COEI-2-HMF 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.     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.

COEI-2-CONGAZ 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 cases

4.1.    Column (for example)

Phase: Molecular sieve Chromosorb 101, Porapak Q

• diameter of particles  5 μm

• granulometry: 80 to 100 mesh

Dimensions: length: 2 m, internal diameter: 2 mm.

4.2.    Vector Gas

• Helium (He), flow: 3 ml/mn
  3.     Oven temperature: 40°C isotherm
  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.

COEI-2-AMIBIO Qualitative method for detection of biogenic amines produced by lactic acid bacteria by thin-layer chromatography (TLC)

1. Principle

This method determines the ability to produce biogenic amines (BA) by bacteria in liquid culture media containing the corresponding amino-acid precursor. The method permits the separation and identification of the amines histamine (HIS), tyramine (TYR), putrescine (PUT), cadaverine (CAD) and phenylethylamine (PEA) using thin layer chromatography (TLC).

2. Reagents

2.1.    Amino acids: L-histidine monohydrochloride, L-tyrosine di-sodium salt, L-ornithine hydrochloride, L-lysine monohydrate and L-phenylalanine;

2.2.    Amines: histamine dihydrochloride, tyramine hydrochloride, 1,4-diaminobutane dihydrochloride , 1,5-diaminopentane dihydrochloride, β-phenylethylamine hydrochloride;

2.3.    Dansyl chloride

2.4.    Acetone

2.5.    Chloroform

2.6.    Triethylamine

2.7.    Isopropanol

2.8.    Triethanolamine

2.9.    Thin-layer chromatography (TLC) plates (10 x 20 precoated plates with 0.20 mm silica gel 60 F₂₅₄)

3. Standard solutions

A stock of standard solutions is prepared by dissolving 0.2 g of each amine (HIS, TYR, PUT, CAD and PEA) in 10mL of 40% ethanol. The working standard solution is prepared by mixing 1 ml of each of these solutions and bringing it to a final volume of 10 mL with water.

Amines are converted to their fluorescent dansyl derivatives as follows: one volume of 250 mM Na₂HPO₄, 0.1 volume of 4N NaOH and 2 volumes of dansyl chloride solution (5 mg/mL dansyl chloride in acetone) are added to one volume of the sample. The mixture is homogenized with a Vortex mixer and incubated at 55° C for 1 hour in the dark.

4. Microorganisms and growth conditions

O. oeni strains are cultured in pH 4.8 MRS broth (Merck), supplemented with 10% tomato juice. Strains of the genera Lactobacillus and Pediococcus are cultured in pH 6.3 MRS broth. All the bacteria are incubated at 30° C.

The broths are supplemented with biogenic-amine precursor amino acids such as histidine (5 mg/mL), tyrosine (5 mg/mL), ornithine (5 mg/mL), lysine (5 mg/mL), and phenylalanine (5 mg/mL). Samples are analysed after 9-12 days of growth.

5. TLC conditions

The amines are fractionated on silica gel plates (silica gel 60 F254s). Amine-derivative extracts (10 μl) are applied 2 cm from the base of the plates with capillary pipettes. The dansylated compounds are separated by ascending development for 17 cm in chloroform:triethylamine (4:1). The spots are visualized under UV by using a transilluminator with a system for image acquisition. If a similar instrument is not available, the plate can be sprayed with isopropanol:triethanolamine (8:2) to enhance the fluorescence and visualized under a classical UV source.

The detection limit for the amines TYR, PUT, CAD and PEA is 0.01 mg/ml and the detection limit for HIS is 1 mg/mL. The method showed less sensitivity to HIS, however this detection level in the TLC method described is also adequate to detect HIS production when the bacteria is growing in a culture media supplemented with 5 mg/mL of histidine, as previously described.

6. Analysis of biogenic amines from bacterial cultures

Bacterial strains are grown as described in section 4. After incubation, the broth media are centrifuged and the supernatants are analysed for BA content. Analysis of amines produced by bacterial strains is performed directly on bacterial supernatants as described above.

The separation order of the resulting amine spots from the top to the bottom of the plate are: PEA, TYR, HIS, CAD, PUT.

7. Bibliography

• CostantiniA., CersosimoM., Del Prete V., Garcia-Moruno E. (2006). Production of biogenic amines by lactic acid bacteria: screening by PCR, TLC and HPLC of strains isolated from wine and must. Journal of food protection, 69 (2): 391-396.
• Garcia-MorunoE., Carrascosa A.V., Muñoz R. (2005). A rapid and inexpensive method for the determination of biogenic amines from bacterial cultures by thin-layer chromatography. Journal of food protection, 68 (3): 625-629.
• Garcia-MorunoE. A method for the determination of biogenic amines from bacterial cultures by thin-layer chromatography (TLC), 2007 OIV FV 1243

COEI-2-AZOTOT 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.
  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.

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.

COEI-2-HYDCAR 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.     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

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).

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.

COEI-2-IBROME 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

2. Solutions

2.1.    Potassium bromate solution 0.016 M

This solution contains for 1000 ml:

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.

2.2.    Iodine solution 0.05 M

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:

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

COEI-2-CADMIU 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:

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

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.

COEI-2-CALCIU 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
  4.     concentrated hydrochloric acid (R): 35% minimum
  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.
  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.

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.

COEI-2-CENDRE 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.

COEI-2-CHLORU 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.

COEI-2-CHROME 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:

2.2.    Adjustments of the automatic sampler

(given as an example)

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.

COEI-2-CUIVRE 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.

2. 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.

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.

COEI-2-FER 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.

COEI-2-METAUX 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.
• 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.

COEI-2-MINERA 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.
  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.     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.

COEI-2-NICKEL 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:

2.2.    Adjustment of automatic sampler (given as an example)

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.

COEI-2-PLOMB 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

2.2.    Adjustments of the automatic sampler

(given as an example)

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 modify: 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.

COEI-2-POTASS 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.     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.

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.

COEI-2-SUCSAC 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.     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.

COEI-2-SODIUM 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.     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.

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 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.

COEI-2-SULFAT 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.

COEI-2-TANTAL Tantalisation of platforms of l’Vov in graphite

1. 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.

2. 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.

3. Reference

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

COEI-2-VISCPE Determination of the ability of an enzymatic preparation to interrupt pectic chains by measuring viscosity

1. Principle

Here, it is proposed to measure the quantity of enzyme needed to halve the viscosity of a standard solution with a given pH, temperature and time.

This is a purely technological measurement designed to test the true clarifying efficiency of the enzyme. It essentially measures the pectinase activity, which cannot be directly deduced from the release of galacturonic acid in the medium.

Comment

To measure the enzyme’s activity, there are two possible approaches:

• Either the time it takes a given concentration of the enzyme to halve the viscosity of the pectin solution,
• Or, the concentration of enzyme needed in order for the pectin solution’s viscosity to be halved in a given period of time.

Tests show that, as long as the substrate is not limiting:

• In the first case, the viscosity logarithm (flow time) is inversely proportional to the reaction time and,
• In the second case, the viscosity logarithm is inversely proportional to the quantity of enzyme in the medium.
• In either case, it is easy to find either the time or the quantity of enzyme needed to halve the viscosity on the basis of a judiciously chosen spectrum.

2. Reagent conditions

70 mmol/l phosphate buffer medium 70 mmol/l and 30 mmol/l citrate

Substrate: 70-75 % esterified apple pectin (e.g. Sigma P 8471), diluted to 10 g/l in the buffer solution.

• pH = 3.5
• Temperature: 30 °C
• Reaction time: 15 minutes.
• Pectinase: spectrum of concentrations covering approximately 10 mg/l of enzyme dry weight in the sample; i.e., for example, 0.5 mg in 50 ml of substrate, which corresponds to the quantity of enzyme that is liable to halve the substrate’s viscosity in 15 minutes in the conditions described above.

3. Apparatus

3.1.    Bath or water circulation thermostat (30 °C 1 °C)

3.2.    Capillary flow viscometer (A.3.1: Fig. 2) with a water value (the time for water to flow between the two marks) of approximately 18 to 20 seconds (i.e. a capillary tube roughly 0.5 to 0.6 mm in diameter)

3.3.    Timer

3.4.    Analytical balance (sensitivity 0,001 g)

3.5.    pH meter

3.6.    Magnetic stirrer, conventional laboratory glassware

3.7.    Rapid paper filters

3.8.    Micro-pipettes or micro-syringes for dispensing volumes from 5 to 500 μl

4. Pure products

4.1.    Pure citric acid (99,5 %)

4.2.    Pure disodium hydrogenophosphate (Na₂ HPO₄·2H₂O) (99,0 %)

4.3.    70-75 % esterified apple pectin with more than 90 % purity (e.g. Sigma P 8471)

4.4.    Distilled or deionized water

4.5.    Pure sodium hydroxide (98 %)

4.6.    Pure hydrochloric acid (11.5 M) (33,5 %)

4.7.    Pectinase the activity of which is to be measured.

5. Solutions

Each solution should be homogenised before using

5.1.    M sodium hydroxide

Weigh out 80 g pure sodium hydroxide (4.5) in a 100-ml volumetric flask and dissolve in deionized water (4.4). Top up to the filler mark after complete dissolution and cooling.

5.2.    2M hydrochloric acid

In a 100-ml volumetric flask half-filled with deionized water, place enough pure hydrochloric acid (4.6) to obtain a 2 M solution, (after having topped up to the filler mark).

5.3.    47 mmol/l phosphate buffer, 53 mmol/l citrate, pH 3.5

5.3.1.  Put 800 ml deionized water (4.4) in a 1,000-ml volumetric flask

5.3.2.  Weigh out 11.22 g citric acid (4.1)

5.3.3.  Weigh out 8.30 g pure disodium hydrogenophosphate (Na₂ HPO₄·2H₂O) (4.2)

5.3.4.  Transfer the quantitatively-weighed chemical products to the 1,000 ml volumetric flask, stirring all the time

5.3.5.  Mix until completely dissolved

5.3.6.  Adjust the pH to 3.50 ± 0.05, at ambient temperature, with 2 M sodium hydroxide (5.1) or 2M hydrochloric acid (5.2), depending on the initial pH

5.3.7.  Top up to the filler mark with deionized water (4.4). Mix

Stability: 8 days at ambient temperature.

5.4.    Substrate: Apple pectin (4.3),

5.4.1.  Put a 400-ml cylindrical container into a bath of water with a temperature of 40° C 3° on a rotating stirrer

5.4.2.  Add 250 ml of buffer with a pH of 3.5 (5.3), measured exactly, to the cylindrical container

5.4.3.  Keep stirring gently at 40 °C

5.4.4.  Weigh out 2,500 g ± 0,01 g of pectin (4.3)

5.4.5.  Slowly add the pectin whilst stirring vigorously

5.4.6.  Then stir slowly for 60 minutes, maintaining the temperature at 40° C

5.4.7.  Stop stirring and cool to 30 °C 3 °C

5.4.8.  Filter with rapid filter paper (3.8) if necessary (if lumpy)

Stability: 24 hours at ambient temperature.

5.5.    100 g/l dry weight pectinase solution (4.7)

5.5.1.  Weigh out 2.50 g 0.01 g of powdered or granulated pectinase

5.5.2.  Transfer to a 25-ml volumetric flask

5.5.3.  Top up to the filler mark with buffer solution at pH 3.5 (5.3)

5.5.4.  Dissolve by stirring for 20 minutes using a magnetic stirrer.

Filter through rapid filter paper if the enzyme is immobilised on an insoluble substance using a rapid filter (3.7)

5.5.5.  In the case of a liquid enzymatic preparation, use it directly.

Stability: 4 hours at ambient temperature.

6. Measurements

6.1.    Put the viscometer in the bath of water at 30 °C or use any device that makes it possible to measure the viscosity at 30 °C.

6.2.    Measure the viscosity (the flow time between the two marks on the viscometer) of the buffer solution at pH 3.5; that is, t_o. This time should be approximately 20 seconds for a capillary tube 0.5 to 0.6 mm in diameter.

6.3.    Measure the flow time of the 10 g/l pectin solution, that is, T_p. This time should be approximately 200 seconds or more.

6.4.    Prepare a series of 4 volumetric flasks containing 50 ml of 10 g/l pectin and put them in the bath of water at 30 °C.

6.5.    Add 5 µl of the 100 g/l enzyme solution to the first flask and homogenize.

Then, approximately every 15 minutes, successively add to the other flasks:

15 μl, 35 μl and 100 μl of the 100 g/l enzyme solution and homogenize.

Measure the time taken by the various solutions to flow between the two marks on the viscometer exactly 15 minutes after adding the enzyme.

7. Graphic representation of the measured valued

Deduct the value corresponding to the buffer at pH 3.5 alone from the flow time.

Produce a graph to represent the flow time logarithm as a function of enzyme concentration.

There must be at least three points in a line corresponding to the strongest dilutions. If this is not the case, use a more diluted enzyme solution - 50 g/l or even 10 g/l, for example.

8. Interpretation of the results

Find the regression line equation passing through the three aligned points:

Deduct from this the necessary concentration of enzyme C to halve the pectin solution’s viscosity ; that is, .

9. Examples

9.1.    Determination of the necessary enzyme concentration to halve the viscosity of the pectin solution. (Table 1)

Flow time of the buffer alone t_o = 19.3 s

Corrected time = flow time – flow time of buffer with a pH of 3.5

* value not taken into consideration regression line equation (Fig.1)

Therefore, 0.044 g/l of enzyme are needed to halve the viscosity of a 10 g/l apple pectin solution at 30 °C during 15 minutes.

It has been shown that 1 g/l of enzyme was sufficient to almost totally reduce the viscosity of the pectin solution in 15 minutes.

9.2.    Reduction in the viscosity of a 10 g/l pectin solution as a function of the reaction time at 30 °C of an enzyme with a concentration of 0.1 g/l. (Fig. 2) – For information only

The buffer flow time was 19.6 seconds.

Table 2:

* Corrected flow time

**Values not taken into consideration since the remaining quantity of pectin limits the reaction.

Interpretation of the results

The values in table 4 show that a T/2 reaction time of 13.3 minutes is needed to halve the viscosity of the 10 g/l pectin solution at 30 °C.

For the calculation, on the basis of the regression line in Fig. 3:

10. Bibliography

• Bertrand A. determination de la capacité d'une préparation enzymatique de type polygalacturonase a couper les chaines pectiques par la mesure de la viscosité OIV FV 1260

COEI-2-ZINC 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.
  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.