/2003

DETERMINATION OF TOTAL NITROGEN

1. APPARATUS

1.1 The apparatus used for separating NH3 is either a distillation apparatus with a
rectifying column or a distillation apparatus under a current of steam (diagram) made up
of:
A 1 l flask A of borosilicate glass used as a boiler with a stopcock funnel for filling.
It can be heated by a gas or electric furnace.
An adapter C which gathers the spent liquid from the bubbler B.
A bubbler B of 500 ml with an inclined neck; the supply tube must reach the
lowest part of the flask. The out-going tube has an anti-entrainment ball that makes up
the top part of the bubbler. A stop-cock funnel E allows to introduce the liquid to be
treated and alkaline lye.
A cooler 30 to 40 cm long, vertical, with a ball with fine dowel bush on the tip.
A 250 ml conical flask for the distillate.

1.2 Mineralisation flask, 300 ml ovoid-shaped flask with a long neck.

2. REAGENTS
Concentrated sulphuric acid (R).
Mineralisation catalyser (R).
Sodium hydroxide solution at 30% (m/m) (R).
Boric acid solution at 4% (R).
Hydrochloric acid solution 0.1 M.
Mixed-based indicator with methyl red (R) and methylene blue.
The boiler must contain acidulated water by 1 per 1 000 of sulphuric acid. It is
advisable to boil this liquid before any operation, with the drain cock P open to let the
CO2 escape.

3. PROCEDURE

In the mineralisation flask, introduce the test sample containing 4 to 50 mg of

nitrogen. Add 5 g of mineralisation catalyser (R) and 10 ml of concentrated sulphuric acid
(R), if the quantity of dry organic matter to be mineralised is below 500 mg. Increase
these quantities if a higher quantity of organic matter must be used.

Heat in an open flame under a hood. The neck of the flask is maintained inclined
until the solution becomes colourless and the walls of the flask are clear of carbonised
products.

After cooling, dilute with 50 ml of water and cool; introduce this liquid in the
bubbler B with the funnel E, then add 40 to 50 ml of sodium hydroxide solution at 30%
(R) in order to obtain frank alkalinisation of the liquid. Entrain the ammoniac with the
vapour by gathering the distillate in 5 ml of boric acid solution (R) placed beforehand in a
receiving conical flask with 10 ml of water, with the tip of the ampoule plunged into the
liquid. Add 1 or 2 drops of mixed-based indicator and gather 70 to 100 ml of distillate.

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 RESOLUTION OENO 18/2003

Titrate the distillate with the hydrochloric acid solution 0.1 M until the indicator
turns pink violet.

1 ml of 0.1 M hydrochloric acid solution corresponds to 1.4 mg of nitrogen.

Apparatus for the distillation of ammoniac

in a current of steam
(PARNAS and WAGNER)

The cocks P and E can be replaced by a plastic

pipe fitting with a Mohr pinch-clamp cock.

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 RESOLUTION OENO 18/2003

DETERMINATION OF MERCURY BY THE GENERATION OF VAPOUR

AND ATOMIC FLUORESCENCE SPECTROMETRY

1 – FIELD OF APPLICATION
This method is applied to the analysis of mercury in oenological products in the
concentration range of 0 to 10 µg/l.

2 – DESCRIPTION OF THE TECHNIQUE

2.1. Principle of the method

2.1.1. Mineralisation by the wet process of the oenological product to be analysed.
2.1.2. Reduction of the permanganate not consumed by hydroxylamine
hydrochloride.
2.1.3. Reduction of mercury(II) into metal mercury by tin chloride (II).
2.1.4. Entrainment of mercury by a current of argon at room temperature.
Detremining mercury in the state of monoatomic vapour by atomic fluorescence
spectrometry, with the wave length at 254 nm: the mercury atoms are excited by a
mercury vapour lamp; the atoms thus excited reemit fluorescent radiation that enables
to quantify the mercury present using a photonic detector placed at 90° in relation to
excitation beam; detection by atomic fluorescence enables to obtain good linearity and
eliminates memory effects.

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

The peristaltic pump draws up the tin chloride (II) solution, the blank (demineralised
water containing 1% nitric acid) and the mineralised sample or calibration.
The metal mercury is entrained in the gas-liquid separator by a current of argon.
After going through the membrane of a dessicator, the mercury is detected by
fluorescence.
Then the gaseous current goes through a potassium permanganate solution in order
to trap the mercury.

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 RESOLUTION OENO 18/2003

3 – REAGENTS AND PREPARATION OF REAGENT SOLUTIONS

3.1. Ultra-pure demineralised water
3.2. Ultra-pure nitric acid at 65%
3.3. Blank: demineralised water (3.1.) containing 1% nitric acid (3.2.)
3.4. Nitric acid solution 5.6 M: introduce 400 ml of nitric acid (3.2.) into a 1000
ml flask; complete to volume with demineralised water (3.1.).
3.5. Sulphuric acid (d = 1.84)
3.6. Sulphuric acid solution 9 M: introduce 200 ml of demineralised water (3.1.)
in a 1000 ml flask, then 500 ml of sulphuric acid (3.5.); after cooling, complete to
volume with demineralised water (3.1.).
3.7. Potassium permanganate KMnO4
3.8. Potassium permanganate solution at 5%: dissolve with demineralised water
(3.1.), 50 g of potassium permanganate (3.7.) in a 1000 ml flask; complete to volume
with demineralised water (3.1.).
3.9. Hydroxylamine hydrochloride NH2OH,HCl
3.10. Reducing solution: weigh 12 g of hydroxylamine hydrochloride (3.9.) and
dissolve in 100 ml of demineralised water (3.1.).
3.11. Tin chloride II (SnCl2,2 H2O)
3.12. Concentrated hydrochloric acid
3.13. Tin (II) chloride solution: weigh 40 g of tin chloride (3.11.) and dissolve in
50 ml of hydrochloric acid (3.12.); complete to 200 ml with demineralised water (3.1.).
3.14 Mercury reference solution at 1 g/l prepared by dissolution of 1.708 g of
Hg(NO3)2.H2O, in 1 l of HNO3 solution at 12% (m/n).
3.15. Mercury calibration solution at 10 mg/l, containing 5 % of nitric acid and
prepared from the reference solution at 1 g/l (3.14).

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 RESOLUTION OENO 18/2003

3.16. Mercury solution at 50 µg/l: place 1 ml of the solution at 10 mg/l (3.14.) in

a 200 ml flask; add 2 ml of nitric acid (3.2.); complete to volume with demineralised
water (3.1.).

4 – APPARATUS
4.1. Glassware:
4.1.1. graduated flasks 100, 200 and 1000 ml (class A)
4.1.2. graduated pipettes 0.5; 1.0; 2.0; 5; 10 and 20 ml (class A)
4.1.3. precautions: before use, the glassware must be washed with nitric acid at
10%, left in contact for 24 hours, then rinsed with demineralised water.
4.2. Mineralisation apparatus (see Compendium of international methods of
analysis of wines and musts)
4.3. Thermostatic heating mantle
4.4. Peristaltic pump
4.5. Cold vapour generator
4.5.1. gas-liquid separator
4.6. Dessicator (hygroscopic membrane) covered by an air current (supplied by a
compressor) and placed before the detector
4.7. Spectrofluorimeter:
4.7.1. mercury vapour lamp, adjusted to the wave length of 254 nm
4.7.2. specific atomic fluorescence detector
4.8. PC:
4.8.1. software that adjusts the parameters of the vapour generator and atomic
fluorescence detector and allows calibration and the analysis of results.
4.8.2. printer that archives results
4.9. Bottle of neutral gas (argon)

5. PREPARATION OF THE SET OF CALIBRATION SOLUTIONS AND SAMPLES

5.1. Set of calibration solutions: 0; 0.25; 0.5 and 1.0 µg/l
Introduce 0; 0.5; 1.0; 2.0 ml of the mercury solution at 50 µg/l (3.15.) in 4 100 ml
flasks; add 1% nitric acid (3.2.); complete to volume with demineralised water (3.1.).
5.2. Samples
Mineralise the samples by wet process The test sample is introduced into the round-
bottomed flask in borosilicate glass placed on a disc with a hole. The neck is inclined.
Add 5 ml of concentrated sulphuric acid (R) and 10 ml of concentrated nitric acid
(R) and gently heat. When the mixture starts to turn brown, add a small quantity of nitric
acid while continuing to heat and so forth until the liquid remains colourless and that the
atmosphere of the flask fills with white smoke of SO3. Allow to cool, take 10 ml of
distilled water and heat again to allow the nitrous fumes to escape until the release of

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 RESOLUTION OENO 18/2003

the white smoke. This operation is repeated; after a third time, boil an instant, cool,
stabilise with several drops (about 10) of potassium permanganate (aqueous sol.) at 5%
(m/m) and add water to the liquid to reach 40 ml.
Filter on filters without cinders. Introduce 10 ml of filtrate into a 50 ml flask. Add
potassium permanganate (3.8.) until persistence of coloration. Solubilise the precipitate
(MnO2) with the reducing solution (3.10.). Complete to volume with demineralised water
(3.1.).
Do a blank test with demineralised water.

6 – PROCEDURE
6.1. Analytical determination
Turn on the fluorimeter; the apparatus is stabilised after 15 minutes.
The peristaltic pump draws up the blank solution (3.3.), the tin chloride (II) solution
(3.13.) and the calibrations or samples (5.1.) or (5.2.).
Check if there is a bubbling in the gas-liquid separator.
Present successively the calibration solutions (5.1.); start the programming of the
vapour generator. The computer software sets up the calibration curve (percentage of
fluorescence depending on the concentration of mercury in µg/l).
Then present the samples (5.2.).
6.2. Self-check
Every five determinations, an analytical blank solution and a calibration are analysed
in order to correct a possible drift of the spectrofluorimeter.

7 – EXPRESSION OF RESULTS
The results are given by the computer software and are expressed in p.p.b. (or µg/l).
The concentration of mercury in oenological products is calculated according to the
test sample and the dilution of the mineralisate. It is expressed in µg/kg.

8 – CONTROL OF RESULTS
The quality control is performed by placing, after the set of calibration solutions and
all five samples, a reference material whose mercury content is known with certainty.
A control card is set up for each reference material used. The control limits are set
at: +/- 2SR intra (SR intra: standard deviation for reproducibility).

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 RESOLUTION OENO 18/2003

10 – BIBLIOGRAPHY
10.1. CAYROL M., BRUN S., 1975. Dosage du mercure dans les vins. Feuillet Vert de
l’O.I.V. n°371.
10.2. REVUELTA D., GOMEZ R., BARDON A., 1976. Dosage du mercure dans le vin
par la méthode des vapeurs froides et spectrométrie d’absorption atomic. Feuillet Vert de
l’O.I.V. n°494.
10.3. CACHO J., CASTELLS J.E., 1989. Determination of mercury in wine by flameless
atomic absorption spectrophotometry. Atomic Spectroscopy, vol. 10, n°3.
10.4. 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.
10.5. SANJUAN J., COSSA D., 1993. Dosage automatique du mercure total dans les
organismes marins par fluorescence atomique. IFREMER, Rapport d’activité.
10.6. AFNOR, 1997. Dosage du mercure total dans les eaux par spectrométrie de
fluorescence atomique. XPT 90-113-2.
10.7. 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.

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 RESOLUTION OENO 18/2003

SEARCH FOR HEAVY METALS

1. Principle of the method

Heavy metals react with the thiol function to form sulphurs. The coloration that
results is compared to a standard.

2. Reagents

2.1 Ammonium acetate,

2.2 Lead nitrate (II),
2.3 Glycerol,
2.4 Methanol,
2.5 Sodium hydroxide, solution at 1 mole NaOH /l,
2.6 Hydrochloric acid at 37%,
2.7 Thioacetamide reagent (R):
2.8 Standard lead solution:
2.8.1 Lead solution at 1000 µg/ml: dissolve 1.598 g of lead nitrate(II) in water and
complete to 1000 ml.
2.8.2 Lead solution at 10 µg/ml. Add 10 ml of the solution 2.8.1 and complete to
1000 ml. To be prepared just before use.

2.9 Buffer solution, pH = 3.5: dissolve 6.25 g of ammonium acetate in 6 ml of

water, add 6.4 ml of hydrochloric acid (2.6) and dilute with water until 25 ml.

3. Procedure

3.1 Test solution: pour 5 ml of buffer solution (2.9), 25.0 g of sample and about 15
ml of water into a 50 ml graduated flask. Complete with water up to the
reference mark.

3.2 Coloured solutions:

3.2.1. Sample solution: mix 12.0 ml of test solution (3.1) and 2.0 ml of buffer
solution (2.9) in a test tube.

3.2.2. Comparative solution: mix 2.0 ml of test solution (3.1), 2.0 ml of buffer
solution (2.9), 0.5 ml of standard lead solution (2.8.2), 4.5 ml of water and
5.0 ml of methanol in a test tube.

3.2.3. Control solution: mix 12.0 ml of test solution (3.1), 2.0 ml of buffer solution
(2.9) and 0.5 ml of standard lead solution (2.8.2) in a test tube.

3.2.4 Comparison of colorations:

add 1.2 ml of thioacetamide reagent (2.7) in the 3 test tubes (3.2.1 to 3), mix
and wait 2 minutes. Compare the coloration vertically in the light of day.

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 RESOLUTION OENO 18/2003

- the sample solution must not be darker than the comparative solution.

- the control solution must not be lighter than the comparative solution.

4. Results:

The conditions described in 3.2.4 are obtained if the heavy metal content is less than 10
mg/l expressed in lead and with a precision of 1 mg/l.

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 RESOLUTION OENO 18/2003

SEARCH FOR SULPHATES

In a 160 × 16 mm test tube, place the volume prescribed of the solution obtained
by the means indicated in each monography; add 1 ml of diluted hydrochloric acid (R);
adjust to 20 ml with water and add 2 ml of barium chloride solution at 10% (R).

Compare the opalescence or any cloudiness to the control sample prepared with 1
ml of solution at 0.100 g of sulphuric acid per litre (i.e. 0.10 mg of H2SO4,) with 1 ml of
diluted hydrochloric acid (R) and water until volume of 20 ml and 2 ml of barium chloride
solution (R). This tube contains 100 µg of H2SO4.

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 RESOLUTION OENO 18/2003

BROMINE INDEX

The bromine index is the quantity of bromine expressed in grammes, that 100 g
of the substance can set.

1. APPARATUS

A graduated flask of 300 to 400 ml with an interior tube welded at the bottom, an
emery stopper and a tube with a handle, compliant with the following diagram

Bromination flask 300 ml in borosilicate glass.

Stopper with ground-glass joints standardised 24/40.

2. SOLUTIONS

2.1 Potassium bromate solution 0.016 M

This solution contains for 1000 ml:

Potassium bromate KBrO3 2.783 g

Weigh exactly 2.783 g of potassium bromate and introduce into a 1000 ml

graduated flask containing about 500 ml of distilled water; shake in order to dissolve and
complete to 20°C with distilled water the volume of 1000 ml of solution. Mix and store in
a flask with a glass stopper.

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 RESOLUTION OENO 18/2003

2.2 Iodine solution 0.05 M

Iodine I 12.69 g
Potassium iodide de KI 18 g
Water q.s.p. 1000 ml
Weigh exactly 12.69 g of iodine, then 18 g of potassium iodide and introduce into
a 1000 ml graduated flask with about 200 ml of distilled water. Allow the dissolution to
operate in cold conditions with the flask being sealed. Add about 500 ml of distilled
water, then shake to absorb the iodine in a vapour state and complete to 20°C with
distilled water, the volume to 1000 ml of solution. Mix and store in a coloured glass flask
with a glass stopper.

2.3 Sodium thiosulphate solution 0.1 M

The 0.1 M sodium thiosulphate solution contains for 1000 ml:

Sodium thiosulphate Na2S2O3.5H2O 24.82 g

Weigh exactly 24.82 g of sodium thiosulphate and introduce into a 1000 ml

graduated flask containing about 600 ml of boiled distilled water. Shake to dissolve and
complete to 20°C with boiled distilled water, the volume to 1000 ml of solution. Mix.
Store away from light. Control the titre of this solution using the 0.05 M iodine solution.

3. TECHNIQUE

Using a tube with a handle, put about 0.50 g of potassium iodide in the recipient
inside the flask; (it is convenient to make a circular mark on the tube corresponding to
the salt’s weight so as not to have to weigh each dosage). Caution has to be taken so as
not to introduce iodide on the external part of the flask. Then introduce the measured
volume of the solution of the product to be measured, dissolved in neutral or alkaline
water, in the external part of the flask, then 25 ml of potassium bromate solution 0.016
M measured with a pipette, and 2 g of pure potassium bromide. Rinse the sides with
water to come to a total volume of about 100 ml, then add 5 ml of concentrated
hydrochloric acid (R); quickly close the flask with the stopper, the joint being humid with
distilled water; by a circular movement homogenise the content and allow to stand the
prescribed time. Shake the flask vigorously so as to put the potassium iodide in contact
with the liquid so as to enable the vapour bromine to react; open the flask while rinsing
the joint and the stopper with a spray of distilled water, and determine iodine using 25
ml of sodium thiosulphate solution 0.1 M; titrate the excess of sodium thiosulphate with
the iodine solution 0.05 M in the presence of starch paste;

Let n be the volume used:

Quantity of bromine (in mg) set by the substance to be dosed = n × 0.008

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 RESOLUTION OENO 18/2003

SEARCH FOR CHLORIDES

In a 160 × 16 mm test tube, place the volume prescribed of the solution obtained
by the means indicated in each monography; add 5 ml of diluted nitric acid (R); complete
to 20 ml and add 0.5 ml of silver nitrate solution at 5% (R).

Compare the opalescence or any cloudiness to the control sample prepared with
0.5 ml of hydrochloric acid at 0.10 g per litre (0.05 mg of HCl) with 5 ml of diluted nitric
acid (R), and adjust to 20 ml with distilled water. Add 0.5 ml of silver nitrate solution at
5% (R). This tube contains 50 µg of HCl.

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 RESOLUTION OENO 18/2003

MINERALISATION METHODS OF SAMPLES BEFORE

DETERMINATION BY ATOMIC ABSORPTION SPECTROMETRY

1. MINERALISATION BY DRY PROCESS

Method applicable for determining the following elements: calcium, magnesium, sodium,
iron, copper, zinc.

1.1 Obtaining cinders

Weigh with precision 5 g of oenological product (or 1 g in the case
of products rich in mineral matters), in a platinum or silice capsule cleaned and tared
beforehand.

Gently burn the sample with the flame of a Bunsen burner under a
hood.
Put the capsule in a muffle oven at 525°C ± 25°C for 12 hours.
Take up the residue with a few ml of demineralised water.
Evaporate water over a water bath at 100°C.
Replace the capsule containing the sample in the oven.
The mineralisation is over when the cinders are white.

1.2 Putting the cinders in a solution

The cinders are solubilised with 2 ml of concentrated hydrochloric
acid (R), bring to volume at 100 ml with demineralised water
Complementary dilutions:
Re-dilute the cinders solution in hydrochloric acid in order to be
compatible with the sensitivity of the apparatus; see separately the method of each
cation.
For the determination of calcium and magnesium, add lanthanum
chloride during this dilution.
Do a blank test.

2. MINERALISATION BY WET PROCESS

Method applicable for determining the following elements: arsenic, cadmium, lead
in oenological products containing water.

2.1 Case of aqueous products

Weigh with precision in a 50 ml polypropylene tube 3 grammes of
pulverised oenological product, add 5 ml of nitric acid at 65%; close with a screw cap;
leave 12 hours at room temperature then after unscrewing the cap place the tube in a
water bath at 90°C for 3 hours under a hood; allow to cool; adjust the volume to 20 ml
with demineralised water; shake; filter on an ashless filter paper (if necessary).
Do a blank test in the same conditions.

2.2 Case of dry products

The mineralisation is similar as for aqueous products but by using a
test sample of 0.5 gramme of oenological product.

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TANTALISATION OF PLATFORMS OF L’Vov IN GRAPHITE

PREPARATION OF TANTALUM SOLUTION AT 6% (m/v) ACCORDING TO THE

ZATKA PROCESS

Three grammes of tantalum powder are put in a 100 ml Teflon  cylindrical vase.
Add 10 ml of hydrofluoric acid diluted to a half, 3 g of dehydrated oxalic acid and 0.5 ml
of hydrogen peroxide at 30 vol.
Heat carefully to dissolve the metal.
Add a few drops of hydrogen peroxide as soon as the reaction slows down; when the
dissolution is complete, add 4 g of oxalic acid and 30 ml of water.
The acid is dissolved and the solution is brought to 50 ml with ultra pure demineralised
water.
Store this solution in a plastic flask.

TREATMENT OF GRAPHITE PLATFORMS

The platform is placed inside the graphite tube or used pyrolytic graphite tube. It is set to
the unit of atomisation of the spectrophotometer.

A volume of 10 µl of tantalum solution is injected on the platform using an automatic

distributor of samples;
Put the tantalum solution in the blank’s position on the sample holder.
The temperature cycle is set according to the following programme:
drying at 100°C for 40 seconds
mineralisation at 900°C for 60 seconds
atomisation at 2600°C for 2.5 seconds
argon is used as an inert gas.

REFERENCE:

Zatka, Anal. Chem., vol 50, n° 3, March 1978.

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MEASURING ARSENIC BY HYDRIDE GENERATION

AND ATOMIC ABSORPTION SPECTROMETRY

1 – FIELD OF APPLICATION
This method applies to the analysis of arsenic in the concentration range of 0 to 200
µg/l with prior mineralisation for oenological products.

2 – DESCRIPTION OF THE TECHNIQUE

2.1. Principle of the method

After reducing arsenic (V) into arsenic (III), arsenic is determined by hydride
generation and atomic absorption spectrometry.

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

The peristaltic pump draws up the borohydride solution, hydrochloric acid solution
and calibration or sample.
The hydride formed in the gas-liquid separator is entrained by a neutral gas (argon).
The gaseous current passes in a dessicator made up of calcium chloride.
The arsenic hydride is analysed in an quartz absorption cell in the flame of a air-
acetylene burner.
The optical path of the hollow-cathode lamp of the atomic absorption spectrometer
passes in the quartz cell.

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 RESOLUTION OENO 18/2003

3 – REAGENTS AND PREPARATION OF REAGENT SOLUTIONS

3.1. Ultra-pure demineralised water
3.2. Ultra-pure nitric acid at 65%
3.3. Potassium iodide KI
3.4. Potassium iodide at 10% (m/v)
3.5. Concentrated hydrochloric acid
3.6. Hydrochloric acid at 10% (m/v)
3.7. Sodium borohydride NaBH4
3.8. Sodium hydroxide NaOH in patches
3.9. Sodium borohydride solution at 0.6% (containing 0.5% of NaOH)
3.10. Calcium chloride CaCl2 (used as a dessicator)
3.11. Silicone antifoam
3.12. Arsenic calibration solution at 1 g/l containing 2% of nitric acid and
prepared from the following acid: H3AsO4½ H20
3.13. Arsenic solution at 10 mg/l: place 1 ml of the calibration solution (3.12.) in
a 100 ml flask; add 1% of nitric acid (3.2.); complete to volume with demineralised
water (3.1.).
3.14. Arsenic solution at 100 µg/l: place 1 ml of the arsenic solution at 10 mg/l
(3.13.) in a 100 ml flask; add 1% of nitric acid (3.2.); complete to volume with
demineralised water (3.1.).

4 – APPARATUS
4.1. Glassware:
4.1.1. graduated flasks 50 and 100 ml (class A)
4.1.2. graduated pipettes 1, 5, 10 and 25 ml (class A)
4.1.3. cylindrical vases 100 ml
4.2. Hot plate with thermostat
4.3. Ashless filter paper
4.4. Atomic absorption spectrophotometer:
4.4.1. air-acetylene burner
4.4.2. hollow-cathode lamp (arsenic)
4.4.3. deuterium lamp
4.5. Accessories:
4.5.1. vapour generator (or gas-liquid separator)
4.5.2. quartz absorption cell placed on the air-acetylene burner
4.5.3. bottle of neutral gas (argon)

5 – PREPARATION OF THE SET OF CALIBRATION SOLUTIONS AND SAMPLES

5.1. Set of calibration solutions 0, 5, 10, 25 µg/l
Place successively 0, 5, 10, 25 ml of the arsenic solution at 100 µg/l (3.14.) in 4, 100
ml flasks; add to each flask 10 ml potassium iodide at 10% (3.4.) and 10 ml of
concentrated hydrochloric acid (3.5.); complete to volume with demineralised water
(3.1.); allow to stand at room temperature for one hour.
5.2. Samples of oenological products
The sample is mineralised by wet process (cf. mineralisation methods of samples
before determination by atomic absorption spectrometry) then filtered. Transfer 10
ml of filtered mineralisate to a 50 ml flask; add 5 ml of potassium iodide at 10%
(3.4.) and 5 ml of concentrated hydrochloric acid (3.5.); add a drop of anti-foam
(3.11.); adjust to volume with demineralised water (3.1.). Allow to stand at room
temperature for one hour. Filter on an ashless filter paper.

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6. PROCEDURE
6.1. Instrumental parameters of the atomic absorption spectrophotometer
(given as an example)
6.1.1. oxidant air-acetylene flame
6.1.2. wave length: 193.7 nm
6.1.3. width of the monochromator’s slit: 1.0 nm
6.1.4. intensity of the hollow-cathode lamp: 7 mA
6.1.5. correction of the non specific absorption with a deuterium lamp

6.2. Analytical determination

The peristaltic pump draws up the reagent solutions (3.6.) and (3.9.) and the
calibrations or samples (5.1.) or (5.2).
Present successively the calibration solutions (5.1.); wait long enough so that the
hydride formed in the gas-liquid separator, passes in the absorption cell; perform an
absorbance reading for 10 seconds; perform two measurements; the spectrometer’s
computer software sets up the calibration curve (absorbance depending on the
concentration of arsenic in µg/l).
Then present the samples (5.2.). Perform two measurements.

6.3. Self-check
Every five determinations, an analytical blank solution and a calibration are analysed
in order to correct a possible deviation of the spectrometer.

7. EXPRESSION OF RESULTS
The results are directly printed by the printer connected to the computer.
The concentration of arsenic in oenological products is expressed in µg/kg while
taking into account the test sample.

8. CONTROL OF RESULTS
The quality control is performed by placing, after the set of calibration solutions and
every five samples, a reference material whose content in arsenic is known with certainty.
A control card is set up for each reference material used. The control limits were set
at: +/- 2SR intra (SR intra : standard deviation of reproductibility).

10. BIBLIOGRAPHY
10.1. PESQUE M., 1982. Dosage de l’arsenic dans le vin. Rapport de stage. Diplôme
d’œnologue. Institut d’œnologie de Bordeaux.
10.2. GAYE J., MEDINA B., 1998. Dosage de l’arsenic dans le vin par spectrométrie
d’absorption atomique. Feuillet Vert de l’O.I.V. n°1069.
10.3. GAYE J., MEDINA B., 1999. Arsenic dans les vins. Feuillet Vert de l’O.I.V.
n°1087.

19
 RESOLUTION OENO 18/2003

DETERMINATION OF CADMIUM BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

The cadmium is determined in solid oenological products after mineralisation by

wet process or directly for liquid oenological products or put in a solution.
The determinations are performed by atomic absorption without a flame (electro-
thermal atomisation in a graphite oven).

2. APPARATUS

2.1 Instrumental parameters (given as an example)

Spectrophotometer equipped with an atomiser with a graphite tube.
wave length: 228.8 nm
hollow-cathode lamp (cadmium)
width of slit: 1 nm
intensity of the lamp: 3 mA
correction of continuum by the Zeeman effect
graphite oven with a tantalised platform
(tantalisation procedure of the platform described above)
adjusting the oven for an analysis:

step temperature time gas flow type of reading

(°C) (s) rate gas of
( / mn signal
1 100 35 3.0 argon no
2 500 10 3.0 argon no
3 500 45 1.5 argon no
4 500 1 0.0 argon no
5 2250 1 0.0 argon yes
6 2250 1 0.0 argon yes
7 2500 2 1.5 argon no
8 1250 10 3.0 argon no
9 75 10 3.0 argon no

2.2 Adjustments of the automatic sampler (given as an example)

volumes injected in µl
solution of Cd blank matrix modifier
at 8 µg/l
blank 0 10 2
calibration N° 1 at 8 µg / l 1 9 2
calibration N° 2 at 16 µg / l 2 8 2
calibration N° 3 at 24 µg / l 3 7 2
calibration N° 4 at 32 µg / l 4 6 2

Sample to be dosed 5 5 2

20
 RESOLUTION OENO 18/2003

3. REAGENTS
Demineralised water
Pure nitric acid for analysis at 65%
Anhydrous palladous chloride (59% in Pd)
Magnesium nitrate with 6 water molecules (ultra pure)
Ammonium dihydrogenophosphate
Matrix modifier: palladous chloride and magnesium nitrate mixture (dissolve 0.25
g of PdCl2 and 0.1 g of Mg(NO3)2.6H2O in 50 ml of demineralised water) or ammonium
dihydrogenophosphate at 6% (dissolve 3 g of NH4H2PO4 in 50 ml of demineralised water).
Cadmium reference solution at 1 g/l, commercial or prepared as follows: dissolve 2.7444
g Cd(NO3)2.4H2O in a solution of HNO3 0.5 M, adjust to 1 l with HNO3 0.5 M.
Cadmium solution at 10 mg/l: place 1 ml of the reference solution in a 100 ml graduated
flask, add 5 ml of pure nitric acid and complete to volume with demineralised water.
Cadmium solution at 0.8 g/l: place 4 ml of the diluted solution in a 50 ml graduated
flask, add 2.5 ml of pure nitric acid and complete to volume with demineralised water.
Calibration range at 0, 8, 16, 24 and 32 µg/l of cadmium.

4. PREPARATION OF SAMPLES

No preparation is necessary for liquid oenological products or in solution form;

solid products are mineralised by wet process.
The blank solution is made up of a pure nitric acid solution for analysis at 1%.

5. PROCEDURE

Each calibration solution is passed right after the blank solution. Perform 2
successive absorbance readings and establish the calibration curve.
Calculate the cadmium content of the samples while taking into account the test
sample of different dilutions.

21
 RESOLUTION OENO 18/2003

DETERMINATION OF CALCIUM BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE
The calcium is directly determined in the liquid oenological product (or in the
mineralisation solution) suitably diluted by atomic absorption spectrometry by air-
acetylene flame after the addition of spectral buffer.

2. APPARATUS
Instrumental parameters (given as an example)
Atomic absorption spectrophotometer
Reducing air-acetylene flame
Hollow-cathode lamp (calcium)
wave length: 422.7 nm
width of slit: 0.2 nm
intensity of the lamp: 5 mA
No correction of non specific absorption.

3. REAGENTS

3.1 demineralised water

3.2 calcium reference solution at 1 g/l, commercial or prepared as follows:
dissolve 5.8919 g of Ca(NO3)2.4H2O in a solution of HNO3 0.5 M, adjust at 1 l with HNO3
0.5 M.
3.3 calcium solution at 100 mg/l:
place 10 ml of the reference solution in a 100 ml graduated flask and 1 ml
of pure nitric acid.
complete to volume with demineralised water
3.4 concentrated hydrochloric acid (R): 35% minimum
3.5 lanthanum solution at 25 g/l:
weigh 65.9 g lanthanum chloride (LaCl3.6H2O) in a 250 ml cylindrical vase,
transfer to a 1000 ml graduated flask with demineralised water; add to the test tube 50
ml of concentrated hydrochloric acid (R); after solubilisation, allow to cool, complete to
volume with demineralised water.
3.6 set of calibration solutions: 0, 2, 4, 6, 8 mg/l of calcium
place successively 0, 1,0, 2,0, 3,0 and 4.0 ml of the solution at 100 mg/l of
calcium in 5, 50 ml graduated flasks, add 10 ml of lanthanum solution at 25 g/l,
complete to volume with demineralised water.

4. PREPARATION OF SAMPLES

4.1 Case of liquid or solution oenological products

In a 50 ml graduated flask place 10 ml of the lanthanum solution and a volume of
sample as after having being completed to volume with demineralised water; the
concentration is below 8 mg/l.

22
 RESOLUTION OENO 18/2003

4.2 Case of solid oenological products

Proceed with mineralisation by dry process;
Put in each solution of the set the same quantity of acid used for putting
cinders in solution or mineralisation (see chapter “Mineralisation”).
Take up cinders and 2 ml of concentrated hydrochloric acid (35% minimum) in a 100 ml
flask; add 20 ml of lanthanum solution at 25 g/l and complete to volume with
demineralised water.
Perform a blank test in the same conditions.

5. PROCEDURE

Pass each solution of the set in ascending order of the concentration of calcium.
For each solution, perform 2 absorbance readings when they are perfectly
stabilised (integration time of signal: 10 seconds).
Pass each sample twice and calculate the calcium content.

23
 RESOLUTION OENO 18/2003

DETERMINATION OF CHROME BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE
The chrome is determined by atomic absorption spectrophotometer without flame.

2. APPARATUS
2.1 Experimental parameters (given as an example)
Atomic absorption spectrophotometer
wave length: 357.9 nm
hollow-cathode lamp (Chrome)
width of slit: 0.2 nm
intensity of the lamp: 7 mA
correction of continuum by the Zeeman effect
introduction in hot conditions of the samples in the graphite oven
measurement of the signal: peak height
time of measurement: 1 second
number of measurements per sample: 2
pyrolytic graphite tube:
pyrolytic graphite oven containing a platform L’Vov tantalised
tantalisation of platform (see above)
inert gas: argon - hydrogen mixture (95%; 5%)
parameters for oven:

step temperature time gas rate type of reading of

(°C) (s) flow gas signal
(l / mn)
1 85 5 3.0 argon + hydrogen no
2 95 40 3.0 argon + hydrogen no
3 120 10 3.0 argon + hydrogen no
4 1000 5 3.0 argon + hydrogen no
5 1000 1 3.0 argon + hydrogen no
6 1000 2 0.0 argon + hydrogen no
7 2600 1.2 0.0 argon + hydrogen yes
8 2600 2 0.0 argon + hydrogen yes
9 2600 2 3.0 argon + hydrogen no
10 75 11 3.0 argon + hydrogen no

24
 RESOLUTION OENO 18/2003

2.2 Adjustments of the automatic sampler

(given as an example)

volumes injected in µl
chrome solution blank matrix modifier
at 50 µg/l
blank 0 17 3
calibration N° 1 at 50 µg/l 5 12 3
calibration N° 2 at 100 µg/l 10 7 3
calibration N° 3 at 150 µg/l 15 2 3

sample to be measured 5 12 3

3. REAGENTS
3.1 pure demineralised water for analysis
3.2 pure nitric acid for analysis at 65%
3.3 anhydrous palladous chloride (59% in Pd)
3.4 pure hexahydrated magnesium nitrate for analysis
3.5 ammonium dihydrogenophosphate
3.6 matrix modifier: mixture of palladium chloride and magnesium nitrate
(dissolve 0.25 g of PdCl2 and 0.1 g of Mg(NO3)2.6H2O in 50 ml of demineralised water)
ammonium dihydrogenophosphate at 6% (dissolve 3 g of NH4H2PO4 in 50 ml of
demineralised water).
3.7 reducing agent: L-ascorbic acid in solution at 1% m/v.
3.8 chrome reference solution at 1 g/l, commercial or prepared as follows:
dissolve 7.6952 g of Cr(NO3)3.9H2O in a solution of HNO3 0.5 M, adjust at 1 l with HNO3
0.5 M
3.9 chrome solution at 10 mg/l: place 1 ml of the reference solution in a 100
ml graduated flask, add 5 ml of nitric acid at 65% and complete to volume with
demineralised water.
3.10 set of calibration solutions: 0, 50, 100 and 150 µg/l of chrome (see
table: adjustments of the automatic sampler).

4. PREPARATION OF SAMPLES

4.1 Case of liquid or solution oenological products

The preparations are performed manually or automatically by the diluter by
following the data from the table “adjustments of the automatic sampler”.

4.2 Case of solid oenological products

Proceed with mineralisation by wet process. Do a blank test.

5. PROCEDURE

Pass each solution of the set in ascending order of the concentration of chrome;
Pass each sample twice and calculate the chrome content while taking into
account the test sample.

25
 RESOLUTION OENO 18/2003

DETERMINATION OF COPPER BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

The copper is determined by atomic absorption spectrometry by flame by using

the method of measured additions.

3. APPARATUS

Instrumental parameters: (given as an example)

Atomic absorption spectrophotometer
flame: oxidant air-acetylene
wave length: 324.7 nm
hollow-cathode lamp (copper)
width of slit: 0.5 nm
intensity of the lamp: 3.5 mA
no correction of non specific absorption.

3. REAGENTS

3.1 pure demineralised water for analysis

3.2 pure nitric acid for analysis at 65%
3.3 reference solution copper at 1 g/l, commercial or prepared as follows:
dissolve 3.8023 g of Cu(NO3)2.3H2O in a solution of HNO3 0.5M, adjust at 1 l with HNO3
0.5M.
3.4 copper solution at 10 mg/l: place 2 ml of the reference copper solution in
a 200 ml graduated flask, add 2 ml of nitric acid at 65% and complete to volume with
demineralised water.

Adjust apparatus using a calibration solution at 0.4 mg/l (2 ml of the copper

solution at 10 mg/l in a 50 ml graduated flask, complete to volume with pure
demineralised water for analysis).

4. PREPARATION OF SAMPLES (METHOD OF MEASURED ADDITIONS)

- Addition of 02 mg/l of copper:

place 5 ml of liquid oenological product or mineralisate of oenological
product obtained by dry process in a flask and add 100 µl of the copper
solution at 10 mg/l
- Addition of 0.4 mg/l of copper:
place 5 ml of liquid oenological product or mineralisate in a flask and add
200 µl of the copper solution at 10 mg/l
- dilution of the sample
Dilution of the sample: the dilution is only necessary if the copper content is more
than 0.5 mg/l of copper.

26
 RESOLUTION OENO 18/2003

5. PROCEDURE

For each sample, pass in order:

- blank solution (demineralised water)
- sample with 0.2 mg/l of copper
- sample with 0.4 mg/l of copper
- sample without addition
the results are obtained automatically or by manual graph.

27
 RESOLUTION OENO 18/2003

DETERMINATION OF IRON BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

The iron is determined by atomic absorption spectrophotometry by flame.

2. APPARATUS
2.1 Instrumental parameters: (given as an example)
atomic absorption spectrophotometry
flame: oxidant air-acetylene
hollow-cathode lamp (iron)
wave length: 248.3 nm
width of slit: 0.2 nm
intensity of the lamp: 5 mA
no correction of non specific absorption.

3. REAGENTS

3.1 pure demineralised water for analysis

3.2 iron solution at 1 g/l, commercial or prepared as follows: dissolve 7.2336 g
of Fe(NO3)2,9H2O in a solution HNO3 0.5 M adjust at 1 l avec HNO3 0.5 M.
3.3 iron solution at 100 mg/l
place 10 ml of the reference iron solution in a 100 ml graduated flask,
complete with demineralised water pure for analysis
3.4 set of calibration solution: 2, 4, 6, 8 mg/l of iron
place successively 1.0, 2.0, 3.0 and 4.0 ml of the solution at 100 mg/l of
iron in 4, 50 ml graduated flasks; complete to volume with pure demineralised water for
analysis

Perform a blank without iron in the same conditions.

4. PREPARATION OF SAMPLES

4.1 Case of liquid or solution oenological products

Each sample is diluted with demineralised water in order to have a
concentration of iron between 0 and 8 mg/l.

4.2 Case of solid oenological products

Proceed with mineralisation by dry process.
Put in each solution of the set of calibration the same quantity of acid used
for putting of cinders in solution; each sample is diluted with demineralised
water in order to have a concentration of iron between 0 and 8 mg/l.

5. PROCEDURE

Pass successively the calibration solutions and the blank which will be
demineralised water or a water-acid solution with concentrations used for samples of
solid oenological products mineralised by dry process and perhaps diluted.

28
 RESOLUTION OENO 18/2003

DETERMINATION OF NICKEL BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

The nickel is directly determined by atomic absorption spectrometry without flame

(electro-thermal atomisation).

2. APPARATUS

2.1 Instrumental parameters: (given as an example)

Atomic absorption spectrophotometer equipped with an atomiser with a
graphite tube.
wave length: 232.0 nm
hollow-cathode lamp (nickel)
width of the slit: 0.2 nm
intensity of the lamp: 4 mA
correction of continuum by the Zeeman effect
introduction in hot conditions of the samples in the graphite oven with an
automatic distributor
rinsing water contains 2 drops of Triton per litre.
measurement of signal: peak height.
Time of measurement: 1 second.
pyrolytic graphite tube:
pyrolytic graphite oven containing a platform of L’Vov tantalised.
tantalisation of a platform: see above.
inert gases: argon and argon + hydrogen mixture (95%: 5%).

parameters for oven:

Parameters for oven for determining nickel
step time gas flow type of gas reading of
temperature rate signal
n° (°C) (s) (l/min)
1 85 5.0 3.0 argon no
2 95 40.0 3.0 argon no
3 120 10.0 3.0 argon no
4 800 5.0 3.0 argon no
5 800 1.0 3.0 argon no
6 800 2.0 0 argon no
7 2 400 1.1 0 argon + hydrogen yes
8 2 400 2.0 0 argon + hydrogen yes
9 2 400 2.0 3.0 argon no
10 75 11.0 3.0 argon no

29
 RESOLUTION OENO 18/2003

2.2 Adjustment of automatic sampler (given as an example)

- Parameters of automatic sampler

volume injected in µl
solution of Ni blank matrix modifier
at 50 µg/l
blank 17 3
calibration 1 5 12 3
calibration 2 10 7 3
calibration 3 15 2 3
sample 5 12 3

3. REAGENTS
3.1 Pure demineralised water for analysis
3.2 Pure nitric acid for analysis at 65%
3.3 Anhydrous palladium chloride (59% in Pd)
3.4 Pure hexahydrated magnesium nitrate for analysis
3.5 Ammonium dihydrogenophosphate
3.6 Matrix modifier: mixture of palladium chloride and magnesium nitrate
(dissolve 0.25 g of PdCl2 and 0.1 g of Mg(NO3)2.6H2O (3.4) in 50 ml of demineralised
water) ammonium dihydrogenophosphate at 6% (dissolve 3 g de NH4H2PO4 in 50 ml of
demineralised water), (3.1).
3.7 L-ascorbic acid
3.8 Analytical blank solution: L-ascorbic acid solution at 1% (m/v).
3.9 Nickel reference solution at 1 g/l (1000 µg/ml) off the shelf or prepared
as follows: dissolve 4.9533 of Ni(NO3)2.6H2O in a solution of HNO3 0.5 M, adjust at 1 l
with HNO3 0.5 M.

4. PROCEDURE
Nickel solution at 10 mg/l: place 1 ml of the reference solution (3.8) in a 100 ml
graduated flask, add 5 ml of nitric acid (3.2); complete to volume with demineralised
water.
Nickel solution at 50 µg/l: place 1 ml of the nickel solution at 10 mg/l in a 200 ml
graduated flask, 10 ml of nitric acid (3.2) and complete with demineralised water.
Set of calibration solution: 0, 50, 100 and 150 µg/l of nickel.
The automatic distributor cycle enables to perform this calibration on the platform
from a nickel solution at 50 µg/l.

5. PREPARATION OF SAMPLES

5.1 Case of liquid or solution samples

No preparation or sample dilution is necessary; the samples are placed
directly in the cups of the automatic injector.

5.2 Case of solid samples

The solid samples are mineralised by dry process.

6. DETERMINATIONS

The calibration graph (absorbance depending on the concentration of nickel) gives

the concentration of nickel in the samples.

30
 RESOLUTION OENO 18/2003

DETERMINATION OF POTASSIUM
BY ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE
The potassium is determined by mineralisation by dry process by atomic
absorption spectrometry.
The addition of a spectral buffer (cesium chloride) to avoid the ionisation of the
potassium is necessary.

2. APPARATUS

2.1 Glassware
100 and 200 ml graduated flasks (class A)
1, 2, 4 and 10 ml graduated pipettes (class A)
100 ml cylindrical vase

2.2 Instrumental parameters (given as an example)

atomic absorption spectrophotometer
oxidant air-acetylene flame (flow rate-air: 3 l/min, flow rate-acetylene: 1.8 l/min.)
Hollow-cathode lamp (potassium)
wave length: 769.9 nm
width of the slit: 0.5 nm
intensity of the lamp: 7 mA
no correction of non specific absorption.

3. REAGENTS

3.1 Pure demineralised water for analysis

3.2 Cesium chloride (CsCl)
3.3 Cesium chloride solution at 5% in cesium: Dissolve 6.330 g of cesium
chloride in 100 ml of demineralised water.
3.4 Potassium reference solution at 1 g/l commercial or prepared as follows:
dissolve 2.5856 g KNO3 in water, adjust to 1 l.
3.5 Diluted potassium solution at 100 mg/l: Place 10 ml of the potassium
reference solution at 1 g/l in a 100 ml graduated flask and 1 ml of pure nitric acid;
complete to volume with pure demineralised water for analysis.
3.6 Set of calibration solution at 0, 2, 4, 6 and 8 mg of potassium per litre:
In a series of 100 ml graduated flasks, introduce 0; 2.0; 4.0; 6.0; 8.0 ml of the
potassium solution at 100 mg/l ; add 2 ml of the cesium chloride solution to all the
graduated flasks; adjust the volume to 100 ml with pure demineralised water for
analysis.
The calibration solutions prepared contain 1 g of cesium per litre.

31
 RESOLUTION OENO 18/2003

4. PREPARATION OF SAMPLES

4.1. Liquid or solution oenological products

In a 50 ml graduated flask, place 1 ml of the cesium chloride solution at 5% and a
volume of a sample as is after having completed to volume with demineralised water; the
concentration of potassium to be measured is below 8 mg/l.

4.2. Solid oenological products

Proceed with mineralisation by dry process (take cinders in 2 ml of
hydrochloric acid in a 100 ml flask, add 2 ml of cesium chloride at 5% and complete to
volume with demineralised water).
Perform a blank test with demineralised water.

5. DETERMINATIONS

Present successively the calibration solutions.

Perform an absorbance reading for 10 seconds; perform two measurements.
Set up the calibration curve (absorbance depending on the concentration in mg/l
of potassium).
Then present the samples, perform an absorbance reading for 10 seconds;
perform two measurements.
Calculate the concentration of potassium in the oenological products in mg/kg.

32
 RESOLUTION OENO 18/2003

DETERMINATION OF LEAD BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

After mineralisation of the sample in an acid medium, the lead is determined by

spectrometry without flame (electro-thermal atomisation).

2. APPARATUS

2.1 Instrumental parameters: (given as an example)

Atomic absorption spectrophotometer equipped with an atomiser with a
graphite tube
wave length: 283.3 nm
hollow-cathode lamp (lead)
width of slit: 0.5 nm
intensity of the lamp: 5 mA
correction of continuum: by Zeeman effect
introduction in hot conditions of the samples in the graphite oven by an
automatic distributor (rinsing water contains 2 drops of Triton per litre)
measurement of signal: peak height
time of measurement: 1 second
number of measurements per sample: 2
pyrolytic graphite tube
pyrolytic graphite oven containing a platform of L’Vov tantalised
(tantalisation of a platform: see above).
parameters for oven

temperature time gas flow type of Reading of

(°C) (s) rate gas signal
(l / min)
150 20.0 3.0 argon no
150 35.0 3.0 argon no
800 15.0 3.0 argon no
800 30.0 3.0 argon no
800 2.0 0.0 argon no
2250 0.8 0.0 argon yes
2250 1.0 0.0 argon yes
2500 1.0 1.5 argon no
1200 9.0 3.0 argon no
75 10.0 3.0 argon no

33
 RESOLUTION OENO 18/2003

2.2 Adjustments of the automatic sampler

(given as an example)

volumes injected in µl
lead solution at 50 blank matrix modifier
µg / l
blank 0 10 2
calibration N° 1 1 9 2
calibration N° 2 2 8 2
calibration N° 3 3 7 2
calibration N° 4 4 6 2
calibration N° 5 6 4 2

Sample to be measured 10 0 2

3. REAGENTS
3.1 Pure demineralised water for analysis
3.2 Pure nitric acid for analysis at 65%
3.3 Ammonium dihydrogenophosphate
3.4 Matrix modifier: ammonium dihydrogenophosphate at 6%.

Introduce 3 g of ammonium dihydrogenophosphate in a 50 ml graduated flask, dissolve

and complete to volume with demineralised water.

Lead reference solution at 1 g/l commercial or prepared as follows: dissolve 1.5985 g of

pure Pb(NO3)2 for analysis in a solution of HNO3 0.5 M, adjust at 1 l avec HNO3 0.5 M.

Lead solution at 10 mg / l: place 1 ml of the reference lead solution at 1 g/l in a 100 ml

graduated flask; add 1 ml of nitric acid at 65% complete to volume with pure
demineralised water for analysis.

Lead solution at 0.1 mg/l: place 1 ml of the lead solution at 10 mg/l in a 100 ml
graduated flask,
add 1 ml of nitric acid at 65%; complete to volume with pure demineralised water
for analysis.

Set of calibration solutions: 0, 50, 100, 150, 200, 300 µg/l of lead.
The automatic distributor cycle allows to directly inject these quantities of
lead on the platform from the lead solution at 0.050 mg/l.

4. PREPARATION OF SAMPLES

The liquid or solution samples must have concentrations between 0 and 300 µg/l
of lead.
The solid samples will be mineralised by wet process (attack by nitric acid).
The blank is made up of pure water for analysis containing 1% of nitric acid at
65%.

5. PROCEDURE

The calibration curve represents the variations of absorbencies depending on the

concentrations enabling to calculate the lead content of the samples.

34
 RESOLUTION OENO 18/2003

DETERMINATION OF SELENIUM BY
ATOMIC ABSORPTION SPECTROMETRY

1. PRINCIPLE

After mineralisation of the sample by wet process, the selenium is determined by

atomic absorption spectrometry without flame (electro-thermal atomisation in the
graphite oven).

2. APPARATUS

2.1 Glassware
Graduated flasks 50, 100 ml (class A)
Graduated pipettes 1, 5 and 10 ml (class A)
Polypropylene tubes 50 ml with screw top.

2.2 Instrumental parameters: (given as an example)

Atomic absorption spectrophotometer equipped with an atomiser
with a graphite tube.
wave length: 196.0 nm
hollow-cathode lamp (selenium)
width of slit: 1.0 nm.
intensity of the lamp: 10 mA
correction of continuum by the Zeeman effect
introduction in hot conditions of the samples in the graphite oven with an
automatic distributor (rinsing water contains 2 drops of Triton
per litre).
measurement of signal: peak height
time of measurement: 1 second
number of measurements per sample: 2
Pyrolytic graphite tube:
Pyrolytic graphite oven containing a platform of L’Vov tantalised.
tantalisation of a platform: see given procedure beforehand.
inert gas: argon.
parameters for oven: table I

Table I - Parameters for oven for determining selenium

step time gas flow type of gas reading of

temperature rate signal
(°C) (s) (l/min)
1 85 5 3.0 argon no
2 95 40 3.0 argon no
3 120 10 3.0 argon no
4 1 000 5 3.0 argon no
5 1 000 1 3.0 argon no
6 1 000 2 0 argon no
7 2 600 0.8 0 argon yes
8 2 600 2 0 argon yes
9 2 600 2 3.0 argon no

35
 RESOLUTION OENO 18/2003

2.3 Automatic sampler parameters (table II)

(given as an example)

Table II - Parameters de automatic sampler.

volumes injected in µl
solution blank matrix modifier
blank 17 3
calibration n°1 50 µg/l 5 12 3
calibration n°2 100 µg/l 10 7 3
calibration n°3 150 µg/l 15 2 3
sample 15 2 3

3. REAGENTS

3.1 Pure demineralised water for analysis

3.2 Pure nitric acid for analysis at 65%
3.3 Anhydrous palladium chloride (59% in Pd)
3.4 Pure hexahydrated magnesium nitrate for analysis
3.5 Ammonium dihydrogenophosphate
3.6 Matrix modifier: mixture of palladium chloride and magnesium nitrate
(dissolve 0.25 g of PdCl2 and 0.1 g of Mg(NO3)2.6H2O in 50 ml of demineralised water)
ammonium dihydrogenophosphate at 6% (dissolve 3 g de NH4H2PO4 in 50 ml of
demineralised water).
3.7 Selenium reference solution at 1 g/l, off the shelf or prepared as follows:
dissolve 1.4052 g SeO2 in a solution of HNO3 0.5 M, adjust at 1 l avec HNO3 0.5 M.
3.8 Selenium solution at 10 mg/l: place 1 ml of the reference solution at 1 g/l
in a 100 ml graduated flask; add 5 ml of nitric acid at 65%; complete to volume with
pure demineralised water for analysis
3.9 Selenium solution at 50 µg/l: place 0.5 ml of the selenium solution at 10
mg/l, 5 ml of nitric acid at 65% in a 100 ml graduated flask; complete to volume
with pure demineralised water for analysis.
3.10 Set of calibration solutions: 0, 50, 100 and 150 µg/l of selenium.
The automatic distributor cycle enables to perform this calibration on the platform
from the selenium solution at 50 µg/l.

4. PREPARATION OF SAMPLES

Weigh with precision a test sample of 1 to 3 g in the graduated tube; add 5 ml of

nitric acid at 65%; close with the screw cap; leave 12 hours at room temperature;
place the tube in a water bath at 90°C for 3 hours (the caps are unscrewed during the
heating); allow to cool; adjust the volume to 20 ml with pure demineralised water for
analysis.

5. DETERMINATIONS

Set up the calibration graph (absorbance depending on the concentration in µg/l

of selenium); determine the concentration of selenium in the samples.
Calculate the concentration of selenium in the mineralisate, then in the sample
in µg/kg.

36
 RESOLUTION OENO 18/2003

DETERMINATION OF SODIUM BY
ABSORPTION ATOMIC SPECTROMETRY

1. PRINCIPLE

The sodium is determined after mineralisation by dry process by atomic

absorption spectrometry.
The addition of a spectral buffer (cesium chloride) to avoid ionisation of sodium is
necessary.

2. APPARATUS

2.1 Glassware
Graduated flasks 50 and 100 ml (class A)
Graduated pipettes 2.0; 5.0; 10.0 ml (class A)
Automatic pipette 1000 µl
Cylindrical vase 100 ml.

2.2 Instrumental parameters: (given as an example)

Atomic absorption spectrophotometer
oxidant air-acetylene flame (rate-air: 3.1 l/mn; rate-acetylene: 1.8 l/mn)
wave length: 589.0 nm
hollow-cathode lamp (sodium)
width of slit: 0.2 nm
intensity of the lamp: 5 mA
no correction of non specific absorption

3. REAGENTS

3.1 Pure demineralised water for analysis

3.2 Pure nitric acid for analysis at 65%
3.3 Cesium chloride solution at 5% in cesium: Dissolve 6.330 g of
cesium chloride in 100 ml of pure demineralised water for analysis.
3.4 Sodium reference solution at 1 g/l commercial or prepared as
follows: dissolve 3.6968 g NaNO3 in water, adjust at 1 l.
3.5 Diluted sodium solution at 10 mg/l:
Place 1 ml of the reference solution at 1 g/l in a 100 ml graduated flask, 1
ml of nitric acid at 65%, complete to volume with pure demineralised water
for analysis.
3.6 Set of calibration solutions 0; 0.25; 0.50; 0.75; 1.00 mg of sodium
per litre:
In a series of 100 ml graduated flasks, place 0; 2.5; 5.0; 7.5; 10 ml of
the diluted sodium solution; in all the graduated flasks add 2 ml of the
cesium chloride solution and adjust the volume at 100 ml with pure
demineralised water for analysis.

The calibration solutions prepared contain 1 g of cesium per litre; they are
stored in polyethylene flasks.

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 RESOLUTION OENO 18/2003

5. PREPARATION OF SAMPLES

4.1. Liquid or solution oenological products

In a 50 ml graduated flask, place 1 ml of the cesium chloride solution at
5% and a volume of sample after having been completed to volume with
demineralised water, the concentration of sodium to be measured is below
at 1 mg/l.

4.2. Solid oenological products

Proceed with a mineralisation by dry process (take up the cinders in 2 ml of
hydrochloric acid in a 100 ml flask, add 2 ml of cesium chloride at 5% and
complete to volume with demineralised water).
Perform a blank test with demineralised water.

5. DETERMINATIONS

Present successively calibration solutions.

Perform an absorbance reading for 10 seconds; perform two measurements.
Set up the calibration curve (absorbance depending on the concentration in mg/l
of sodium).
Then present the samples; determine the concentration of sodium of the diluted
samples in mg/l.
Calculate the concentration of sodium in the oenological products in mg/kg.
The dosages of air-acetylene flame are performed manually.

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 RESOLUTION OENO 18/2003

DETERMINATION OF ZINC BY
ATOMIC ABSORPTION SPECTROMETRY

1 .PRINCIPLE
The zinc is determined directly by atomic absorption spectrometry by flame.

2. APPARATUS ()
Instrumental parameters: (given as an example)
atomic absorption spectrometer
oxidant air-acetylene flame
wave length: 213.9 nm
hollow-cathode lamp (zinc)
width of slit: 0.5 nm
intensity of the lamp: 3.5 mA
correction of the non specific absorption with a deuterium lamp.

3. REAGENTS
3.1 Pure demineralised water for analysis
3.2 Pure nitric acid for analysis at 65%
3.3 Zinc reference solution at 1 g/l commercial or prepared as follows:
dissolve 4.5497 g of Zn(NO3)2. 6H2O in a solution of HNO3 0.5 M, adjust at 1 l with HNO3
0.5 M.
3.4 Zinc solution at 10 mg/l:
place 1 ml of the zinc reference solution in a 100 ml graduated flask, 1 ml of
nitric acid (3.2) and complete to volume with pure demineralised water for analysis.
3.5 Set of calibration solution: 0.2; 0.4; 0.6; 0.8; 1.0 mg/l: place successively
1, 2, 3, 4, 5 ml of the zinc solution at 10 mg/l in 5, 50 ml graduated flasks, complete to
volume with pure demineralised water for analysis.

4. PREPARATION OF SAMPLES
The liquid or solution samples must have concentrations between 0 and 1
mg/l of zinc.
The solid samples are mineralised by dry process.
The blank solution is made up of pure water for analysis containing 1% of
nitric acid at 65%.

5. PROCEDURE
Pass successively the blank, the calibration solutions and the samples of
oenological products.
The absorbency readings are performed for 10 seconds and the measurements
are duplicated.
The concentrations of zinc in the samples are obtained from absorbency values.

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 RESOLUTION OENO 18/2003

ANALYSES OF GAS CONTROL BY GASEOUS CHROMATOGRAPHY

1. PRINCIPLE
The gases are controlled by chromatography in gaseous phase using a
"molecular sieve" type column and detection by catharometer or flame ionisation.

2. SAMPLING
Either use
- a stainless steel flask for sampling gas
- a Teflon sampling bag for gas.

3. INJECTION METHOD

Use of a unheated gas valve with a 250 µl ring.

4. SEPARATION OF LIGHT GASES, H2, O2, N2, CO, CH4.

4.1 Column (for example)

Phase: Molecular sieve Chromosorb 101, Porapak Q
diameter of particles 5µm
granulometry: 80 to 100 mesh
Dimensions: length: 2 m, internal diameter: 2 mm.

4.2 Vector Gas

Helium (He), flow: 3 ml/mn

4.3 Oven temperature: 40°C isotherm

4.4 Detector: Catharometer, Intensity 190 µA

5. SEPARATION OF LIGHT HYDROCARBONS

5.1 Column (for example)

Wide bore

Phase: apolar, diameter of particles: 5 µm

Length: 30 m, internal diameter: 0.53 mm

5.2 Vector gas

Nature: Helium, Flow: 3 ml/mn

Oven temperature 35°C to 200°C rise: 10°C/mn

5.3 Detector: Flame ionisation, temperature 220°C.

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 RESOLUTION OENO 18/2003

AROMATIC POLYCYCLIC HYDROCARBONS

DETERMINATION OF BENZO[a]PYRENE IN OENOLOGICAL CHARBONS BY HPLC

1. PRINCIPLE

Polycyclic aromatic hydrocarbons including benzo[a]pyrene are extracted by

hexane; the solvent is evaporated and the residue is taken up by the methanol-
tetrahydrofuran for analysis by HPLC.

2. APPARATUS AND REAGENTS

2.1 Reagents and calibrations

Acetonitrile for HPLC
Hexane for pesticide residues
Tetrahydrofuran for HPLC (THF)
Deionised and microfiltered water
Benzo[a]pyrene for HPLC.

2.2 Apparatus and chromatographic conditions

octadecyl type HPLC column

fluorimetric detector adjusted to the following detection conditions:
excitation wave length: 300 nm,
emission wave length: 416 nm.

Mobile phase:
solvent A: Deionised and microfiltered water

solvent B: acetonitrile

variations in the composition of the solvent

TIME % solvent A % solvent B

in min
0 50 50
15 20 80
40 0 100
45 50 50

Flow 1.0 ml/mn

2.3 Preparation of reference solutions

Benzo[a]pyrene reference solution at about 100 mg/l in a methanol/THF mixture

(50/50) stored for 3 years maximum in cold conditions.
Daughter solution at about 20 µg/l, prepared extemporaneous (0.5 ml of reference
solution in 50 ml of methanol/THF then 1 ml of this intermediate solution in 50 ml de
methanol/THF).

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 RESOLUTION OENO 18/2003

2.4 Preparation of samples

2 g of oenological charbon are mixed in a 50 ml volumetric flask with 30 ml of

hexane.
The polycyclic aromatic hydrocarbons are extracted for 5 min using a magnetic
stirrer. The organic phase recovered by filtration is gathered in a evaporating flask and
evaporated. The extract is taken up by 2 ml of a methanol/THF mixture (1/1, v/v) and
injected.

3. RESULTS

The benzo[a]pyrene content must not be higher than 1 µg/kg.

REMARK: It is also possible to determine benzo[a]pyrene by chromatography in

gaseous phase by an apolar capillary column with detection by mass
spectrometry.

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 RESOLUTION OENO 18/2003

DETERMINATION OF 5-(HYDROXYMETHYL)FURFURAL

1. PRINCIPLE

The 5-(hydroxymethyl)furfural (HMF) is determined by HPLC (sharing liquid

chromatography in reverse phase).

2. APPARATUS AND SOLUTIONS

2.1 Instrumental parameters (for example)

Chromatograph in liquid phase
UV/visible detector
column: octadecyl type grafted silica (C18), (length: 20 cm; internal
diameter: 4.6 mm; granulometry of phase: 5 µm)
mobile phase: ultra filtered demineralised water - methanol - acetic acid
(80, 10, 3: v/v/v)
flow: 0.5 ml/mn
detection wave length: 280 nm
injected volume: 20 µl

2.2 Preparation of calibration solutions

Solution HMF at 20 mg/l:
In a 100 ml graduated flask, introduce 20 mg of HMF weighed within 0.1
mg and complete to the graduated line with ultra filtered demineralised
water,
introduce 10 ml of this solution in a 100ml graduated flask and complete
with ultra filtered demineralised water;
the solution HMF at 20 mg/l is to be prepared each day.

3. PREPARATION OF SAMPLES

The samples and the calibration solution HMF are injected after filtration on a 0.45
µm membrane.

4. PROCEDURE

The chromatographic column is stabilised with the mobile phase for about 30 min.
Calculate the concentration of HMF of the sample from the peak surfaces.

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 RESOLUTION OENO 18/2003

GRAPE SUGAR:

DETERMINATION OF SACCHAROSE BY HPLC

1. PRINCIPLE

The samples diluted or put in solution are analysed by high performance

liquid chromatography: Separation on column of grafted silica NH2 and detection
using a differential refractometer.

2. APPARATUS AND ANALYTICAL CONDITIONS (for example)

2.1 Chromatograph

- Grafted silica column NH2 (length 20 cm, internal diameter 4 mm

granulometry 5 µm)
- A pumping system
- An auto-sampler (maybe)
- Microfiltres with porosity 0.45 µm
- Differential refractometry detector

2.2 Chromatographic conditions (given as an example)

The water used is deionised and microfiltered.
The acetonitrile is of HPLC quality
The composition of the mobile phase is the following:
- If the column is new: acetonitrile/water (75/25)
- When the fructose - glucose resolution starts to deteriorate, the mobile
phase is then a acetonitrile/water 80/20 mixture.

The flow is 1 ml/min.

3. REAGENTS AND CALIBRATION SOLUTIONS

3.1 Preparation of the reference solution

The chemicals used for the reference solution preparation are of "pure for
analysis" quality.
The composition of this solution is about 10 g/l for each sugar (fructose, glucose
and saccharose).
The reference solution is prepared every two weeks (maximum) and stored in
the refrigerator in the 100 ml graduated flask used for the preparation.

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 RESOLUTION OENO 18/2003

SULPHURIC CINDERS

The sulphuric cinders result from the calcination after being in contact with air
after being attacked by sulphuric acid.

Heat a silica or platinum crucible of low form for 30 min until red; allow to cool in
a vacuum dessicator and tare the crucible. Place the exactly weighed test sample in the
crucible and wet it with a sufficient quantity of concentrated sulphuric acid (R) diluted
beforehand by an equal volume of water. Heat until dry evaporation, then in a muffle
oven, first carefully until red without exceeding the temperature of 600°C ± 25°C.
Maintain calcination until the black particles disappear, allow to cool, add 5 drops of
sulphuric acid diluted to half to the residue, then evaporate and calcinate as previously
until constant weight; weigh after cooling in the desiccator.

Calculate the rate of sulphuric cinders referring to 100 g of substance.

TOTAL CINDERS

The total cinders result from the calcination of the product after contact with air.

Heat a silica or platinum crucible of low form for 30 min until red. Allow to cool in
a vacuum dessicator and tare the crucible. Dispose homogenously the exactly weighed
test sample in the crucible. Desiccate for an hour in the incubator at 100°C-105°C.
Incinerate in the muffle oven, first carefully to avoid that the sample catches fire, then
until red at a temperature of 600°C ± 25°C. Maintain the calcination until the black
particles disappear. For 30 min allow to cool in a vacuum desiccator. Weigh. Continue the
calcination until constant mass.
If the black particles persist, take up the cinders in hot distilled water. Filter these
cinders on an ashless filter paper (porosity 10 µm). Incinerate the filter and residue until
constant mass. Group the new cinders with the filtrate. Evaporate the water. Incinerate
the residue until constant mass.

Calculate the rate of total cinders by referring to 100 g of substance.

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