1990R2676 — EN — 09.03.2005 — 010.001

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This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents

►B	COMMISSION REGULATION (EEC) No 000/90 of 17 September 1990 determining Community methods for the analysis of wines (OJ L 272, 3.10.1990, p.1)

Amended by:

		Official Journal
		No	page	date
►M1	COMMISSION REGULATION (EEC) No 2348/91 of 29 July 1991	L 214	39	2.8.1991
M3	COMMISSION REGULATION (EEC) No 2645/92 of 11 September 1992	L 266	10	12.9.1992
M4	COMMISSION REGULATION (EC) No 60/95 of 16 January 1995	L 11	19	17.1.1995
►M5	COMMISSION REGULATION (EC) No 69/96 of 18 January 1996	L 14	13	19.1.1996
►M6	COMMISSION REGULATION (EC) No 822/97 of 6 May 1997	L 117	10	7.5.1997
►M7	COMMISSION REGULATION (EC) No 761/1999 of 12 April 1999	L 99	4	14.4.1999
►M8	COMMISSION REGULATION (EC) No 1622/2000 of 24 July 2000	L 194	1	31.7.2000
►M10	COMMISSION REGULATION (EC) No 440/2003 of 10 March 2003	L 66	15	11.3.2003
►M11	COMMISSION REGULATION (EC) No 128/2004 of 23 January 2004	L 19	3	27.1.2004
►M12	COMMISSION REGULATION (EC) No 355/2005 of 28 February 2005	L 56	3	2.3.2005

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▼B

COMMISSION REGULATION (EEC) No 000/90

of 17 September 1990

determining Community methods for the analysis of wines

Article 1

1.  The Community methods for the analysis of wine making it possible, in the context of commercial transactions and all control operations, to:

are those set out in the Annex to this Regulation.

2.  For substances for which reference methods and usual methods are prescribed, the results obtained by the use of the reference methods shall prevail.

Article 2

For the purposes of applying this Regulation:

The term ‘single test result’ shall be the value obtained when the standardized test method is applied fully and once to a single sample.

Unless otherwise stated, the probability shall be 95 %.

Article 3

1.  Automated analytical methods shall be acceptable, under the responsibility of the Director of a laboratory on condition that the accuracy, repeatability and reproducibility of the results are at least equivalent to those of the results obtained by the analytical methods listed in the Annex.

Where a dispute arises, the methods listed in the Annex may not be replaced by automated methods.

▼M12 —————

▼B

Article 4

Wherever mention is made of water for solution, dilution, or washing purposes, this shall mean distilled water or demineralized water of equivalent purity. All chemicals shall be of analytical reagent quality except where otherwise specified.

Article 5

Regulation (EEC) No 1108/82 is repealed.

However Article 1 (4) of that Regulation shall apply until 31 December 1990.

Article 6

This Regulation shall enter into force on the day of its publication in the Official Journal of the European Communities.

It shall apply with effect from 1 October 1990.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

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ANNEX

1.   DENSITY AND SPECIFIC GRAVITY AT 20 °C

1.   DEFINITIONS

The density is the mass per unit volume of wine or must at 20 °C. It is expressed in grams per millilitre and denoted by the symbol ρ 20 °C.

The specific gravity at 20 °C (or the 20 °C/20 °C relative density) is the ratio, expressed as a decimal number, of the density of a certain volume of the wine or must at 20 °C to the density of same volume water at the same temperature. It is denoted by the symbol

.

2.   PRINCIPLE OF METHODS

The density and specific gravity at 20 °C are measured on a test sample:

Note:

For very accurate measurement, the density must be corrected for the effect of sulphur dioxide by using the formula:

ρ 20 °C = ρ′ 20 °C − 0,0006 × S

where ρ 20 °C

=

corrected density

ρ′ 20 °C

=

observed density

S

=

total quantity of sulphur dioxide in grams per litre

3.   PRELIMINARY TREATMENT OF SAMPLE

If the wine or must contains appreciable quantities of carbon dioxide, remove most of it by stirring 250 ml of the wine in a 1-litre flask or by filtration under reduced pressure through 2 g of cotton wool placed in an extension tube.

4.   REFERENCE METHOD

4.1.   Apparatus:

Normal laboratory equipment, and in particular:

4.1.1.

A Pyrex pycnometer ( 4 ), of approximately 100 ml capacity, with a removable ground-glass jointed thermometer calibrated in tenths of a degree from 10 to 30 °C (Figure 1). The thermometer must be standardized. Figure 1 The pycnometer and its tare The pycnometer has a side tube 25 mm in length and 1 mm (maximum) in internal diameter, ending in a conical ground joint. This side tube may be capped by a ‘reservoir stopper’ consisting of a conical ground-glass joint tube and terminating in a drawn-out section. This stopper serves as an expansion chamber. The two ground joints of the apparatus should be prepared with great care.

4.1.2.

A tare bottle, consisting of a vessel with the same outside volume (to within at least 1 ml) as the pycnometer and with a mass equal to the mass of the pycnometer filled with a liquid of specific gravity 1,01 (sodium chloride solution 2,0 % m/v). A thermally insulated container, exactly fitting the body of the pycnometer.

4.1.3.

A twin-pan balance with a range of at least 300 g and a sensitivity of 0,1 mg, or a single-pan balance with a range of at least 200 g and a sensitivity of 0,1 mg.

4.2.   Calibration of pycnometer

Calibration of the pycnometer involves a determination of the following quantities:

4.2.1.    Method using a twin-pan balance

Place the tare bottle on the left-hand pan of the balance and the clean and dry pycnometer, fitted with its ‘reservoir stopper’, on the right-hand pan. Add weights to the pan holding the pycnometer and record the weight required to establish equilibrium: let it be p grams.

Carefully fill the pycnometer with distilled water at the ambient temperature and fit the thermometer; carefully wipe the pycnometer dry and place it in the thermally insulated container. Mix it by inverting the container until the temperature reading on the thermometer is constant. Accurately adjust the level to the upper rim of the side tube. Wipe the side tube dry and put the reservoir stopper on; read the temperature t °C carefully, possibly correcting it for the inaccuracy in the temperature scale. Weigh the water-filled pycnometer against the tare and record the weight p′ in grams required to establish equilibrium.

Calculation ( 5 ):

Taring of empty pycnometer:

tare of empty pycnometer = p + m,

where m

=

mass of air contained in the pycnometer,

m

=

0,0012 (p − p′).

Volume at 20 °C:

V 20 °C = (p + m − p′) · Ft ,

where Ft

=

a factor taken from Table I for the temperature t °C.

V 20 °C should be known to within ± 0,001 ml.

Mass of water at 20 °C:

M 20 °C = 0,998203 V 20 °C,

where 0,998203 is the density of water at 20 °C.

4.2.2.   Method using a single-pan balance

Determine:

Calculation (5) :

Taring of empty pycnometer:

tare of empty pycnometer = P − m,

where m

=

mass of air contained in the pycnometer,

m

=

0,0012 (P 1 − P).

Volume at 20 °C:

V 20 °C = [P 1 − (P − m)] × Ft

where Ft

=

a factor taken from Table I for the temperature t °C.

The volume at 20 °C should be known to within ± 0,001 ml.

Mass of water at 20 °C:

M 20 °C = 0,998203 V 20 °C,

where 0,998203 is the density of water at 20 °C.

4.3.   Method of measurement (5) 

4.3.1.   Method using twin-pan balance

Fill the pycnometer with the prepared test sample, following the procedure described in 4.2.1 above.

Let p″ be the weight in grams required to establish equilibrium at t °C.

Mass of liquid contained in the pycnometer = p + m − p″.

Apparent density at t °C:

Calculate the density at 20 °C using one of the correction tables given later, in accordance with the nature of the liquid being measured: dry wine (Table II), natural or concentrated must (Table III), sweet wine (Table IV).

The 20 °C/20 °C specific gravity of the wine is calculated by dividing its density at 20 °C by 0,998203.

4.3.2.   Method using a single-pan balance ( 6 )

Weigh the tare bottle and let its mass be T.

Calculate dT = T 1 − T 0.

Mass of the empty pycnometer at the time of measurement = P − m + dT.

Weigh the pycnometer filled with the prepared test sample, following the procedure described in 4.2.1 above. Let its mass at t °C be P 2.

Mass of liquid contained in pycnometer at t °C = P 2 − (P − m + dT)

Apparent density at t °C:

Calculate the density at 20 °C of the liquid under test (dry wine, natural or concentrated must, or sweet wine) as indicated in 4.3.1 above.

The 20 °C/20 °C specific gravity is calculated by dividing the density at 20 °C by 0,998203.

4.3.3.

The repeatability of the density measurements for dry and semi-sweet wines: r = 0,00010, and for sweet wines: r = 0,00018.

4.3.4.

The reproducibility of the density measurements: for dry and semi-sweet wines: R = 0,00037, and for sweet wines: R = 0,00045.

▼M8 —————

▼B

6.   EXAMPLE OF THE CALCULATION OF THE DENSITY AT 20 °C AND THE 20 °C/20 °C SPECIFIC GRAVITY (REFERENCE METHOD)

6.1.   Pycnometry using a twin-pan balance

6.1.1.   Standardization of the pycnometer:

1.	Weighing of clean dry pycnometer: Tare = pycnometer + p p = 104,9454 g

2.	Weighing of pycnometer filled with water at t °C: Tare = pycnometer + water + p′ p′ = 1,2396 g at t = 20,5 °C

3.	Calculation of mass of air contained in the pycnometer: m = 0,0012 (p − p′) m = 0,0012 (104,9454 − 1,2396) m = 0,1244 g

4.

Characteristic values to be retained: Tare of empty pycnometer, p + m: p+ m = 104,9454 + 0,1244 p + m = 105,0698 g Volume at 20 °C = F(p + m − p′)t°C F 20,5 °C = 1,001900 V 20 °C = (105,0698 − 1,2396) × 1,001900 V 20 °C = 104,0275 ml Mass of water at 20 °C = M 20 °C = V 20 °C · 0,998203 M 20 °C = 103,8405 g

6.1.2.   Determination of the density and specific gravity at 20 °C/20 °C of a dry wine

ρ″ = 1,2622 at 17,80 °C

ρ 17,80 °C = 0,99788 g/ml

Table II enables ρ 20 °C to be calculated from ρ t°C using the relationship:

For t = 17,80 °C and for an alcoholic strength of 11 % vol. c = 0,54.

ρ20 °C = 0,99734 g/ml

6.2.   Pycnometry using a single-pan balance

6.2.1.   Standardization of the pycnometer:

1.	Weight of clean dry pycnometer: P = 67,7913 g

2.	Weight of pycnometer filled with water at t °C: P 1 = 169,2715 at 21,65 °C

3.	Mass of air contained in the pycnometer: m = 0,0012 (P 1 − P) m = 0,0012 × 101,4802 m = 0,1218 g

4.

Characteristic values to be retained: Tare of empty pycnometer, P − m: P− m = 67,7913 − 0,1218 P− m = 67,6695 g Volume at 20 °C = [P 1 − (P − m)]F t°C F 21,65 °C = 1,002140 V 20 °C = 1,002140 (169,2715 − 67,6695) V 20 °C = 101,8194 ml Mass of water at 20 °C: M 20 °C = V 20 °C × 0,998203 M 20 °C = 101,6364 g Mass of tare bottle, T o: T o = 171,9160 g

6.2.2.   Determination of the density and specific gravity at 20° C of a dry wine

T 1 = 171,9178 g

dT = 171,9178 − 171,9160 = 0,0018 g

P − m + dT = 67,6695 + 0,0018 = 67,6713 g

P 2 = 169,2799 at 18 °C

ρ 18 °C = 0,99793 g/ml

Table II enables ρ 20 °C to be calculated from ρ t °C using the relationship:

For t = 18 °C and an alcoholic strength of 11 % vol. c = 0,49.

ρ20 °C = 0,99744 g/ml

TABLE I

F factors by which the mass of water contained in the Pyrex pycnometer at t °C has to be multiplied in order to calculate the pycnometer volume at 20 °C

TABLE II

Temperature corrections c to the density of alcohol-free dry wines measured with a Pyrex glass pycnometer at t °C to relate the result to 20 °C

Note: This table may be used to convert the specific gravity

to the specific gravity

.

TABLE III

Temperature corrections c to the density of natural musts and of concentrated musts measured with a Pyrex glass pycnometer at t °C to relate the result to 20 °C

Note: This table may be used to convert the specific gravity

to the specific gravity

.

TABLE IV

Temperature corrections c to the density of wines of 13 % vol and above containing residual sugar measured with a Pyrex glass pycnometer at t °C to relate the result to 20 °C

TABLE V

Temperature corrections c to the density of dry wines and alcohol-free dry wines measured with an ordinary glass pycnometer or hydrometer at t °C to relate the result to 20 °C

Note: This table may be used to convert the specific gravity

to the specific gravity

.

TABLE VI

Temperature corrections c to the density of natural musts and of concentrated musts measured with a pycnometer or hydrometer of ordinary glass at t °C to relate the result to 20 °C

Note: This table may be used to convert the specific gravity

to the specific gravity

.

TABLE VII

Temperature corrections c to the density of wines of 13 % vol and above containing residual sugar measured with a hydrometer or pycnometer of ordinary glass at t °C to relate the result to 20 °C

2.   EVALUATION BY REFRACTOMETRY OF THE SUGAR CONCENTRATION IN GRAPE MUSTS, CONCENTRATED GRAPE MUSTS AND RECTIFIED CONCENTRATED GRAPE MUSTS

1.   PRINCIPLE OF THE METHOD

The refractive index at 20 °C, expressed either as an absolute value or as a percentage by mass of sucrose, is given in the appropriate table to provide a means of obtaining the sugar concentration in grams per litre and in grams per kilogram for grape musts, concentrated grape musts and rectified concentrated grape musts.

2.   APPARATUS

2.1.   Abbé refractometer

The refractometer used must be fitted with a scale giving:

The refractometer must be equipped with a thermometer having a scale extending at least from + 15 °C to + 25 °C and with an arrangement for circulating water enabling measurements to be made at a temperature of 20 ± 5 °C.

The operating instructions for this instrument must be strictly adhered to, particularly with regard to calibration and the light source.

3.   PREPARATION OF THE SAMPLE

3.1.   Must and concentrated must

Pass the must, if necessary, through a dry gauze folded into four and, after discarding the first drops of the filtrate, carry out the determination on the filtered product.

3.2.   Rectified concentrated must

Depending on the concentration, use either the rectified concentrated must itself or a solution obtained by making up 200 g of rectified concentrated must to 500 g with water, all weighings being carried out accurately.

4.   PROCEDURE

Bring the sample to a temperature close to 20 °C. Place a small test sample on the lower prism of the refractometer, taking care (because the prisms are pressed firmly against each other) that this test sample covers the glass surface uniformly. Carry out the measurement in accordance with the operating instructions of the instrument used.

Read off the percentage by mass of sucrose to within 0,1 % or read the refractive index to four decimal places.

Carry out at least two determinations on the same prepared sample.

Note the temperature t °C.

5.   CALCULATION

5.1.   Temperature correction

5.1.1.

Instruments graduated in percentage by mass of sucrose: use Table I to obtain the temperature correction.

5.1.2.

Instruments graduated in refractive index: find the index measured at t °C in Table II to obtain (column 1) the corresponding value of the percentage by mass of sucrose at t °C. This value is corrected for temperature and expressed as a concentration at 20 °C by means of Table I.

5.2.   Sugar concentration in must and concentrated must

Find the percentage by mass of sucrose at 20 °C in Table II and read from the same row the sugar concentration in grams per litre and grams per kilogram. The sugar concentration is expressed in terms of invert sugar to one decimal place.

5.3.   Sugar concentration in rectified concentrated must

Find the percentage by mass of sucrose at 20 °C in Table III and read from the same row the sugar concentration in grams per litre and grams per kilogram. The sugar concentration is expressed in terms of invert sugar to one decimal place.

If the measurement was made on diluted rectified concentrated must, multiply the result by the dilution factor.

5.4.   Refractive index of must, concentrated must and rectified concentrated must

Find the percentage by mass of sucrose at 20 °C in Table II and read from the same row the refractive index at 20 °C. This index is expressed to four decimal places.

TABLE I

Correction to be made when the percentage by mass of sucrose has been determined at a temperature different from 20 °C

Temperatures must not differ from 20 °C by more than ± 5 °C.

TABLE II

Table giving the sugar concentration ( 7 ) in must and concentrated must in grams per litre and grams per kilogram, determined by means of a refractometer graduated either in percentage by mass of sucrose at 20 °C or in refractive index at 20 °C. The density at 20 °C is also given

TABLE III

Table giving the sugar ( 8 ) concentration in rectified concentrated must in grams per litre and grams per kilogram, determined by means of a refractometer graduated either in percentage by mass of sucrose at 20 °C or in refractive index at 20 °C. The density at 20 °C is also given

3.   ALCOHOLIC STRENGTH BY VOLUME

1.   DEFINITION

The alcoholic strength by volume is the number of litres of ethanol contained in 100 litres of wine, both volumes being measured at a temperature of 20 °C. It is expressed by the symbol ‘% vol’.

Note:

Homologues of ethanol, together with the ethanol and ethanol homologues in ethyl esters, are included in the alcoholic strength since they occur in the distillate.

2.   PRINCIPLE OF METHODS

2.1.	Distillation of wine made alkaline by a suspension of calcium hydroxide. Measurement of the alcoholic strength of the distillate.

▼M12

2.2.	Reference methods: — measurement of the alcoholic strength of the distillate using a pycnometer, — measurement of the alcoholic strength of wines using a hydrostatic balance, — measurement of the alcoholic strength of wines by electronic densimetry using a frequency oscillator.

▼B

2.3.

Usual methods: 2.3.1. Measurement of the alcoholic strength of the distillate with a hydrometer. ▼M11 ————— ▼B 2.3.3. Measurement of the alcoholic strength of the distillate by refractometry. Note: To obtain the alcoholic strength from the density of the distillate, use Tables I, II and III in Appendix II to this section of the Annex. These have been calculated from the International Tables of Alcoholic Strength published in 1972 by the International Legal Metrology Organization in its Recommendation 22 and adopted by the OIV (General Assembly, 1974). Table I gives the general formula relating the alcoholic strength by volume and the density of alcohol-water mixtures as a function of temperature.

3.   METHOD OF OBTAINING DISTILLATE

3.1.   Apparatus

3.1.1.

Distillation apparatus, consisting of: — a round-bottomed 1-litre flask with ground-glass joints, — a rectifying column about 20 cm in height or any apparatus to prevent splashing, — a source of heat; any pyrolysis of extracted matter must be prevented by a suitable arrangement, — a condenser terminated by a drawn-out tube taking the distillate to the bottom of a graduated receiving flask containing several ml of water.

3.1.2.

Steam-distillation apparatus consisting of: 1. a steam-generator; 2. a steam pipe; 3. a rectifying column; 4. a condenser. Any type of distillation or steam-distillation apparatus may be used provided that it satisfies the following test: Distil an ethanol-water mixture with an alcoholic strength of 10 % vol five times in succession. The distillate should have an alcoholic strength of at least 9,9 % vol after the fifth distillation, i.e. the loss of alcohol during each distillation should not be more than 0,02 % vol.

3.2.   Reagents

3.2.1.

A 2 M suspension of calcium hydroxide, obtained by carefully pouring 1 litre of water at 60 to 70 °C on to 120 g of quicklime (CaO).

3.3.   Preparation of sample

Remove the bulk of any carbon dioxide from young and sparkling wines by stirring 250 to 300 ml of the wine in a 500-ml flask.

3.4.   Procedure

Measure out 200 ml of the wine using a graduated flask.

Record the temperature of the wine.

Transfer the wine to the distillation flask and introduce the steam-pipe of the steam-distillation apparatus. Rinse the graduated flask four times with successive 5-ml washings of water added to the flask or the steam-pipe. Add 10 ml of (3.2.1) calcium hydroxide and several pieces of inert porous material (pumice, etc).

Collect the distillate in the 200-ml graduated flask used to measure the wine.

Collect a volume of about three-quarters of the initial volume if distillation is used and a volume of 198 to 199 ml of distillate if steam distillation is used. Make up to 200 ml with distilled water, keeping the distillate at a temperature within 2 °C of the initial temperatures.

Mix with great care, using a circular motion.

Note:

In the case of wines containing particularly large concentrations of ammonium ions, the distillate may be redistilled under the conditions described above, but replacing the suspension of calcium hydroxide with 1 M sulphuric acid diluted to 10 parts in 100 (vol/vol).

▼M12

4.   REFERENCE METHODS

4–A   Measurement of the alcoholic strength of the distillate using a pycnometer

▼B

4.1.   Apparatus

4.1.1.

Use the standardized pycnometer as described in the chapter ‘Density and specific gravity’ (Annex, chapter 1).

4.2.   Procedure

Measure the apparent density of the distillate (3.4) at t °C as described in the chapter ‘Density and specific gravity’ (Annex, chapter 1, sections 4.3.1 and 4.3.2). Let this density be ρt .

4.3.   Expression of results

4.3.1.   Method of calculation

Find the alcoholic strength at 20 °C using Table I. In the horizontal line of this table corresponding to the temperature T (expressed as a whole number) immediately below t °C, find the smallest density greater than ρt . Use the tabular difference just below this density to calculate the density ρ at this temperature T.

On the line of the temperature T, find the density ρ immediately above ρ′ and calculate the difference between the densities ρ and ρ′. Divide this difference by the tabular difference just to the right of the density ρ′. The quotient gives the decimal part of the alcoholic strength, while the whole number part of this strength is shown at the head of the column in which the density ρ′ is located.

An example of the calculation of an alcoholic strength is given in Appendix I to this chapter of the Annex.

This temperature correction has been incorporated in a computer program and might possibly be carried out automatically.

4.3.2.

Repeatability, r: r = 0,10 % vol.

4.3.3.

Reproducibility, R: R = 0,19 % vol.

▼M12

4–B   Measurement of the alcoholic strength of wines using a hydrostatic balance

▼M11

1.   METHOD OF MEASUREMENT

1.1.   Introduction

Alcoholic strength by volume must be measured before marketing, principally to comply with labelling rules.

Alcoholic strength by volume is equal to the number of litres of ethanol contained in 100 litres of wine measured at 20 °C. Referred to as ‘ % vol.’.

1.2.   Object and field of application

The method of measurement described is densimetry using a hydrostatic balance.

For the purposes of the regulatory provisions in force, the trial temperature is set at 20 °C.

1.3.   Principle and definitions

The principle of this method is based on distilling wine volume by volume. The distilling method is described in this Chapter. Distillation eliminates non-volatile substances. Homologues of ethanol, together with the ethanol and ethanol homologues in ethyl esters, are included in the alcoholic strength since they occur in the distillate.

The density of the distillate obtained is then measured. The density of a liquid at a given temperature is equal to the quotient of the mass over its volume: ρ2 = m/V; for wine, it is expressed in g/ml.

The alcoholic strength of wines can be measured by densimetry using a hydrostatic balance based on Archimedes' principle, according to which a body immersed in a liquid receives a vertical upward thrust from the liquid equal to the weight of liquid displaced.

1.4.   Reagents

During the analysis, unless otherwise is stated, use only reagents of recognised analytical grade and water of at least grade 3 as defined in ISO 3696:1987.

1.4.1.   Float-cleaning solution (sodium hydroxide, 30 % w/v)

To prepare 100 ml, weigh 30 g sodium hydroxide and make up to volume using 96 % volume ethanol.

1.5.   Apparatus and equipment

Usual laboratory apparatus and in particular the following:

1.5.1.

Single-pan hydrostatic balance with a sensitivity of 1 mg.

1.5.2.

Float with a volume of at least 20 ml, specially adapted to the balance, suspended with a thread of diameter not exceeding 0,1 mm.

1.5.3.

Measuring cylinder bearing a level mark. The float must be capable of being contained completely within the volume of the cylinder located below the mark; the surface of the liquid may be penetrated only by the supporting thread. The measuring cylinder must have an internal diameter at least 6 mm larger than that of the float.

1.5.4.

Thermometer (or temperature-measuring probe) graduated in degrees and tenths of a degree from 10 °C to 40 °C, calibrated to 0,05 °C.

1.5.5.

Weights, calibrated by a recognised certifying body.

1.6.   Procedure

The float and measuring cylinder must be cleaned between each measurement with distilled water, dried with soft laboratory paper which does not shed fibres and rinsed with the solution whose density is to be determined. Measurements must be made as soon as the apparatus has reached stability so as to restrict alcohol loss by evaporation.

1.6.1.   Calibration of the balance

Although balances usually have an internal calibration system, the hydrostatic balance must be capable of calibration with weights checked by an official certifying body.

1.6.2.   Calibration of the float

1.6.2.1.

Fill the measuring cylinder to the mark with double-distilled water (or water of equivalent purity, e.g. microfiltered water with a conductivity of 18,2 MΩ/cm) at a temperature between 15 °C and 25 °C but preferably at 20 °C.

1.6.2.2.

Immerse the float and the thermometer, stir, read off the density of the liquid from the apparatus and, if necessary, correct the reading so that it is equal to that of the water at measurement temperature.

1.6.3.   Control using a water-alcohol solution

1.6.3.1.

Fill the measuring cylinder to the mark with a water-alcohol mixture of known strength at a temperature between 15 °C and 25 °C but preferably at 20 °C.

1.6.3.2.

Immerse the float and the thermometer, stir, read off the density of the liquid (or the alcoholic strength if this is possible) from the apparatus. The alcoholic strength thus established should be equal to the previously determined alcoholic strength.

Note: This solution of known alcoholic strength can also be used to calibrate the float instead of double-distilled water.

1.6.4.   Measurement of the density of a distillate (or of its alcoholic strength if the apparatus allows)

1.6.4.1.

Pour the test sample into the measuring cylinder up to the graduation mark.

1.6.4.2.

Immerse the float and the thermometer, stir, read off the density of the liquid (or the alcoholic strength if this is possible) from the apparatus. Note the temperature if the density is measured at t °C ρt.

1.6.4.3.

Correct ρt using the table of densities ρt for water-alcohol mixtures (Table II of Annex II to this Chapter).

1.6.5.   Cleaning of float and measuring cylinder

1.6.5.1.

Immerse the float in the float-cleaning solution in the measuring cylinder.

1.6.5.2.

Allow to soak for one hour spinning the float periodically.

1.6.5.3.

Rinse with copious amounts of tap water followed by distilled water.

1.6.5.4.

Dry with soft laboratory paper which does not shed fibres. Carry out this procedure when the float is first used and then regularly as required.

1.6.6.   Result

Using the density ρ20, calculate the real alcoholic strength using the table giving the value of the alcoholic strength by volume ( % vol.) at 20 °C as a function of the density at 20 °C of water-alcohol mixtures, i.e. the international table adopted by the International Legal Metrology Organisation in its Recommendation No 22.

2.   COMPARISON OF MEASUREMENTS MADE USING A HYDROSTATIC BALANCE WITH PROCEDURES OBTAINED USING AN ELECTRONIC DENSIMETER

Using samples with an alcoholic strength between 4 % and 18 % vol., repeatability and reproducibility were measured following an inter-laboratory ring test. The alcoholic strength of different samples as measured using the hydrostatic balance and the electronic densimeter and the repeatability and reproducibility values derived from an extensive multiannual inter-comparison exercise were compared.

2.1.   Samples

Wines of different density and alcoholic strength prepared each month on industrial scale, taken from a properly stored stock of bottles and delivered as anonymous products to the laboratories.

2.2.   Laboratories

Laboratories participating in the monthly ring test organised by the Unione Italiana Vini (Verona, Italy) according to ISO 5725 (UNI 9225) rules and the International Protocol of Proficiency Testing for chemical analysis laboratories established by AOAC, ISO and IUPAC and ISO 43 and ILAC G13 guidelines. An annual report is to be supplied by this company to all participants.

2.3.   Equipment

2.3.1.

Electronic hydrostatic balance (accurate to 5 decimal places), if possible with a data processing device:

2.3.2.

Electronic densimeter, if possible with autosampler.

2.4.   Analysis

According to the rules for the validation of methods, each sample was analysed twice consecutively to determine the alcoholic strength.

2.5.   Result

Table 1 shows the results of the measurements obtained by the laboratories using the hydrostatic balance.

Table 2 shows the results obtained by the laboratories using an electronic densimeter.

2.6.   Evaluations of the results

2.6.1.

The trial results were examined for evidence of individual systematic error (p < 0,025) using Cochran's and Grubb's tests successively, by procedures described in the internationally agreed Protocol for the Design, Conduct and Interpretation of Method-Performance Studies.

2.6.2.

Repeatability (r) and reproducibility (R) Calculations for repeatability (r) and reproducibility (R) as defined by that protocol were carried out on those results remaining after the removal of outliers. When assessing a new method there is often no validated reference or statutory method with which to compare precision criteria, hence it is useful to compare the precision data obtained from a collaborative trial with ‘predicted’ levels of precision. These ‘predicted’ levels are calculated from the Horwitz equation. Comparison of the trial results and the predicted levels give an indication as to whether the method is sufficiently precise for the level of analyte being measured. The Horwitz predicted value is calculated from the Horwitz equation. RSDR = 2(1-0,5 logC) where C = measured concentration of analyte expressed as a decimal (e.g. 1 g/100 g = 0,01). The Horrat value gives a comparison of the actual precision measured with the precision predicted by the Horwitz equation for a method measuring at that particular level of analyte. It is calculated as follows: HoR = RSDR(measured)/RSDR(Horwitz )

2.6.3.

Interlaboratory precision A Horrat value of 1 usually indicates satisfactory inter-laboratory precision, whereas a value of 2 usually indicates unsatisfactory precision, i.e. one that is too variable for most analytical purposes or where the variation obtained is greater than that expected for the type of method employed. Hor is also calculated, and used to assess intra-laboratory precision, using the following approximation: RSDr(Horwitz) = 0,66 RSDR(Horwitz) (this assumes the approximation r = 0,66 R). Table 3 shows the differences between the measurements obtained by laboratories using electronic densimetry and those using a hydrostatic balance. Excluding sample 2000/3, which had very low alcohol strength and for which both techniques show poor reproducibility, a very good agreement is generally observed for all other samples.

2.6.4.

Fidelity parameters Table 4 shows the average overall fidelity parameters computed from all monthly trials carried out from January 1999 until May 2001. In particular: Repeatability (r) = 0,074 ( % vol.) for the hydrostatic balance and 0,061 ( % vol.) for electronic densimetry; Reproducibility (R) = 0,229 ( % vol.) for the hydrostatic balance and 0,174 ( % vol.) for electronic densimetry.

2.7.   Conclusion

The results of determining the alcoholic strength of a wide range of wines show the comparability of measurements carried out with the hydrostatic balance and the electronic density-meter using a frequency oscillator and demonstrate that the values of the validation parameters are similar for both methods.

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4–C   Measurement of the alcoholic strength of wines by electronic densimetry using a frequency oscillator

1.   Method of measurement

1.1.   Title and introduction

The alcoholic strength by volume of wines must be measured before marketing, principally to comply with labelling rules.

Alcoholic strength by volume is defined in paragraph 1 of this chapter.

1.2.   Purpose and scope

The method of measurement described is electronic densimetry using a frequency oscillator.

For the purposes of the regulatory provisions in force, the trial temperature is set at 20 °C.

1.3.   Principle and definitions

The principle of this method is based on distilling wine volume by volume. The distilling method is described in paragraph 3 of this Chapter. Distillation eliminates non-volatile substances. Homologues of ethanol, together with ethanol and ethanol homologues in ethyl esters, are included in the alcoholic strength since they occur in the distillate.

The density of the distillate obtained is then measured. The density of a liquid at a given temperature is equal to the quotient of the mass over its volume:

ρ = m/V, for wine, it is expressed in g/ml.

For a hydroalcoholic solution such as a distillate, where the temperature is known, tables can be used to map density and alcoholic strength. This alcoholic strength corresponds to that of wine (distillation volume by volume).

In this method the density of the distillate is measured by electronic densimetry using a frequency oscillator. The principle consists of measuring the period of oscillation of a tube containing the sample subject to electromagnetic excitation. The density can then be calculated – it is linked to the period of oscillation by the following formula:

(1)

ρ

=

density of the sample

T

=

period of vibration induced

M

=

mass of the empty tube

C

=

spring constant

V

=

volume of the sample in vibration

This link takes the form ρ = A T2 – B (2); hence there is a linear relationship between the density and the period squared. The constants A and B are specific to each oscillator and are estimated by measuring the period of fluids of known density.

1.4.   Reagents and products

1.4.1.   Reference fluids

Two reference fluids are used to adjust the densimeter. The densities of the reference fluids must encompass those of the distillates to be measured. A difference in the density of the reference fluids greater than 0,01000 g/ml is recommended. Their density must be known with a degree of uncertainty below +/- 0,00005 g/ml, at a temperature of 20,00 °C +/- 0,05 °C.

To measure the alcoholic strength by volume using an electronic densimeter, the reference fluids are:

1.4.2.   Cleaning and drying products

1.5.   Apparatus

1.5.1.   Electronic densimeter using a frequency oscillator

The electronic densimeter comprises the following:

The densimeter is placed on a perfectly stable stand that is insulated from all vibrations.

1.5.2.   Controlling the temperature in the measuring cell

The measuring tube is placed in a thermostatic chamber. The temperature stability must be +/- 0,02 °C or better.

Where possible, the temperature of the measuring cell should be checked as this has a strong impact on the measurement results. The density of a hydroalcoholic solution of 10 % vol is 0,98471 g/ml at 20 °C and 0,98447 g/ml at 21 °C, or a difference of 0,00024 g/ml.

The trial temperature is set at 20 °C. The temperature is measured in the cell using a thermometer with a resolution of below 0,01 °C in line with national standards. It should guarantee a temperature measurement with under +/- 0,07 °C uncertainty.

1.5.3.   Calibration of the apparatus

The apparatus must be calibrated before its first use, then every six months or if the check is unsatisfactory. Two reference fluids should be used to calculate the constants A and B (see above formula 2). Calibration should be carried out in line with the operating instructions for the apparatus. In principle, calibration is carried out using dry air (taking into account atmospheric pressure) and extremely pure water (twice distilled and/or micro-filtered with extremely high resistivity > 18 MΩ).

1.5.4.   Checking the calibration

To check the calibration, measure the density of the reference fluids.

The air density should be checked every day. A difference between the theoretical and the actual density greater than 0,00008 g/ml may indicate that the tube is blocked. It should therefore be cleaned. After cleaning, check the air density once again. If this check is not conclusive, the apparatus must be adjusted.

Check the water density also. If the difference between the theoretical and the actual density is greater than 0,00008 g/ml, the apparatus should be adjusted.

If it is difficult to check the temperature of the cell, the density of a hydroalcoholic solution with an alcoholic strength by volume comparable to that of the distillates analysed can be checked directly.

1.5.5.   Checks

If the difference between the theoretical density of a reference solution (known with under 0,00005 g/ml uncertainty) and the measurement is greater than 0,00008 g/ml, the temperature of the cell must be checked.

1.6.   Sampling and preparation of the samples

(see point 3 of this Chapter, ‘Method of obtaining distillate’).

1.7.   Procedure

After obtaining the distillate, measure its density or alcoholic strength by volume using densimetry.

First ensure the temperature stability of the measuring cell. The distillate in the densimeter cell must not contain air bubbles and must be homogeneous. If a lighting system is available which can help verify the absence of bubbles, it should quickly be switched off after carrying out the checks as the heat generated by the bulb affects the measuring temperature.

If the apparatus only gives the period, the density should be calculated using the constants A and B (see 1.3). If the apparatus does not give the alcoholic strength by volume directly, this can be obtained from the tables.

1.8.   Expression of results

The alcoholic strength by volume of the wine is that obtained for the distillate. It is expressed in ‘% vol’.

If the temperature conditions cannot be complied with, correct the result to express it at 20 °C. Give the result to two decimal places.

1.9.   Remarks

The volume placed in the measuring cell must be large enough to avoid any contamination by the previous sample. Consequently, at least two measurements should be carried out. If these do not give results within the repeatability limit, a third measurement is needed. The results of the last two measurements are normally homogeneous and the first value is eliminated.

1.10.   Precision

For samples with an alcoholic strength by volume of between 4 and 18 % vol

2.   Interlaboratory trials. Precision and accuracy on adjuncts

The method performance characteristics shown in paragraph 1.10 were obtained from an interlaboratory test carried out in accordance with established international procedures on six samples and by eleven laboratories.

All the details and repeatability and reproducibility calculations carried out in this test are described in the Chapter ‘TITRE ALCOOMETRIQUE VOLUMIQUE’ (Alcoholic strength by volume) (point 4.B.2) of the OIV’s ‘Recueil International des Méthodes d’Analyse’ (compendium of analysis methods – 2004 edition).

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6.   EXAMPLE OF THE CALCULATION OF THE ALCOHOLIC STRENGTH OF A WINE

6.1.   Measurement by pycnometer on a twin-pan balance

6.1.1.

The constants of the pycnometer have been determined and calculated as described in chapter 1, ‘Density and specific gravity’, section 6.1.1.

6.1.2.

Weighing of pycnometer filled with distillate     Numeric example Tare = pycnometer + distillate at t °C + p″ t °C = 18,90 °C t °C corrected = 18,70 °C p″ = 2,8074 g p + m − p″ = mass of distillate at t °C 105,0698 − 2,8074 = 102,2624 g Apparent density at t °C

6.1.3.

Calculation of alcoholic strength Refer to the table of apparent densities of water-alcohol mixtures at different temperatures, as indicated above On the line 18 °C of the table of apparent densities, the smallest density greater than the observed density of 0,983076 is 0,98398 in column 11 % vol The density at 18 °C is: (98 307,6 + 0,7 × 22) 10−5 = 0,98323 0,98398 − 0,98323 = 0,00075 The decimal portion of the % vol of alcoholic strength is 75/114 = 0,65 The alcoholic strength is 11,65 % vol

6.2.   Measurement by pycnometer on a single pan balance

6.2.1.

The constants of the pycnometer have been determined and calculated as described in chapter 1, ‘Density and specific gravity’, section 6.2.1.

6.2.2.

Weighing of the pycnometer filled with distillate Weight of tare bottle at the time of measurement in grams: T 1 = 171,9178 Pycnometer filled with distillate at 20,50 °C in grams: P 2 = 167,8438 Variation in the buoyancy of air: dT = 171,9178 − 171,9160   = + 0,0018 Mass of the distillate at 20,50 °C: Lt = 167,8438 − (67,6695 + 0,0018)   = 100,1725 Apparent density of the distillate:

6.2.3.

Calculation of alcoholic strength Refer to the table of apparent densities of water-alcohol mixtures at different temperatures, as indicated above On the line 20 °C of the table of apparent densities, the smallest density greater than observed density of 0,983825 is 0,98471 in column 10 % vol The density at 20 °C is: (98 382,5 + 0,5 × 24) 10−5 = 0,983945 0,98471 − 0,983945 = 0,000765 The decimal portion of the % vol of alcoholic strength is 76,5/119 = 0,64 The alcoholic strength is 10,64 % vol

FORMULA FROM WHICH TABLES OF ALCOHOLIC STRENGHTS OF ETHANOL-WATER MIXTURES ARE CALCULATED

The density ρ in kilograms per metre cubed (kg/m3) of an ethanol-water mixture at temperature t in degrees Celsius is given by the formula below as a function of:

The formula is valid for temperatures between − 20 and +40 °C.

Numerical coefficients in the formula

TABLE I

INTERNATIONAL ALCOHOLIC STRENGTH AT 20 °C

Table of apparent densities of ethanol-water mixtures — Pyrex pycnometer

Densities at t °C corrected for air buoyancy

TABLE II

INTERNATIONAL ALCOHOLIC STRENGTH AT 20 °C

Table of corrections to be applied to the apparent alcoholic strength to correct for the effect of temperature

Add or subtract from the apparent alcoholic strength at t °C (ordinary glass alcoholmeter) the correction indicated below

TABLE III

INTERNATIONAL ALCOHOLIC STRENGTH AT 20 °C

Table of apparent densities of ethanol-water mixtures — Ordinary glass apparatus

Densities at t °C corrected for air buoyancy

TABLE IV

Table giving the refractive indices of pure ethanol-water mixtures and distillates at 20 °C and the corresponding alcoholic strengths at 20 °C

4.   TOTAL DRY EXTRACT

Total dry matter

1.   DEFINITION

The total dry extract or the total dry matter includes all matter which is non-volatile under specified physical conditions. These physical conditions must be such that the matter forming the extract undergoes as little alteration as possible while the test is being carried out.

The sugar-free extract is the difference between the total dry extract and the total sugars.

The reduced extract is the difference between the total dry extract and the total sugars in excess of 1 g/l, potassium sulphate in excess of 1 g/l, any mannitol present and any other chemical substances which may have been added to the wine.

The residual extract is the sugar-free extract less the fixed acidity expressed as tartaric acid.

The extract is expressed in grams per litre and it should be determined to within the nearest 0,5 g.

2.   PRINCIPLE OF THE METHOD

Single method: measurement by a densimeter

The total dry extract is calculated indirectly from the specific gravity of the must and, for wine, from the specific gravity of the alcohol-free wine.

This dry extract is expressed in terms of the quantity of sucrose which, when dissolved in water and made up to a volume of one litre, gives a solution of the same specific gravity as the must or the alcohol-free wine. This quantity is shown in Table I.

3.   METHOD OF CALCULATION

The 20/20 specific gravity dr of the ‘alcohol-free wine’ is calculated using the following formula:

d r = d v − d a + 1,000

where:

d v

=

specific gravity of the wine at 20 °C (corrected for volatile acidity) ( 10 ),

d a

=

specific gravity at 20 °C of a water-alcohol mixture of the same alcoholic strength as the wine.

d r may also be calculated from the densities at 20 °C, ρ v of the wine and ρ a of the water-alcohol mixture of the same alcoholic strength by the formula:

d r = 1,0018 (ρv − ρa) + 1,000

where the coefficient 1,0018 approximates to 1 when ρv is below 1,05, which is most often the case.

4.   EXPRESSION OF RESULTS

Table I should be used for calculating the total dry extract in g/l from the 20/20 specific gravity d r of the alcohol-free wine or from the specific gravity of the must.

The total dry extract is expressed in g/l to one decimal place.

TABLE I

for the calculation of the content total dry extract (g/l)

5.   REDUCING SUGARS

1.   DEFINITION

Reducing sugars comprise all the sugars exhibiting ketonic and aldehydic functions and are determined by their reducing action on an alkaline solution of a copper salt.

2.   PRINCIPLE OF THE METHODS

2.1.   Clarification

2.1.1.

Reference method: after neutralization and removal of alcohol, the wine is passed through an ion exchange column in which its anions are exchanged for acetate ions, followed by clarification with neutral lead acetate.

2.1.2.

Usual methods: the wine is treated with one of the following reagents: 2.1.2.1. Neutral lead acetate; 2.1.2.2. Zinc 2-hexacyanoferrate.

2.2.	Determination 2.2.1. Single method: the clarified wine or must is reacted with a specific quantity of an alkaline copper salt solution and the excess copper ions are then determined iodometrically.

3.   CLARIFICATION

The sugar content of the liquid in which sugar is to be determined must lie between 0,5 and 5 g/l.

Dry wines should not be diluted during clarification; sweet wines should be diluted during clarification in order to bring the sugar level to within the limits prescribed in the following table:

3.1.   Reference method

3.1.1.   Reagents

3.1.1.1.

1 M solution hydrochloric acid (HCl);

3.1.1.2.

1 M solution sodium hydroxide (NaOH);

3.1.1.3.

4 M solution acetic acid (CH3COOH);

3.1.1.4.

2 M solution sodium hydroxide (NaOH);

3.1.1.5.

Anion exchange resin (Dowex 3 (20-50 mesh) or equivalent resin). Place a small plug of glass wool and 15 ml of the anion exchange resin (3.1.1.5) in the bottom of the burette. Before the resin is used, subject it to two complete cycles of regeneration by passing alternately the 1 M solutions of hydrochloric acid (3.1.1.1) and sodium hydroxide (3.1.1.2) through it. After rinsing with 50 ml of distilled water, transfer the resin to a beaker, add 50 ml of the 4 M solution of acetic acid (3.1.1.3) and stir for five minutes. Refill the burette with resin and pour 100 ml of the 4 M acetic acid solution (3.1.1.3) through the column. (It is preferable to have a stock of the resin stored in a bottle filled with this 4 M acetic acid solution.) Wash the column with distilled water until the effluent is neutral. Pour 150 ml of a 2 M sodium hydroxide solution through the resin to remove acids and most of the pigments fixed to the resin. Rinse with 100 ml of water, and then pour 100 ml of 4 M acetic acid solution through it. Wash the column until the effluent is neutral.

3.1.1.6.

Neutral lead acetate solution (approximately saturated) Neutral lead acetate [Pb (CH3 COO)2 · 3 H2O], 250 g; very hot water to 500 ml; stir until dissolved.

3.1.1.7.

Calcium carbonate (Ca CO3)

3.1.2.   Procedure

3.1.2.1.   Dry wines

Place 50 ml of the wine in a beaker having a diameter of about 10 to 12 cm together with ½ (n − 0,5) ml of 1 M sodium hydroxide solution (3.1.1.2) (n being the volume of 0,1 M sodium hydroxide solution used for determining total acidity in 10 ml of wine), and evaporate over a boiling water bath in a stream of hot air until the liquid is reduced to about 20 ml.

Pour this liquid through an anion exchange resin column in acetate form (3.1.1.5) at a rate of 3 ml every two minutes. Collect the effluent in a 100 ml volumetric flask. Wash the vessel and column six times using 10 ml of distilled water each time. Stirring all the time, add 2,5 ml of saturated lead acetate solution (3.1.1.6) and 0,5 g of calcium carbonate (3.1.1.7) to the effluent: shake several times and allow to stand for at least 15 minutes. Make up to the mark with water. Filter.

1 ml of this filtrate corresponds to 0,5 ml of wine.

3.1.2.2.   Musts, mistelles, sweet and semi-sweet wines:

The dilutions below are given for guidance.

1.	Musts and mistelles: prepare a 10 % solution of the liquid to be analysed and take 10 ml of the diluted sample.

2.	Sweet wines, whether fortified or not, having a density between 1,005 and 1,038: prepare a 20 % solution of the liquid to be analysed and take 20 ml of the diluted sample.

3.

Semi-sweet wines having a density at 20 °C between 0,997 and 1,005: take 20 ml of the undiluted wine. Allow the abovementioned volume of wine or must to flow through an anion exchange column in acetate form at a rate of 3 ml every two minutes. Collect the effluent in a 100 ml volumetric flask, and rinse the column with water until about 90 ml of the effluent is obtained. Add 0,5 g calcium carbonate and 1 ml saturated lead acetate solution to the effluent. Stir and allow to stand for 15 minutes, stirring occasionally. Make up to the mark with water. Filter. In case: 1. 1 ml of filtrate corresponds to 0,01 ml of must or mistelle. 2. 1 ml of filtrate corresponds to 0,04 ml of sweet wine. 3. 1 ml of filtrate corresponds to 0,20 ml of semi-sweet wine.

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4.   DETERMINATION OF SUGARS

4.1.   Reagents

4.1.1   Alkaline copper salt solution:

Dissolve the copper sulphate in 100 ml of water, the citric acid in 300 ml of water and the sodium carbonate in 300 to 400 ml of hot water. Mix the citric acid and sodium carbonate solutions. Add the copper sulphate solution and make up to one litre.

4.1.2.   30 % potassium iodide solution:

Store in a coloured glass bottle.

4.1.3.   25 % sulphuric acid:

Add the acid slowly to the water, allow to cool and make up to 100 ml with water.

4.1.4.   5 g/l starch solution:

Mix 5 g of starch in with about 500 ml of water. Bring to the boil, stirring all the time, and boil for 10 minutes. Add 200 g of sodium chloride (NaCl). Allow to cool and then make up to one litre with water.

Place the following into a 200 ml volumetric flask:

Heat the flask in a water-bath maintained at 60 °C until the temperature of the solution reaches 50 °C; then keep the flask and solution at 50 °C for 15 minutes. Allow the flask to cool naturally for 30 minutes and then immerse it in a cold water-bath. Transfer the solution to a one-litre volumetric flask and make up to one litre. This solution keeps satisfactorily for a month. When it is to be used, neutralize the test sample (the solution being approximately 0,06 M acid) with sodium hydroxide solution.

4.2.   Procedure

Mix 25 ml of the alkaline copper salt solution, 15 ml water and 10 ml of the clarified solution in a 300 ml conical flask. This volume of sugar solution must not contain more than 60 mg of invert sugar.

Add a few small pieces of pumice stone. Fit a reflux condenser to the flask and bring the mixture to the boil within two minutes. Keep the mixture boiling for exactly 10 minutes.

Cool the flask immediately in cold running water. When completely cool, add 10 ml of 30 % potassium iodide solution (4.1.2), 25 ml of 25 % sulphuric acid (4.1.3) and 2 ml of starch solution (4.1.4).

Titrate with 0,1 M sodium thiosulphate solution (4.1.5) Let n be the number of ml used.

Also carry out a blank titration in which the 10 ml of sugar solution is replaced by 10 ml of distilled water. Let n′ be the number of ml of sodium thiosulphate used.

4.3.   Expression of results

4.3.1.   Calculations

The quantity of sugar, expressed as invert sugar, contained in the test sample is given in the table below as a function of the number (n′ − n) of ml of sodium thiosulphate used.

The sugar content of the wine is to be expressed in grams of invert sugar per litre to one decimal place, account being taken of the dilution made during clarification and of the volume of the test sample.

4.3.2.   Repeatability

r

=

0,015 xi

xi

=

concentration of inverted sugar in g/l per sample

4.3.3.   Reproducibility

R

=

0,058 xi

xi

=

concentration of inverted sugar in g/l per sample

6.   SUCROSE

1.   PRINCIPLE OF METHODS

I.	For qualitative testing by thin-layer chromatography: sucrose is separated from other sugars using thin-layer chromatography on plate coated with cellulose. The developing agent is urea-hydrochloric acid at 105 ° C.

II.

For testing and determination by high-performance liquid chromatography: the sucrose is separated in a column of alkylamine-bonded silica and detected by refractometry. The result is quantified by reference to an external standard analysed under the same conditions. Note: Authentication of a must or of a wine may be checked by the method using NMR of deuterium described for detecting the enrichment of musts, rectified concentrated musts and wines. For testing and determination of sucrose, chromatography in gaseous phase may also be used, as described in chapter 42, point (f).

2.   QUALITATIVE TESTING BY THIN-LAYER CHROMATOGRAPHY

2.1.   Equipment

2.1.1.

Chromatograhic plates covered with a desired thickness of cellulose powder (e.g. MN 300) (20 × 20).

2.1.2.

Chromatography tank.

2.1.3.

Micrometric syringe or micropipette.

2.1.4.

Oven with regulation to 105 ± 2 °C.

2.2.   Reagents

2.2.1.

Decolourizing charcoal.

2.2.2.

Mobile phase: Dichloromethane — glacial acetic acid (p20 — 1,05 g/ml) — ethanol — methanol — water (50:25:9:6:10).

2.2.3.

Developing agent Urea 5 g Hydrochloric acid 2 M 20 ml Ethanol 100 ml

2.2.4.

Reference solutions Glucose 35 g Fructose 35 g Sucrose 0,5 g Distilled water 1 000 ml

2.3.   Procedure

2.3.1.   Preparation of sample

When the must or wine is strongly coloured, decolourize it by treating it with activated charcoal.

For rectified concentrated musts, use the solution with a sugar concentration of 25 % by mass (25 ° Brix) prepared as described in the chapter ‘pH of wine and must’, section 4.1.2, and dilute it with water to a quarter of its concentration by making 25 ml up to 100 ml in a volumetric flask.

2.3.2.   Obtaining the chromatogram

Place on a parallel line 2,5 cm from the bottom edge of the plate:

Place the plate in the tank, previously saturated with the vapour from the mobile phase. Allow the mobile phase to migrate to within 1 cm of the top of the plate. Remove the plate and dry it in a current of warm air. Repeat the migration two more times, drying the plate each time. Spray the plate uniformly with 15 ml of colouring agent and place in the oven at 105 °C for approximately five minutes.

2.4.   Results

Saccharose and fructose appear as a deep blue spot on a white background: glucose gives a less intense green spot.

3.   TESTING AND DETERMINATION BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The chromatographic conditions are given for guidance

3.1.   Equipment

3.1.1.

High-performance liquid chromatograph equipped with: 1. 10 µl loop injector, 2. a detector: a differential refractometer or an interferometer refractometer, 3. an alkylamine-bonded silica column (length 25 cm, internal diameter 4 mm), 4. a guard column filled with the same phase, 5. an arrangement for insulating the guard column and analytical columns or for maintaining their temperature (30 ° C), 6. a recorder and, if required, an integrator, 7. mobile phase flow rate: 1 ml/min.

3.1.2.

Arrangement for membrane filtration (0,45 µm).

3.2.   Reagents

3.2.1.

Doubly distilled water.

3.2.2.

HPLC quality acetonitrile (CH3CN).

3.2.3.

Mobile phase: acetonitrile-water, previously subjected to membrane filtration (0,45 µm), (80:20 v/v). This mobile phase must be outgassed before being used.

3.2.4.

Standard solution: 1,2 g/l aqueous sucrose solution. Filter using a 0,45 µm membrane filter.

3.3.   Procedure

3.3.1.   Preparation of sample:

3.3.2.   Chromatographic determination

Inject in turn into the chromatograph 10 µl of the standard solution and 10 µm of the sample prepared as described in 3.3.1. Repeat these injections in the same order.

Record the chromatogram.

The retention time of the sucrose is approximately 10 minutes.

3.4.   Calculations

For the calculation, use the average of two results for the standard solution and the sample.

3.4.1.

For wines and musts: calculate the concentration in g/l.

3.4.2.

For rectified concentrated musts: let C be the sucrose concentration in g/l of the 40 % (m/v) solution of rectified concentrated must. The sucrose concentration in g/kg of the rectified concentrated must is then: 2,5C.

3.5.   Expression of results

The sucrose concentration in wines, musts and rectified concentrated musts is expressed in grams per litre for wines and musts and in grams per kilogram for rectified concentrated musts, each to one place of decimals.

7.   GLUCOSE AND FRUCTOSE

1.   DEFINITION

Glucose and fructose may be determined individually by an enzymatic method, with the sole aim of calculating the glucose/fructose ratio.

2.   PRINCIPLE OF THE METHOD

Glucose and fructose are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalysed by hexokinase (HK), and produce glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P):

glucose + ATP G6P + ADP

fructose + ATP F6P + ADP

The glucose 6-phosphate is first oxidized to gluconate 6-phosphate by nicotinamide adenine dinucleotide phosphate (NADP) in the presence of the enzyme glucose 6-phosphate dehydrogenase (G6PDH). The quantity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced corresponds to that of glucose 6-phosphate and thus to that of glucose.

G6P + NADP+ gluconate 6-phosphate + NADPH + H+

The reduced nicotinamide adenine dinucleotide phosphate is determined from its absorption at 340 nm.

At the end of this reaction, the fructose 6-phosphate is converted into glucose 6-phosphate by the action of phosphoglucose isomerase (PGI):

F6P G6P

The glucose 6-phosphate again reacts with the nicotinamide adenine dinucleotide phosphate to give gluconate 6-phosphate and reduced nicotinamide adenine dinucleotide phosphate, and the latter is then determined.

3.   APPARATUS

4.   REAGENTS

4.1.

Solution 1: buffer solution (0,3 M triethanolamine, pH 7,6, 4 × 10−3 M in Mg2+): dissolve 11,2 g triethanolamine hydrochloride ((C2H5)3N · HCl) and 0,2 g MgSO4 · 7H2O in 150 ml of doubly distilled water, add about 4 ml of 5 M sodium hydroxide (NaOH) solution to obtain a pH value of 7,6 and make up to 200 ml. This buffer solution may be kept for four weeks at + 4 °C.

4.2.	Solution 2: nicotinamide adenine dinucleotide phosphate solution (about 11,5 × 10−3 M): dissolve 50 mg disodium nicotinamide adenine dinucleotide phosphate in 5 ml of doubly distilled water. This solution may be kept for four weeks at +4 °C.

4.3.	Solution 3: adenosine 5′-triphosphate solution (about 81 × 10−3 M): dissolve 250 mg disodium adenosine 5′-triphosphate and 250 mg sodium hydrogencarbonate (NaHCO3) in 5 ml of doubly distilled water. This solution may be kept for four weeks at +4 °C.

4.4.	Solution 4: hexokinase/glucose 6-phosphate dehydrogenase: mix 0,5 ml hexokinase (2 mg of protein/ml or 280 U/ml) with 0,5 ml glucose 6-phosphate dehydrogenase (1 mg of protein/ml). This mixture may be kept for a year at about +4 °C.

4.5.	Solution 5: phosphoglucose isomerase (2 mg of protein/ml or 700 U/ml). The suspension is used undiluted. This may be kept for a year at about +4 °C. Note: All solutions used above are available commercially.

5.   PROCEDURE

5.1.   Preparation of sample

Depending on the estimated amount of glucose + fructose per litre, dilute the sample as follows:

5.2.   Determination

With the spectrophotometer adjusted to the 340 nm wavelength, make measurements using air (no cell in the optical path) or water as reference.

Temperature between 20 and 25 °C.

Into two cells with 1 cm optical paths, place the following:

Mix, and after about three minutes read off the absorbence of the solutions (A 1). Start the reaction by adding:

Mix; wait 15 minutes; read off the absorbence and check that the reaction has stopped after a further two minutes (A 2).

Add immediately:

Mix; read off the absorbence after 10 minutes and check that the reaction has stopped after a further two minutes (A 3).

Calculate the differences in the absorbences:

A 2 − A 1 corresponding to glucose,

A 3 − A 2 corresponding to fructose,

for the reference and sample cells.

Calculate the differences in absorbence for the reference cell (ΔA R) and the sample cell (ΔA S) and then obtain:

for glucose: ΔA G = ΔA S − ΔA R

for fructose: ΔA F = ΔA S − ΔA R

Note:

The time needed for the completion of enzyme activity may vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch.

5.3.   Expression of results

5.3.1.   Calculation

The general formula for calculating the concentrations is:

where

V

=

volume of the test solution (ml)

ν

=

volume of the sample (ml)

M

=

molecular mass of the substance to be determined

d

=

optical path in the cell (cm)

ε

=

absorption coefficient of NADPH at 340 nm (= 6,3 mM−1 × l × cm−1)

and

V

=

2,92 ml for the determination of glucose

V

=

2,94 ml for the determination of fructose

ν

=

0,20 ml

M

=

180

d

=

1

so that:

For glucose: C (g/l) = 0,417 ΔA G

For fructose: C (g/l) = 0,420 ΔA F

If the sample was diluted during its preparation, multiply the result by the dilution factor F.

Note:

If the measurements are made at 334 or 365 nm, then the following expressions are obtained:

5.3.2.   Repeatability (r)

r = 0,056 xi

5.3.3.   Reproducibility (R)

R

=

0,12 + 0,076 xi

xi

=

concentration of glucose or fructose in g/l.

8.   DETECTING ENRICHMENT OF GRAPE MUSTS, CONCENTRATED GRAPE MUSTS, RECTIFIED CONCENTRATED GRAPE MUSTS AND WINES BY APPLICATION OF NUCLEAR MAGNETIC RESONANCE OF DEUTERIUM (SNIF-NMR/RMN-FINS)

1.   DEFINITION

The deuterium contained in the sugars and the water in grape must will be redistributed after fermentation in molecules I, II, III and IV of the wine:

The addition of exogenous sugar (sugaring in the dry) before the must ferments will have an effect on the distribution of the deuterium.

As compared with the figures for parameters for a natural control wine from the same region, the enrichment of an exogenous sugar will lead to the following variations:

(D/H)I

:

Isotope ratio associated with molecule I

(D/H)II

:

Isotope ratio associated with molecule II

:

Isotope ratio of the water in the wine.

R

=

2(D/H)II/(D/H)I expresses the relative distribution of deuterium in molecules I and II; R is measured directly from the h-intensities of the signals and then R = 3hII/hI.

(D/H)I mainly characterizes the vegetable species which synthesized the sugar and to a lesser extent the geographical location of the place of harvest (type of water used during photosynthesis).

(D/H)II represents the climatology of the place of production of the grapes (type of rainwater and weather conditions) and to a lesser extent the sugar concentration of the original must.

represents the climatology of the place of production and the sugar content of the original must.

2.   PRINCIPLE

The parameters defined above (R, (D/H)I, (D/H)II) are determined by nuclear magnetic resonance of the deuterium in the ethanol extracted from the wine or from the fermentation products of the must, the concentrated must or the rectified concentrated must obtained under given conditions; they may be supplemented by determining the isotope ratio of the water extracted from the wine,

and by determining the ratio 13C/ 12C in the ethanol.

▼M1 —————

▼B

3.   PREPARATION OF THE SAMPLE FOR ANALYSIS

3.1.   Extraction of ethanol and water in the wine

Note:

Any method for ethanol extraction can be used as long as 98 to 98,5 % of the total alcohol in the wine is recovered in a distillate which contains 92 to 93 % mas (95 % vol).

3.1.1.   Apparatus and reagents

Apparatus for extracting ethanol (Figure 1) comprising:

Reagents for the determination of water by the Karl Fischer method (e.g. Merck 9241 and 9243).

3.1.2.   Procedure

3.1.2.1.

Determine the alcoholic strength of the wine (t v) to better than the nearest 0,05 % vol.

3.1.2.2.

Extraction of the ethanol Introduce a homogeneous sample of 500 ml of wine of alcoholic strength t v into the flask in the distillation apparatus with a constant reflux ratio of about 0,9. Place a 125 ml ground conical flask, calibrated beforehand, to receive the distillate. Collect the boiling liquid between 78,0 and 78,2 °C, i.e. approximately 40 to 60 ml. If the temperature exceeds 78,5 °C, discontinue collection for five minutes. When the temperature returns to 78 °C, recommence collecting the distillate until 78,5 °C and repeat this operation until the temperature, after discontinuing collection and operating within a closed circuit, remains constant. Complete distillation lasts approximately five hours. This procedure enables between 98 and 98,5 % of the total alcohol in the wine to be recovered in a distillate with a strength of between 92 and 93 % mas (95 % vol), a strength for which the NMR conditions described in section 4 have been established. The ethanol collected is weighed. A homogeneous 60 ml sample of the residues is kept in a 60 ml flask and represents the water in the wine. Its isotope ratio may be determined if required. If a spectrometer fitted with a 10 mm probe is available (cf. section 4), a homogeneous test sample of 300 ml of wine is sufficient.

3.1.2.3.

Determination of the alcoholic strength of the alcohol extracted The water content (p′ g) is determined by the Karl Fischer method using a sample of about 0,5 ml of alcohol of exactly known mass p. Figure 1 Distillation apparatus for the extraction of ethanol The strength by mass of the alcohol is given by

3.2.   Fermentation of musts, concentrated musts and rectified concentrated musts

3.2.1.   Apparatus and reagents

If the isotope ratio of the must is known the yeast can be reactivated prior to use for 15 minutes in a minimum amount of lukewarm non-distilled water, so that the isotope ratio is similar to that of the must.

If the isotope ratio of the must is not known it is better to use fresh/direct.

Fermentation vessel of a capacity of 1,5 litres fitted with a device to keep it airtight and to condense alcohol vapour, since no loss of ethanol during fermentation must be tolerated. The rate of conversion of fermentable sugars into ethanol should be greater than 98 %.

3.2.2.   Procedure

3.2.2.1.   Musts

3.2.2.2.   Concentrated musts

Place V ml of concentrated must containing a known amount of sugar (approximately 170 g) into the fermentation vessel. Top up to one litre with (1 000 − V) ml of water from the normal water supply of same isotope ratio as natural must samples. Add (3.2.1) dry yeasts (1 g) and 3 g of DIFCO Bacto Yeast Nitrogen Base without amino acids. Homogenize and proceed as before.

3.2.2.3.   Rectified concentrated musts

Proceed as described in 3.2.2.2, topping up to one litre with (1 000 − V) ml of water from the normal water supply of same isotope ratio but also containing 3 g dissolved tartaric acid.

Retain 50 ml of sample of must or sulphur dioxide treated must or concentrated must or rectified concentrated must with a view to the possible extraction of the water and the determination of its isotope ratio

. The extraction of the water contained in the must may be very simply carried out by azotropic distillation using toluene.

3.3.   Preparation of alcohol sample for NMR measurement

3.3.1.   Reagents

N, N-tetramethyl urea (TMU); use a sample of standard TMU with a given, monitored isotope ratio D/H. This sample may be supplied by:

3.3.2.   Procedure

3.4.   Preparation of a water sample for the NMR measurement, for the purpose of a possible determination of its isotope ratio

3.4.1.   Reagents

N, N-tetramethyl urea (TMU): see 3.3.1.

3.4.2.   Procedure

Place 3 ml of water obtained as in 3.1.2 or 3.2 (note) into a tared flask and weigh to the nearest 0,1 mg (m′E). Place 4 ml of internal standard (TMU) and weigh to the nearest 0,1 mg (m′st). Homogenize by shaking.

Note:

If the laboratory has a mass spectrometer for determining isotope ratios, the measurement may be carried out on this instrument to reduce the load on the NMR spectrometer. It is necessary to standardize the ratio Trv (5.2) for each series of wines examined.

4.   RECORDING OF 2H NMR SPECTRA OF THE ALCOHOL AND THE WATER

Determination of isotope parameters.

4.1.   Apparatus

4.2.   Standardization of spectrometer and checks

4.2.1.   Standardization

Carry out customary standardization for homogeneity and sensitivity according to the manufacturer's specifications.

4.2.2.   Checking the validity of the standardization

Use standard ethanols, designated by the letters C, V and B, having isotope concentrations that are different but accurately standardized. They carry the following meaning:

These samples are supplied by the Community Bureau of References.

Following the procedure described in 4.3, determine the isotope values of these alcohols, denoting them Cmeas, Vmeas, Bmeas (see 5.3).

Compare them with the given corresponding standard values, denoted by a superscript Cst, Bst, Vst (see 5.3).

Figure 2a

2H NMR spectrum of an ethanol from wine with an internal standard (TMU: N, N-tetramethylurea)>

Figure 2b

2H spectrum of ethanol taken under the same conditions as those of Figure 2a, but without exponential multiplication (LB = 0)

The standard deviation for repeatability obtained on an average of 10 repetitions of each spectrum must be less than 0,01 for the ratio R and less than 0,3 ppm for (D/H)I and (D/H)II.

The average values obtained for the various isotopic parameters (R, (D/H)I, (D/H)II) must be within the corresponding standard deviation of repeatability given for those parameters for the three standard alcohols by the Community Bureau of References. If they are not, carry out the checks again.

4.3.   Conditions for obtaining NMR spectra

Place a sample of alcohol prepared as in 3.3 (or the water sample, prepared as in 3.4) in a 15-mm or 10-mm tube and introduce it into the probe.

The conditions for obtaining NMR spectra are as follows:

For each spectrum, carry out a number of accumulations NS sufficient to obtain the signal-to-noise ratio given in 4.1 and repeat this set of NS accumulations NE = 10 times. The values of NS depend on the types of spectrometer and probe used (cf. section 4). Examples of the possible choices are:

5.   EXPRESSION OF RESULTS

5.1.   Ethanol

For each of the 10 spectra (see NMR spectrum for ethanol, Figure 2a), determine:

with

The use of peak heights instead of peak area, which is less precise, supposes that peak width at half height is identical and is a reasonable approximation if applicable (Figure 2b).

5.2.   Water

When the isotope ratio of water is determined by NMR from the water-TMU mixture, the following relationship is used:

with

5.3.

For each of the isotope parameters, calculate the average of 10 determinations and the confidence interval. Optional software (e.g. SNIF-NMR) suitable for the spectrometer computer enables such calculations to be carried out on-line. Note: If, after standardization of the spectrometer, there is a systematic difference between the average values obtained for the characteristic isotopes of the standard alcohols (4.2.2) and the values indicated by the Community Bureau of References, to within the standard deviation, the following corrections may be applied to obtain the true value for any sample X. The interpolation will be made by taking the values for the standard sample which straddle that of the sample X. Let be the measured value and be the corrected value. This will give: = + α [ − ] where Example: Standard samples supplied and standardized by the Community Bureau of References: = 102,0 ppm = 91,95 ppm Standard samples measured by the laboratory: = 102,8 ppm = 93,0 ppmSuspect uncorrected sample: = 100,2 ppmα = 1,0255 and = 99,3 ppm are calculated.

6.   INTERPRETATION OF RESULTS

Compare the value R X obtained for the R ratio of the suspect sample with the ratios obtained for the control wines. If R X differs by more than two standard deviations from the average R T value obtained for the control wine, adulteration may be assumed.

6.1.   Addition of beet sugar, cane sugar or maize glucose

6.1.1.   Wines

R X higher than R T: beet sugar is assumed to have been added.

R X less than R T: cane sugar or maize sugar is assumed to have been added.

Note that

and

are increased.Consider

:

6.1.2.   Musts, concentrated musts and rectified concentrated masts

The values of the isotopic parameters for the alcohol extracted as described in 3.1 from the fermented product obtained (3.2) from must, concentrated must and rectified concentrated must are examined according to the instructions given in 6 under ‘Interpretation of results’ (6.1.1) and compared with the alcohol extracted from the fermentation product of musts.

The enrichment, E % vol, expresses the volume of alcohol added to the fermented product. Knowing the dilution that may have been carried out prior to fermentation (concentrated musts and rectified concentrated musts), assuming that 16,83 g of sugar yield 1 % vol of alcohol, calculate the amount of sugar (mass) added per litre of must, concentrated must or rectified concentrated must.

6.2.   Addition of a mixture of beet sugar and cane sugar or maize glucose

The isotope ratios (D/H)I and R are changed less than when only one type of sugar is added.

(D/H)II is higher, as is

.

These additions may be confirmed by determining the 13C/12C ratio of the ethanol by mass spectrometry; in that case the ratio is higher.

9.   ASH CONTENT

1.   DEFINITION

The ash content is defined to be all those products remaining after igniting the residue left from evaporation of the wine. The ignition is carried out in such a way that all the cations (excluding the ammonium cation) are converted into carbonates or other anhydrous inorganic salts.

2.   PRINCIPLE OF THE METHOD

The wine extract is ignited at a temperature between 500 and 550 °C until complete combustion (oxidation) of organic material has been achieved.

3.   APPARATUS

3.1.	boiling water-bath;

3.2.	balance sensitive to 0,1 mg;

3.3.	hot-plate or infra-red evaporator;

3.4.	temperature-controlled electric muffle furnace;

3.5.	desiccator;

3.6.	flat-bottomed platinum dish 70 mm in diameter and 25 mm in height.

4.   PROCEDURE

Pipette 20 ml of wine into the previously tared platinum dish (original weight P° g). Evaporate on the boiling water-bath, and heat the residue on the hot-plate at 200 °C or under the infra-red evaporator until carbonization begins. When no more fumes are produced, place the dish in the electric muffle furnace maintained at 525 ± 25 °C. After 15 minutes of carbonization, remove the dish from the furnace, add 5 ml of distilled water, evaporate on the water-bath or under the infra-red evaporator, and again heat the residue to 525 °C for 10 minutes.

If combustion (oxidation) of the carbonized particles is not complete, repeat the operations of washing the carbonized particles, evaporation of water and ignition.

For wines with a high sugar content, it is advantageous to add a few drops of pure vegetable oil to the extract before the first ashing to prevent excessive foaming.

After cooling in the desiccator, the dish is weighed (P 1 g).

The weight of the ash in the sample (20 ml) is then P = (P 1 − P o) g.

5.   EXPRESSION OF RESULTS

5.1.	Method of calculation The weight P of the ash in grams per litre will be given to two decimal places by the expression: P = 50p

10.   ALKALINITY OF THE ASH

1.   DEFINITION

The alkalinity of the ash is defined as the sum of cations, other than the ammonium ion, combined with the organic acids in the wine.

2.   PRINCIPLE OF THE METHOD

The ash is dissolved in a known (excess) amount of a hot standardized acid solution; the excess is determined by titration using methyl orange as an indicator.

3.   REAGENTS AND APPARATUS

3.1.	0,05 M sulphuric acid solution (H2SO4);

3.2.	0,1 M sodium hydroxide solution (NaOH);

3.3.	methyl orange, 0,1 % solution in distilled water;

3.4.	boiling water-bath.

4.   PROCEDURE

Add 10 ml of the 0,05 M sulphuric acid solution (3.1) to the ash from 20 ml of wine contained in the platinum dish. Place the dish on the boiling water-bath for about 15 minutes, breaking up and agitating the residue with a glass rod to speed up the dissolution. Add two drops of methyl orange solution and titrate the excess sulphuric acid against 0,1 M sodium hydroxide (3.2) until the colour of the indicator changes to yellow.

5.   EXPRESSION OF RESULTS

Method of calculation

The alkalinity of the ash, expressed in milliequivalents per litre to one decimal place, is given by

A = 5 (10 − n)

where n ml is the volume of 0,1 M sodium hydroxide used.

11.   CHLORIDES

1.   PRINCIPLE

Chlorides are determined directly in the wine by potentiometry using an Ag/AgCl electrode.

2.   APPARATUS

2.1.	pH/mV meter graduated at intervals of at least 2 mV.

2.2.	Magnetic stirrer.

2.3.	Ag/AcCl electrode with a saturated solution of nitrate potassium as electrolyte.

2.4.	Microburette graduated in 1/100 ml.

2.5.	Chronometer.

3.   REAGENTS

3.1.	Standard chloride solution: 2,1027 g of potassium chloride, KCl (max. 0,005 % Br), dried before use by leaving in a desiccator for several days, are diluted in distilled water and made up to one litre. 1 ml of this solution contains 1 mg Cl−.

3.2.	Silver nitrate titrating solution: 4,7912 g of analytical grade silver nitrate, AgNO3 are diluted in a 10 % (v/v) alcohol solution and made up to one litre. 1 ml of this solution corresponds to 1 mg Cl−.

3.3.	Nitric acid, of at least 65 % purity ( ρ20 = 1,40 g/ml).

4.   PROCEDURE

4.1.

5,0 ml of standard chloride solution are measured into a 150 ml cylindrical vessel placed on a magnetic stirrer, diluted with distilled water to approximately 100 ml and acidified with 1,0 ml of nitric acid (at least 65 %). After immersing the electrode, titrate by adding the silver nitrate titrating solution with the microburette, with moderate stirring. Begin by adding 1,00 ml for the first 4 ml and read the corresponding millivolt values. Add the next 2 ml in fractions of 0,20 ml. Finally, continue the addition in fractions of 1 ml until a total of 10 ml has been added. After each addition, wait for approximately 30 seconds before reading the corresponding millivolts. Transfer the values thus obtained onto graph paper against the corresponding millilitres of titrating solution and determine the potential of the equivalence point on the basis of the singular point on the curve obtained.

4.2.

5 ml of the standard chloride solution are measured into a 150 ml cylindrical vessel with 95 ml of distilled water and 1 ml of nitric acid (at least 65 %). Immerse the electrode and titre, whilst stirring, until the potential of the equivalence point is obtained. This determination is repeated until a good degree of agreement in the results is obtained. This check must be carried out before each series of measurements of chlorides in the samples.

4.3.	50 ml of wine for analysis are measured into a 150 ml cylindrical vessel. Add 50 ml of distilled water and 1 ml of nitric acid (at least 65 %) and titrate using the procedure described in 4.2.

5.   EXPRESSION OF RESULTS

5.1.   Calculations

If n represents the number of millilitres of silver nitrate titrating solution, the chloride content in the tested liquid is:

5.2.   Repeatability (r):

r

=

1,2 mg Cl per litre

r

=

0,03 meg per litre

r

=

2,0 mg NaCl per litre

5.3.   Reproducibility (R):

R

=

4,1 mg Cl per litre

R

=

0,12 meg per litre

R

=

6,8 mg NaCl per litre

6.

Note: For very precise determination. Refer to the complete titration curve obtained during determination of the test liquid with the silver nitrate solution. (a) Measure 50 ml of the wine to be analysed into a 150 ml cylindrical vessel. Add 50 ml of distilled water and 1 ml of nitric acid (at least 65 %). Titrate using the silver nitrate solution, adding 0,5 ml at a time and recording the corresponding potential in millivolts. Derive from this first titration the approximate volume of silver nitrate solution required. (b) Recommence determination in the same conditions. Begin by adding 0,5 ml of titrating solution at a time until the volume added is 1,5 to 2 ml less that the volume determined in (a). Hereafter add 0,2 ml at a time. Continue to add the solution beyond the approximately located equivalence point in a symmetrical manner, i.e. by adding 0,2 ml and then 0,5 ml at a time. The end point of the measurement and the exact volume of silver nitrate consumed are obtained: — either by drawing the curve and determining the equivalence point, — or by the following calculation: —  — Where: —   V = volume of titrating solution at equivalence point; V′ = volume of titrating solution before the largest potential change; Δ Vi = constant volume of the increments of titrating solution, i.e. 0,2 ml; ΔΔ E1 = second difference in potential before the largest potential change; ΔΔ E2 = second difference in potential after the largest potential change. Example: Volume of AgNO3 titrating solution E potential in mV Difference Δ E Second difference ΔΔ E 0 204         4   0,2 208   0     4   0,4 212   2     6   0,6 218   0     6   0,8 224   0     6   1,0 230   2     8   1,2 238   4     12   1,4 250   10     22   1,6 272   22     44   1,8 316   10     34   2,0 350   8     26   2,2 376   6     20   2,4 396     In this example, the end point of the titration is between 1,6 and 1,8 ml: the largest potential change (Δ E = 44 mV) occurs in this interval. The volume of silver nitrate titrating solution consumed to measure the chlorides in the test sample is:

12.   SULPHATES

1.   PRINCIPLE

1.1.   Reference method

Precipitation of barium sulphate and weighing. The barium phosphate precipitated in the same conditions is eliminated by washing the precipitate in hydrochloric acid.

In the case of musts or wine rich in sulphur dioxide, prior de-sulphiting by boiling in an airtight vessel is recommended.

1.2.   Quick test method

Wines are classified into several categories using the so-called limits method, based on the precipitation of barium sulphate using a barium ion titrant.

2.   REFERENCE METHOD

2.1.   Reagents

2.1.1.

2 M solution of hydrochloric acid.

2.1.2.

Barium chloride solution of 200 g/l of BaCl2 · 2H2O.

2.2.   Procedure

2.2.1.   General procedure:

Measure 40 ml of the analysis sample into a 50 ml centrifuge tube; add 2 ml of 2 M hydrochloric acid and 2 ml of barium chloride solution at 200 g/l. Stir with a glass stirrer; rinse the stirrer with a little distilled water and leave to stand for five minutes. Centrifuge for five minutes, then carefully decant the supernatant liquid.

Next wash the barium sulphate precipitate as follows: add 10 ml of 2 M hydrochloric acid, place the precipitate in suspension and centrifuge for five minutes, then carefully decant the supernatant liquid. Repeat the washing procedure twice in the same conditions using 15 ml distilled water each time.

Quantitatively transfer the precipitate, by rinsing with distilled water, into a tared platinum capsule and place over a water bath at 100 °C until fully evaporated. The dried precipitate is calcined several times briefly over a flame until a white residue is obtained. Leave to cool in a desiccator and weigh.

Let m = the mass in milligrams of barium sulphate obtained.

2.2.2.

Special procedure: sulphited must and wine with a high sulphur dioxide content. Beforehand, eliminate the sulphur dioxide. Measure 25 ml of water and 1 ml of pure hydrochloric acid ( ρ 20 = 1,15 to 1,18 g/ml) into a 500 ml conical flask equipped with a dropping funnel and an outlet tube. Boil the solution to remove the air and introduce 100 ml of wine through the dropping funnel. Continue boiling until the volume of liquid in the flask has been reduced to about 75 ml and quantitatively transfer it, after cooling, to a 100 ml volumetric flask. Make up to mark with water. Determine the sulphates in a 40 ml sample as indicated in 2.2.1.

2.3.   Expression of results

2.3.1.   Calculations:

The sulphate content, expressed in milligrams per litre of potassium sulphate, K2SO4 is:

18,67 × m

The sulphate content in musts or wine is expressed in milligrams per litre of potassium sulphate, with no decimal point.

2.3.2.   Repeatability

up to 1 000 mg/l: r = 27 mg/l

about 1 500 mg/l: r = 41 mg/l

2.3.3.   Reproducibility

up to 1 000 mg/l: R = 51 mg/l

about 1 500 mg/l: R = 81 mg/l

▼M8 —————

▼B

13.   TOTAL ACIDITY

1.   DEFINITION

The total acidity of the wine is the sum of its titratable acidities when it is titrated to pH 7 against a standard alkaline solution.

Carbon dioxide is not included in the total acidity.

2.   PRINCIPLE OF THE METHOD

Potentiometric titration or titration with bromothymol blue as an indicator and comparison with an end-point colour standard.

3.   REAGENTS

3.1.	Buffer solution pH 7,0: —  monopotassium phosphate, (KH2PO4) … 107,3 g —  1 M sodium hydroxide (NaOH) solution … 500 ml —  water to … 1 000 ml Alternatively, ready made buffer solutions are available commercially.

3.2.	0,1 M sodium hydroxide (NaOH) solution.

3.3.	4 g/l bromothymol blue indicator solution: —  bromothymol blue (C27H28Br2O5S) … 4 g —  neutral ethanol, 96 % vol … 200 ml Dissolve and add: —  water free of CO2 … 200 ml —  1 M sodium hydroxide solution sufficient to produce blue-green colour (pH 7) … 7,5 ml approximately —  water to … 1 000 ml

4.   APPARATUS

4.1.	Water vacuum pump.

4.2.	500 ml vacuum flask.

4.3.

Potentiometer with scale graduated in pH values, and electrodes. The glass electrode must be kept in distilled water. The calomel/saturated potassium chloride electrode must be kept in a saturated potassium chloride solution. A combined electrode is most frequently used: it should be kept in distilled water.

4.4.	Measuring cylinders 50 ml (wine), 100 ml (rectified concentrated must).

5.   PROCEDURE

5.1.   Preparation of sample:

5.1.1.   Wines

Elimination of carbon dioxide. Place about 50 ml of wine in a vacuum flask; apply vacuum to the flask with the water pump for one to two minutes, whilst shaking continuously.

5.1.2.   Rectified concentrated musts

Introduce 200 g of accurately weighed rectified concentrated must. Make up to the mark with 500 ml water. Homogenize.

5.2.   Potentiometric titration

5.2.1.   Calibration of pH meter

The pH meter is now calibrated for use at 20 °C, according to the manufacturer's instructions, with the pH 7,00 buffer solution at 20 °C.

5.2.2.   Method of measurement

Into a measuring cylinder (4.4), introduce a volume of the sample, prepared as described in 5.1, equal to 10 ml in the case of wine and 50 ml in the case of rectified concentrated must. Add about 10 ml of distilled water and then add the 0,1 M sodium hydroxide solution (3.2) from the burette until the pH is equal to 7 at 20 °C. The sodium hydroxide must be added slowly and the solution stirred continuously. Let n ml be the volume of 0,1 M NaOH added.

5.3.   Titration with indicator (bromothymol blue)

5.3.1.   Preliminary test: end-point colour determination.

Into a measuring cylinder (4.4) place 25 ml of boiled distilled water, 1 ml of bromothymol blue solution (3.3) and a volume prepared as in (5.1) equal to 10 ml in the case of wine and 50 ml in the case of rectified concentrated must. Add the 0,1 M sodium hydroxide solution (3.2) until the colour changes to blue-green. Then add 5 ml of the pH 7 buffer solution (3.7).

5.3.2.   Measurement

Into a measuring cylinder (4.4) place 30 ml of boiled distilled water, 1 ml of bromothymol blue solution (3.3) and a volume of the sample, prepared as described in 5.1, equal to 10 ml in the case of wine and 50 ml in the case of rectified concentrated must. Add 0,1 M sodium hydroxide solution (3.2) until the same colour is obtained as in the preliminary test above (5.3.1). Let n ml be the volume of 0,1 M sodium hydroxide added.

6.   EXPRESSION OF RESULTS

6.1.   Method of calculation

6.1.1.   Wines

The total acidity expressed in milliequivalents per litre is given by:

A = 10n.

It is recorded to one decimal place

The total acidity expressed in grams of tartaric acid per litre is given by:

A′ = 0,075A

It is recorded to one decimal place.

6.1.2.   Rectified concentrated musts

6.2.   Repeatability (r) for titration with the indicator:

r

=

0,9 meq/litre

r

=

0,07 g tartaric acid/litre

for white, rosé and red wines.

6.3.   Reproducibility (R) for titration with the indicator (5.3):

For white and rosé wines:

R

=

3,6 meq/litre

R

=

0,3 g tartaric acid/litre

For red wines:

R

=

5,1 meq/litre

R

=

0,4 g tartaric acid/litre

14.   VOLATILE ACIDITY

1.   DEFINITION

The volatile acidity is formed from the acids of the acetic series present in wine in the free state and combined as a salt.

2.   PRINCIPLE OF THE METHOD

Titration of the volatile acids separated from the wine by steam distillation and titration of the distillate.

Carbon dioxide is first removed from the wine.

The acidity of free and combined sulphur dioxide distilled under these conditions should be deducted from the acidity of the distillate.

The acidity of any sorbic acid which may have been added to the wine must also be deducted.

Note: Part of the salicylic acid used in some countries to stabilize the wines before analysis is present in the distillate. This must be determined and deducted from the acidity. The method of determination is given in section 7 of this chapter.

3.   REAGENTS

3.1.	Crystalline tartaric acid (C4H6O6).

3.2.	0,1 M sodium hydroxide solution (NaOH).

3.3.	1 % phenolphthalein solution in 96 % vol neutral alcohol.

3.4.	Hydrochloric acid (ρ 20 = 1,18 to 1,19 g/ml) diluted 1/4 (v/v).

3.5.	0,005 M iodine (I2) solution.

3.6.	Crystalline potassium iodide (KI).

3.7.	5 g/l starch solution. Mix 5 g of starch with about 500 ml of water. Bring to the boil, stirring continuously and boil for 10 minutes. Add 200 g sodium chloride. When cool, make up to one litre.

3.8.	Saturated solution of sodium borate (Na2B4O7 · 10H2O), i.e. about 55 g/l at 20 °C.

4.   APPARATUS

4.1.

Steam distillation apparatus consisting of: 1. a steam generator; the steam must be free of carbon dioxide; 2. a flask with steam pipe; 3. a distillation column; 4. a condenser. This equipment must pass the following three tests: (a) Place 20 ml of boiled water in the flask. Collect 250 ml of the distillate and add to it 0,1 ml of 0,1 M sodium hydroxide solution (3.2) and two drops of the phenolphthalein solution (3.3). The pink colouration must be stable for at least 10 seconds (i.e. steam to be free of carbon dioxide). (b) Place 20 ml of a 0,1 M acetic acid solution in the flask. Collect 250 ml of the distillate. Titrate with the 0,1 M sodium hydroxide solution (3.2): the volume oft his used must be at least 19,9 ml (i.e. at least 99,5 % of the acetic acid entrained with the steam). (c) Place 20 ml of 1 M lactic acid solution in the flask. Collect 250 ml of the distillate and titrate the acid with the 0,1 M sodium hydroxide solution (3.2). The volume of sodium hydroxide solution added must be less than or equal to 1,0 ml (i.e. not more than 0,5 % of lactic acid is distilled). Any apparatus or procedure which passes these tests satisfactorily fulfils the requirements of official international apparatus or procedures.

4.2.	Water pump.

4.3.	Vacuum flask.

5.   PROCEDURE

5.1.	Preparation of sample: elimination of carbon dioxide. Place about 50 ml of wine in a vacuum flask; apply vacuum to the flask with the water pump for one to two minutes, shaking continuously.

5.2.	Steam distillation Place 20 ml of wine, freed from carbon dioxide as in 5.1, in the flask. Add about 0,5 g of tartaric acid (3.1). Collect at least 250 ml of the distillate.

5.3.

Titration Titrate with the 0,1 M sodium hydroxide solution (3.2) using two drops of phenolphthalein (3.3) as indicator. Let n ml be the volume of sodium hydroxide used. Add four drops of 1/4 dilute hydrochloric acid (3.4), 2 ml starch solution (3.3) and a few crystals of potassium iodide (3.6). Titrate the free sulphur dioxide with the 0,005 M iodine solution (3.5). Let n″ ml be the volume used. Add the saturated sodium borate solution (3.8) until the pink coloration reappears. Titrate the combined sulphur dioxide with the 0,005 M iodine solution (3.5). Let n″ ml be the volume used.

6.   EXPRESSION OF RESULTS

6.1.   Method of calculation

The volatile acidity, expressed in milliequivalents per litre to one decimal place, is given by:

A = 5 (n − 0,1 n′ − 0,05 n″).

The volatile acidity, expressed in grams of acetic acid per litre to two decimal places, is given by:

0,300 (n − 0,1 n′ − 0,05 n″).

6.2.   Repeatability (r)

r

=

0,7 meq/litre

r

=

0,04 g acetic acid/litre.

6.3.   Reproducibility (R)

R

=

1,3 meq/litre

R

=

0,08 g acetic acid/litre.

6.4.   Wine with sorbic acid present

Since 96 % of sorbic acid is steam distilled with a distillate volume of 250 ml, its acidity must be deducted from the volatile acidity, knowing that 100 mg of sorbic acid corresponds to an acidity of 0,89 milliequivalents or 0,053 g of acetic acid and knowing the concentration of sorbic acid in mg/l as determined by other methods.

7.   DETERMINATION OF SALICYLIC ACID ENTRAINED IN THE DISTILLATE FROM THE VOLATILE ACIDITY

7.1.   Principle

After the determination of the volatile acidity and the correction for sulphur dioxide, the presence of salicylic acid is indicated, after acidification, by the violet colouration that appears when an iron (III) salt is added.

The determination of the salicylic acid entrained in the distillate with the volatile acidity is carried out on a second distillate having the same volume as that on which the determination of volatile acidity was carried out. In this distillate, the salicylic acid is determined by a comparative colorimetric method. It is deducted from the acidity of the volatile acidity distillate.

7.2.   Reagents

7.2.1.

Hydrochloric acid (HCl) (ρ 20 = 1,18 to 1,19 g/l).

7.2.2.

Sodium thiosulphate, (Na2S2O3 · 5H2O) in a 0,1 M solution.

7.2.3.

10 % (m/v) solution of iron (III) ammonium sulphate (Fe2(SO4)3 · (NH4)2SO4 · 24H2O).

7.2.4.

0,01 M solution of sodium salicylate. Solution containing 1,60 g/l of sodium salicylate (NaC7H5O3).

7.3.   Procedure

7.3.1.   Identification of salicylic acid in the volatile acidity distillate.

Immediately after the determination of the volatile acidity and the correction for free and combined sulphur dioxide, introduce into a conical flask 0,5 ml hydrochloric acid (7.2.1), 3 ml of the 0,1 M sodium thiosulphate solution (7.2.2) and 1 ml of the iron (III) ammonium sulphate solution (7.2.3).

If salicylic acid is present, a violet coloration appears.

7.3.2.   Determination of salicylic acid

On the above conical flask, indicate the volume of the distillate by a reference mark. Empty and rinse the flask.

Subject a new test sample of 20 ml wine to steam distillation and collect the distillate in the conical flask up to the reference mark. Add 0,3 ml pure hydrochloric acid (7.2.1), and 1 ml of the iron (III) ammonium sulphate solution (7.2.3). The contents of the conical flask turn violet.

Into a conical flask identical to that carrying the reference mark, introduce distilled water up to the same level as that of the distillate. Add 0,3 ml pure hydrochloric acid (7.2.1) and 1 ml of the iron (III) ammonium sulphate solution (7.2.3). From the burette run in the 0,01 M sodium salicylate solution (7.2.4) until the violet coloration obtained has the same intensity as that of the conical flask containing the wine distillate.

Let n″ ml be the volume of solution added from the burette.

7.3.3.   Correction to the volatile acidity

Subtract the volume 0,1 × nXXX ml from the volume n ml of 0,1 M sodium hydroxide solution used to titrate the acidity of the distillate during the determination of volatile acidity.

15.   FIXED ACIDITY

1.   PRINCIPLE

The fixed acidity is calculated from the difference between total acidity and volatile acidity.

2.   EXPRESSION OF RESULTS

The fixed acidity is expressed in:

16.   TARTARIC ACID

1.   PRINCIPLE OF METHODS

1.1.   Reference method

Tartaric acid is precipitated in the form of calcium (±)tartrate and determined gravimetrically. This determination may be completed by a volumetric procedure for comparison. The conditions for precipitation (pH, total volume used, concentrations of precipitating ions) are such that precipitation of the calcium (±)tartrate is complete whereas the calcium D(−) tartrate remains in solution.

When mesotartaric acid has been added to the wine, which causes the precipitation of the calcium (±)tartrate to be incomplete, it must first be hydrolysed.

1.2.   Usual method

The tartaric acid, separated using an ion exchange column, is determined colorimetrically in the eluate by measurement of the red colour produced on reaction with vanadic acid. The eluate also contains lactic and malic acids which do not interfere.

2.   REFERENCE METHOD

2.1.   Gravimetric method

2.1.1.   Reagents

2.1.1.1.

Calcium acetate solution containing 10 g of calcium per litre: calcium carbonate (CaCO3) … 25 g acetic acid glacial (CH3COOH) (ρ20 = 1,05 g/ml) … 40 ml water to … 1 litre

2.1.1.2.

Calcium (±)tartrate, crystallized: CaC4O6H4 · 4H2O: Place 20 ml of L(+) tartaric acid solution (5 g/l) into a 400 ml beaker. Add 20 ml of ammonium D(−) tartrate solution (6,126 g/l) and 6 ml of calcium acetate solution containing 10 g of calcium per litre (2.1.1.1). Allow to stand for two hours to precipitate. Collect the precipitate in a sintered glass crucible of porosity No 4, and wash it three times with about 30 ml of distilled water. Dry to constant weight in the oven at 70 °C. Using the quantities of reagent indicated above, about 340 mg of crystallized calcium (±)tartrate is obtained. Store in a stoppered bottle.

2.1.1.3.

Precipitation solution (pH 4,75): —  D(−) tartaric acid … 122 mg —  25 % (v/v) ammonium hydroxide solution (ρ20 = 0,97 g/ml) … 0,3 ml —  calcium acetate solution (10 g calcium/litre) … 8,8 ml —  water to … 1 000 ml Dissolve the D(−) tartaric acid, add the ammonium hydroxide and make up to about 900 ml; add 8,8 ml of calcium acetate solution (2.1.1), make up to a litre and adjust the pH to 4,75 with acetic acid. Since calcium (±)tartrate is slightly soluble in this solution, add 5 mg of calcium (±)tartrate per litre, stir for 12 hours and filter.

2.1.2.   Procedure

2.1.2.1.   Wines with no added mesotartaric acid

Place 500 ml of precipitation solution and 10 ml of wine into a 600 ml beaker. Mix and initiate precipitation by rubbing the sides of the vessel with the tip of a glass rod. Leave to precipitate for 12 hours (overnight).

Filter the liquid and precipitate through a weighed sintered glass crucible of porosity No 4 fitted on a clean vacuum flask. Rinse the vessel in which precipitation took place with the filtrate to ensure that all precipitate is transferred.

Dry to constant weight in an oven at 70 °C. Weigh. Let p be the weight of crystallized calcium (±)tartrate (CaC4O6H4 · 4H2O) obtained.

2.1.2.2.   Wines to which mesotartaric acid has been added

When analysing wines to which mesotartaric acid has been or is suspected of having been added, proceed by first hydrolysing this acid as follows:

Place 10 ml of wine and 0,4 ml of glacial acetic acid (CH3COOH, ρ20 = 1,05 g/ml) into a 50-ml conical flask. Place a reflux condenser on top of the flask and boil for 30 minutes. Allow to cool and then transfer the solution in the conical flask to a 600-ml beaker. Rinse the flask twice using 5 ml of water each time and then continue as described above.

Mesotartaric acid is calculated and included as tartaric acid in the final result.

2.1.3.   Expression of results

One molecule of calcium (±)tartrate corresponds to half a molecule of L(+) tartaric acid in the wine.

The quantity of tartaric acid per litre of wine, expressed in milliequivalents is equal to 384,5 p.

It is quoted to one decimal place.

The quantity of tartaric acid per litre of wine, expressed in grams of tartaric acid is equal to 28,84 p.

It is quoted to one decimal place.

The quantity of tartaric acid per litre of wine, expressed in grams of potassium acid tartrate is equal to 36,15 p.

It is quoted to one decimal place.

2.2.   Comparative volumetric analysis

2.2.1.   Reagents

2.2.1.1.

Hydrochloric acid (HCl) (1:5 v/v) (ρ20 = 1,18 to 1,19 g/ml)

2.2.1.2.

EDTA solution, 0,05 M: EDTA (ethylenediaminetetraacetic acid disodium salt:     (C10H14N2O8Na2 · 2H2O) … 18,61 g distilled water to … 1 000 ml

2.2.1.3.

Sodium hydroxide solution, 40 % (m/v): sodium hydroxide (NaOH) … 40 g distilled water to … 100 ml

2.2.1.4.

Complexometric indicator: 1 % (m/m) 2-hydroxy-1-(2-hydroxy-4-sulpho-1-naphthylazo)-3-naphthoic acid (C21H14N2O7S · 3H2O) … 1 g anhydrous sodium sulphate (Na2SO4) … 100 g

2.2.2.   Procedure

After weighing, replace the sintered glass crucible containing the precipitate of calcium (±)tartrate on the vacuum flask and dissolve the precipitate with 10 ml of dilute hydrochloric acid (2.2.1.1). Wash the sintered glass crucible with 50 ml of distilled water.

Add 5 ml of 40 % sodium hydroxide solution (2.2.1.3) and about 30 mg of indicator (2.2.1.4). Titrate with 0,05 M EDTA (2.2.1.2). Let the number of ml used be n.

2.2.3.   Expression of results

The quantity of tartaric acid per litre of wine, expressed in milliequivalents is equal to 5 n.

It is quoted to one decimal place.

The quantity of tartaric acid per litre of wine, expressed in grams of tartaric acid is equal to 0,375 n.

It is quoted to one decimal place.

The quantity of tartaric acid per litre of wine, expressed in grams of potassium acid tartrate is equal to 0,470 n.

It is quoted to one decimal place.

▼M8 —————

▼B

17.   CITRIC ACID

1.   PRINCIPLE OF THE METHOD

Citric acid is converted into oxaloacetate and acetate in a reaction catalysed by citrate-lyase (CL):

citrate oxaloacetate + acetate

In the presence of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), the oxaloacetate and its decarboxylation derivative, pyruvate, are reduced to L-malate and L-lactate by reduced nicotinamide adenine dinucleotide (NADH):

oxaloacetate + NADH + H+ L-malate + NAD+

pyruvate + NADH + H+ L-lactate + NAD+

The amount of NADH oxidized to NAD+ in these reactions is proportional to the amount of citrate present. The oxidation of NADH is measured by the resultant decrease in absorbence at a wavelength of 340 nm.

2.   REAGENTS

2.1.

Buffer solution pH 7,8. (0,51 M glycylglycine; pH 7,8; Zn2+ (0,6 × 10− 3M): dissolve 7,13 g of glycylglycine in approximately 70 ml of doubly distilled water. Adjust the pH to 7,8 with approximately 13 ml of 5 M sodium hydroxide solution, add 10 ml of zinc chloride (ZnCl2 80 mg in 100 ml H2O) solution and make up to 100 ml with doubly distilled water.

2.2.	Reduced nicotinamide adenine dinucleotide (NADH) solution (approximately 6 × 10− 3 M): dissolve 30 mg NADH and 60 mg NaHCO3 in 6 ml of doubly distilled water.

2.3.	Malate dehydrogenase/lactate dehydrogenase solution (MDH/LDH, 0,5 mg MDH/ml, 2,5 mg LDH/ml): mix together 0,1 ml MDH (5 mg MDH/ml), 0,4 ml ammonium sulphate solution (3,2 M and 0,5 ml LDH (5 mg/ml). This suspension remains stable for at least a year at 4 °C.

2.4.	Citrate-lyase (CL, 5 mg protein/ml): dissolve 168 mg lyophilisate in 1 ml ice-cold water. This solution remains stable for at least a week at 4 °C and for at least four weeks if frozen. It is recommended that, prior to the determination, the enzyme activity should be checked.

2.5.	Polyvinylpolypyrrolidone (PVPP) Note: All the reagents above are available commercially.

3.   APPARATUS

3.1.

A spectrophotometer permitting measurement to be made at 340 nm, the wavelength at which absorption by NADH is at a maximum. Failing that, a spectrophotometer, with a discontinuous spectrum source permitting measurements to be made at 334 nm or 365 nm, may be used. Since absolute absorbence measurements are involved (i.e. calibration curves are not used but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbence of the apparatus must be checked.

3.2.	Glass cells with optical path lengths of 1 cm or single-use cells.

3.3.	Micropipettes for pipetting volumes in the range 0,02 to 2 ml.

4.   PREPARATION OF THE SAMPLE

Citrate determination is normally carried out directly on the wine, without preliminary removal of pigmentation (colouration) and without dilution provided that the citric acid content is less than 400 mg/l. If this is not so, dilute the wine until the citrate concentration lies between 20 and 400 mg/l (i.e. between 5 and 80 µg of citrate in the test sample).

With red wines that are rich in phenolic compounds, preliminary treatment with PVPP is recommended:

Form a suspension of about 0,2 g of PVPP in water and allow to stand for 15 minutes. Filter using a fluted filter.

Place 10 ml of wine in a 50 ml conical flask, add the moist PVPP removed from the filter with a spatula. Shake for two to three minutes. Filter.

5.   PROCEDURE

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbence using the 1 cm cells, using air as the zero absorbence (reference) standard (no cell in the optical path). Place the following in the 1 cm cells:

Mix, and after about five minutes read the absorbence of the solutions in the reference and sample cells (A 1).

Add:

Mix; wait until the reaction is completed (about five minutes) and read the absorbences of the solutions in the reference and sample cells (A 2).

Calculate the absorbence difference (A 2 − A 1) for the reference and sample cells, ΔAR and ΔAS .

Finally, calculate the difference between those differences:

ΔA = ΔAs − ΔAR

Note:

The time needed for the completion of enzyme activity can vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch.

6.   EXPRESSION OF RESULTS

Citric acid concentration is given in milligrams per litre to the nearest whole number.

6.1.   Method of calculation

The general formula for calculating the concentration in mg/l is:

where V

=

volume of test solution in ml (here 3,14 ml)

ν

=

volume of the sample in ml (here 0,2 ml)

M

=

molecular mass of the substance to be determined (here, for anhydrous citric acid, M = 192,1)

d

=

optical path in the cell in cm (here, 1 cm)

ε

=

absorption coefficient of NADH, (at 340 nm, ε = 6,3 mmol − 1 × l × cm − 1),

so that

C = 479 × ΔA

If the sample was diluted during its preparation, multiply the result by the dilution factor.

Note:

At 334 nm: C = 488 × ΔA (= 6,2 m mol − 1 × l × cm − 1).

At 365 nm: C = 887 × ΔA (= 3,4 m mol − 1 × l × cm − 1).

6.2.   Repeatability (r)

Citric acid concentration less than 400 mg/l: r = 14 mg/l.

Citric acid concentration greater than 400 mg/l: r = 28 mg/l.

6.3.   Reproducibility (R)

Citric acid concentration less than 400 mg/l: R = 39 mg/l.

Citric acid concentration greater than 400 mg/l: R = 65 mg/l.

18.   LACTIC ACID

1.   PRINCIPLE OF THE METHOD

Total lactic acid (L-lactate and D-lactate) is oxidized by nicotinamide adenine dinucleotide (NAD) to pyruvate in a reaction catalysed by L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH).

The equilibrium of the reaction normally lies more strongly in favour of the lactate. Removal of the pyruvate from the reaction mixture displaces the equilibrium towards the formation of pyruvate.

In the presence of L-glutamate, the pyruvate is transformed into L-alanine in a reaction catalysed by glutamate pyruvate transaminase (GPT):

The amount of NADH formed, measured by the increase in absorbence at the wavelength of 340 nm, is proportional to the quantity of lactate originally present.

Note:

L-lactic acid may be determined independently by using reactions (1) and (3), while D-lactic acid may be similarly determined by using reactions (2) and (3).

1.2.   Usual method

The lactic acid, separated by passage through an ion exchange resin column, is oxidized to ethanal and determined by colorimetry after reacting with sodium nitroprusside and piperidine.

2.   REFERENCE METHOD

2.1.   Reagents

2.1.1.

Buffer solution, pH 10 (glycylglycine 0,6 mol/l; L-glutamate 0,1 mol/l): dissolve 4,75 g of glycylglycine and 0,88 g of L-glutamic acid in approximately 50 ml of doubly distilled water; adjust the pH to 10 with a few millilitres of 10 M sodium hydroxide and make up to 60 ml with doubly distilled water. This solution will remain stable for at least 12 weeks at 4 °C.

2.1.2.

Nicotinamide adenine dinucleotide (NAD) solution, approximately 40 × 10− 3 M: dissolve 900 mg of NAD in 30 ml of doubly distilled water. This solution will remain stable for at least four weeks at 4 °C.

2.1.3.

Glutamate pyruvate transaminase (GPT) suspension, 20 mg/ml. The suspension remains stable for at least a year at 4 °C.

2.1.4.

L-lactate dehydrogenase (L-LDH) suspension, 5 mg/ml. This suspension remains stable for at least a year at 4 °C.

2.1.5.

D-lactate dehydrogenase (D-LDH) suspension, 5 mg/ml. This suspension remains stable for at least a year at 4 °C. It is recommended that, prior to the determination, the enzyme activity should be checked. Note: All the reagents are available commercially.

2.2.   Apparatus

2.2.1.

A spectrophotometer permitting measurements to be made at 340 nm, the wavelength at which absorption by NADH is at a maximum. Failing that, a spectrophotometer with a discontinuous spectrum source permitting measurements to be made at 334 or 365 nm may be used. Since absolute absorbence measurements are involved (i.e. calibration curves are not used, but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbence of the apparatus must be checked.

2.2.2.

Glass cells with optical path lengths of 1 cm or single-use cells.

2.2.3.

Micropipettes for pipetting sample volumes in the range 0,02 to 2 ml.

2.3.   Preparation of the sample

Preliminary note: No part of the glassware that comes into contact with the reaction mixture should be touched with the fingers, since this could introduce L-lactic acid and thus give erroneous results.

Lactate determination is normally carried out directly on the wine, without prior removal of pigmentation (colouration) and without dilution provided that the lactic acid concentration is less than 100 mg/l. If, however, the lactic acid concentration lies between:

2.4.   Procedure

2.4.1.   Determination of total lactic acid

The buffer solution must be at a temperature between 20 and 25 °C before proceeding to the measurement.

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbence using the cells having optical paths of 1 cm, with air as the zero absorbence (reference) standard (no cell in the optical path) or with water as the standard.

Place the following in the cells having 1 cm optical paths:

Mix using a glass stirrer or a rod of synthetic material with a flattened end; after about five minutes, measure the absorbences of the solutions in the reference and sample cells (A 1).

Add 0,02 ml of solution 2.1.4 and 0,05 ml of solution 2.1.5, homogenize, wait for the reaction to be completed (about 30 minutes) and measure the absorbences of the solutions in the reference and sample cells (A 2).

Calculate the differences (A 2 − A 1) in the absorbences of the solutions in the reference and sample cells, ΔA R and ΔA S.

Finally, calculate the difference between those differences:

ΔA = ΔAS − ΔAR

2.4.2.   Determination of L-lactic acid and D-lactic acid

Determinations of the L-lactic acid or D-lactic acid can be carried out independently by applying the procedure for total lactic acid up to the determination of A 1 and then continuing as follows:

Add 0,02 ml of solution 2.1.4, homogenize, wait until the reaction is complete (about 20 minutes) and measure the absorbences of the solutions in the reference and sample cells (A 2).

Add 0,05 ml of solution 2.1.5, homogenize, wait until the reaction is complete (about 30 minutes) and measure the absorbences of the solutions in the reference and sample cells (A 3).

Calculate the differences (A 2 − A 1) for L-lactic acid and (A 3 − A 2) for D-lactic acid between the absorbences of the solutions in the reference and sample cells, ΔAR and ΔAS .

Finally, calculate the difference between those differences:

ΔA = ΔAS − ΔAR

Note:

The time needed for the completion of enzyme activity can vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch. When determining the L-lactic acid alone, the incubation time may be reduced to 10 minutes.

2.5.   Expression of results

Lactic acid concentration is given in grams per litre to one decimal place.

2.5.1.   Method of calculation

The general formula for calculating the concentration in g/l is:

where

V

=

volume of test solution in ml (V = 2,24 ml for L-lactic acid, V = 2,29 ml for D-lactic acid and total lactic acid)

ν

=

volume of the sample in ml (here 0,2 ml)

M

=

molecular mass of the substance to be determined (here, for DL-lactic acid, M = 90,08)

d

=

optical path in the cell in cm (here, 1 cm)

ε

=

absorption coefficient of NADH, (at 340 nm, ε = 6,3 mmol− 1 × l × cm− 1).

2.5.1.1.   Total lactic acid and D-lactic acid

C = 0,164 × ΔA

If the sample was diluted during its preparation, multiply the result by the dilution factor.

Measurement at 334 nm: C = 0,167 × ΔA, (ε = 6,2 m mol − 1 × l × cm − 1).

Measurement at 365 nm: C = 0,303 × ΔA, (ε = 3,4 m mol − 1 × l × cm − 1).

2.5.1.2.   L-lactic acid

C = 0,160 × ΔA

If the sample was diluted during its preparation, multiply the result by the dilution factor.

Measurement at 334 nm: C = 0,163 ΔA, (ε = 6,2 m mol − 1 × l × cm − 1).

Measurement at 365 nm: C = 0,297 ΔA, (ε = 3,4 m mol − 1 × l × cm − 1).

2.5.2.   Repeatability (r)

r = 0,02 + 0,07xi g/l

xi is the lactic acid concentration in the sample in g/l.

2.5.3.   Reproducibility (R)

R = 0,05 + 0,125xi g/l

xi is the lactic acid concentration in the sample in g/l.

▼M8 —————

▼B

19.   L-MALIC ACID

1.   PRINCIPLE OF THE METHOD

L-malic acid (L-malate) is oxidized by nicotinamide adenine dinucleotide (NAD) to oxaloacetate in a reaction catalysed by L-malate dehydrogenase (L-MDH).

The equilibrium of the reaction normally lies more strongly in favour of the malate. Removal of the oxaloacetate from the reaction mixture displaces the equilibrium towards the formation of oxaloacetate. In the presence of L-glutamate, the oxaloacetate is transformed into L-aspartate in a reaction catalysed by glutamate oxaloacetate transaminase (GOT):

The amount of NADH formed, measured by the increase in absorbence at the wavelength of 340 nm, is proportional to the quantity of L-malate originally present.

2.   REAGENTS

2.1.

Buffer solution, pH 10 (glycylglycine 0,6 M; L-glutamate 0,1 M): dissolve 4,75 g of glycylglycine and 0,88 g of L-glutamic acid in approximately 50 ml of doubly distilled water; adjust the pH to 10 with about 4,6 ml of 10 M sodium hydroxide and make up to 60 ml with doubly distilled water. This solution will remain stable for at least 12 weeks at 4 °C.

2.2.	Nicotinamide adenine dinucleotide (NAD) solution, approximately 47 × 10− 3 M: dissolve 420 mg of NAD in 12 ml of doubly distilled water. This solution will remain stable for at least four weeks at 4 °C.

2.3.	Glutamate oxaloacetate transaminase (GOT) suspension, 2 mg/ml. The suspension remains stable for at least a year at 4 °C.

2.4.	L-malate dehydrogenase (L-MDH) solution, 5 mg/ml. This solution remains stable for at least a year at 4 °C. Note: All the reagents above are available commercially.

3.   APPARATUS

3.1.

A spectrophotometer permitting measurement to be made at 340 nm, the wavelength at which absorption by NADH is at a maximum. Failing that, a spectrophotometer, with a discontinuous spectrum source permitting measurements to be made at 334 or 365 nm, may be used. Since absolute measurements of absorbence are involved (i.e. calibration curves are not used, but standardization is made by consideration of the extinction coefficient of NADH), the wavelength scales and spectral absorbence of the apparatus must be checked.

3.2.	Glass cells with optical path lengths of 1 cm or single-use cells.

3.3.	Micropipettes for pipetting sample volumes in the range 0,01 to 2 ml.

4.   PREPARATION OF THE SAMPLE

L-malate determination is normally carried out directly on the wine, without prior removal of pigmentation (colouration) and without dilution provided that the L-malic acid concentration is less than 350 mg/l (measured at 365 mg/l). If this is not so, dilute the wine with doubly distilled water until the L-malate concentration lies between 30 and 350 mg/l (i.e. amount of L-malate in the test sample lies between 3 and 35 µg).

If the malate concentration in the wine is less than 30 mg/l, the volume of the test sample may be increased up to 1 ml. In this case, the volume of water to be added is reduced in such a way that the total volumes in the two cells are equal.

5.   PROCEDURE

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbence using the cells having optical paths of 1 cm, with air as the zero absorbence (reference) standard (no cell in the optical path) or with water as the standard.

Place the following in the cells having 1 cm optical paths:

Mix; after about three minutes, measure the absorbences of the solutions in the reference and sample cells (A 1).

Add:

Mix; wait for the reaction to be completed (about 5 to 10 minutes) and measure the absorbences of the solutions in the reference and sample cells (A 2).

Calculate the differences (A 2 − A 1) in the absorbences of the solutions in the reference and sample cells, ΔAR and ΔAS .

Finally, calculate the difference between those differences: ΔA = ΔAS − ΔAR

Note: The time needed for the completion of enzyme activity can vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch.

6.   EXPRESSION OF RESULTS

L-malic acid concentration is given in grams per litre to one decimal place.

6.1.   Method of calculation

The general formula for calculating the concentration in g/l is:

where

V

=

volume of test solution in ml (here 2,22 ml)

ν

=

volume of the sample in ml (here 0,1 ml)

M

=

molecular mass of the substance to be determined (here, for L-malic acid, M = 134,09)

d

=

optical path in the cell in cm (here, 1 cm)

ε

=

absorption coefficient of NADH, (at 340 nm, ε = 6,3 m mol− 1 × l × cm− 1),

so that for L-malate:

C = 0,473 × ΔA g/l

If the sample was diluted during its preparation, multiply the result by the dilution factor.

Note:

Measurement at 334 nm: C = 0,482 × ΔA

Measurement at 365 nm: C = 0,876 × ΔA

6.2.   Repeatability (r)

r = 0,03 + 0,034xi

xi is the malic acid concentration in the sample in g/l.

6.3.   Reproducibility (R)

R = 0,05 + 0,071xi

xi is the malic acid concentration in the sample in g/l.

▼M7

20.   D-MALIC ACID

(enzymatic method)

1.   PRINCIPLE

In the presence of D-malate dehydrogenase (D-MDH), D-malic acid (D-malate) is oxidised by nicotinamide adenine dinucleotide (NAD) to oxaloacetate. The oxaloacetate formed is split into pyruvate and carbon dioxide.

D-malate + NAD+ pyruvate + CO2 + NADH + H+

The quantity of NADH formed is proportional to the concentration of D-malic acid and is measured at a wavelength of 334, 340 or 365 nm.

2.   REAGENTS

Test combination for approximately 30 determinations:

Preparation of the solutions

Stability of the solutions

The content of bottle 1 is stable for at least one year if stored at +4 °C; solution 2 is stable for three weeks if stored at +4 °C, and for two months if stored at − 20 °C; solution 3 is stable for five days if stored at +4 °C.

3.   APPARATUS

4.   PREPARATION OF THE SAMPLE

D-malate analysis is normally carried out directly on the wine, without preliminary decolorisation.

The amount of D-malate in the cuvette should be between 2 and 50 μg. The wine therefore must be diluted to yield a D-malate concentration between 0,02 and 0,5 g/l or 0,02 and 0,3 g/l, respectively (depending on the apparatus used).

Dilution table:

5.   PROCEDURE

With the spectrophotometer adjusted to a wavelength of 340 nm, determine the absorbance using the 1 cm cuvettes, either using air to set zero absorbance (no cuvette in the optical path) or using water.

Pipette into the cuvettes:

Mix, and after about six minutes measure the absorbance of the reference and test solutions (A1).

Add:

Mix; wait until the reaction is completed (about 20 minutes) and measure the absorbances of the reference and test solutions (A2).

Calculate the absorbance difference (A2 − A1) for the reference (ΔΑT) and test (ΔΑE) solutions. Finally, calculate the difference between those differences:

.

Note: The time needed for the completion of enzyme activity can vary from one batch to another. The above time is given only for guidance and it is recommended that it be determined for each batch.

D-malic acid reacts rapidly. The enzyme also transforms L-tartaric acid, although very much more slowly. This explains the slight side reaction, which can be corrected by means of extrapolation (see Appendix A).

6.   EXPRESSION OF THE RESULTS

The general formula for calculating the concentration in mg/l is:

where:

V

=

volume of test solution in ml (2,95 ml)

ν

=

volume of the sample in ml (0,1 ml)

PM

=

molecular mass of the substance to be determined (for D-malic acid, PM = 134,09)

d

=

optical path of the cuvette in cm (1 cm)

ε

=

absorption coefficient of NADH: at 340 nm = 6,3 (1 mmol−1 cm−1) at 365 nm = 3,4 (1 mmol−1 cm−1) at 334 nm = 6,18 (1 mmol−1 cm−1).

If the sample was diluted during its preparation, multiply the result by the dilution factor.

The D-malic acid concentration is given in milligrams per litre (mg/l), with no decimal places.

7.   ACCURACY

Details of the interlaboratory trial on the accuracy of the method are summarised in Appendix B. The values derived from the interlaboratory trial may not be applicable to ranges of analyte concentration and matrices other than those in Appendix B.

7.1.   Repeatability

The absolute difference between two individual results obtained on identical matter submitted to a trial by an operator using the same apparatus, within the shortest time interval, will not exceed repeatability value r in more than 5 % of cases.

r

=

11 mg/l.

7.2.   Reproducibility

The absolute difference between two individual results obtained on identical matter submitted to a trial in two different laboratories will not exceed reproducibility value R in more than 5 % of cases.

R

=

20 mg/l.

▼M10

8.   DOSAGE OF D-MALIC ACID (D(+)-MALIC ACID) IN WINES WITH LOW LEVELS

8.1.   Field of application

The method described is applied to the dosage, by enzymatic means, of D-malic acid of wines with levels under 50 mg/l.

8.2.   Principle

The principle of the method is described in point 1. The formation of NADH after the introduction into the cuvette of 50 mg/l of D-malic acid is proportional to the quantity of D-malate present and is measured on the basis of the increase in absorbance at a wavelength of 340 nm.

8.3.   Reagents

A 0,199 g/l D-malic acid solution plus the reagents indicated in point 2.

8.4.   Apparatus

Apparatus indicated in point 3.

8.5.   Preparation of the sample

As indicated in point 4.

8.6.   Procedure

The procedure is as described in point 5, but with the introduction into the measuring cuvette of 50 mg/l of D-malic acid. (Introduction of 0,025 ml of 0,199 g/l D-malic acid solution, displacing the equivalent volume of water); the values obtained are decreased by 50 mg/l.

8.7.   Internal validation

The table below summarises the internal validation file on the method for determining the dosage of D(+)-malic acid after the addition of 50 mg/l of the isomer.

▼M7

Appendix A

How to deal with side reactions

Side reactions are generally due to secondary reactions of the enzyme, to the presence of other enzymes in the sample matrix, or to interaction of one or more elements of the matrix with a co-factor in the enzymatic reaction.

With a normal reaction, absorbance reaches a constant value after a certain time, generally 10 to 20 minutes, depending on the speed of the specific enzymatic reaction. However, when secondary reactions occur, absorbance does not reach a constant value, but increases regularly with time. This type of process is commonly called a ‘side reaction’.

When side reaction occurs, the absorbance of the solution should be measured at regular intervals (every two to five minutes) after the required time for the standard solution to reach its final absorbance has elapsed. If the absorbance increases regularly, five or six measurements should be made, and extrapolated back by means of a graph or of calculation, to determine the absorbance that would have been observed when the final enzyme was added (T0). The substrate concentration is calculated on the basis of the difference in absorbance extrapolated at that time (Af − Ai).

Figure 1. Side reaction

Appendix B

▼B

21.   TOTAL MALIC ACID

1.   PRINCIPLE

Malic acid, separated by means of an anion exchange column, is determined colorimetrically in the eluent by measuring the yellow coloration it forms with chromotropic acid in the presence of concentrated sulphuric acid. A correction for interfering substances is made by subtracting the absorbence, obtained using 86 % sulphuric and chromotropic acid respectively (malic acid does not react at these acid concentrations), from the absorbence obtained from using 96 % strength acids.

2.   APPARATUS

2.1.	Glass column approximately 250 mm in length and 35 mm internal diameter, fitted with drain tap.

2.2.	Glass column approximately 300 mm in length and 10 to 11 mm internal diameter, fitted with drain tap.

2.3.	Thermostatically controlled water bath at 100 °C.

2.4.	Spectrophotometer set to measure absorbence at 420 nm using 10 mm cells.

3.   REAGENTS

3.1.	A strongly basic ion exchange resin (e.g. Merck III).

3.2.	Sodium hydroxide 5 % (m/v).

3.3.	Acetic acid 30 % (m/v).

3.4.	Acetic acid 0,5 % (m/v).

3.5.	Sodium sulphate solution 10 % (m/v).

3.6.	Concentrated sulphuric acid 95 to 97 % (m/m).

3.7.	Sulphuric acid 86 % (m/m).

3.8.	Chromotropic acid 5 % (m/v) Prepare fresh solution before each determination by dissolving 500 mg sodium chromotropate, (C10H6Na2O8S2.2H20) in 10 ml distilled water.

3.9.	DL-Malic acid solution 0,5 g/l. Dissolve 250 g malic acid (C4H6O5) in sodium sulphate solution (10 %), make up to 500 ml with sodium sulphate solution (10 %) (3.5).

4.   PROCEDURE

4.1.   Preparation of ion exchange resin

Place a plug of cotton wool impregnated with distilled water at the bottom of the column (35 × 250 mm) above the tap. Pour a suspension of the anion exchange resin into the glasscolumn. The level of the liquid should be 50 mm above the top of the resin. Rinse with 1 000 ml of distilled water. Wash the column with sodium hydroxide solution (5 %), allow to drain to within 2 to 3 mm of the top of the resin and repeat with two further washings of sodium hydroxide 5 % and leave for one hour. Wash the column with 1 000 ml of distilled water. Refill the column with acetic (30 %) acid, allow to drain to within 2 to 3 mm from the top of the column and repeat with two further washings of acetic acid (30 %). Leave for at least 24 hours before use. Keep the ion exchange resin in acetic acid (30 %) for the subsequent analyses.

4.2.   Preparation of ion exchange column

Place a plug of cotton wool at the bottom of the column (11 × 300 mm) above the tap. Pour in the suspension of ion exchange resin (prepared in 4.1) to a height of 10 cm. Open the tap and allow the acetic acid solution (30 %) to drain to within 2 to 3 mm of the top of the resin. Wash with a 50 ml portion of acetic acid (0,5 %).

4.3.   Separation of DL-malic acid

Pour onto the column (prepared in 4.2) 10 ml of wine or must. Allow to drain one drop at a time (average rate of one drop per second) and stop the flow 2 to 3 mm from the top of the resin. Wash the column with 50 ml acetic acid (0,5 %) then with 50 ml of distilled water and allow to drain at the same rate as previously, stopping the flow 2 to 3 mm from the top of the resin.

Elute the acids absorbed on the exchange resin with a 10 % sodium sulphate solution (3.5). Collect the eluate in a 100 ml volumetric flask.

The column can be regenerated using the procedure desribed in (4.1).

4.4.   Determination of malic acid

Label two wide necked 30-ml tubes (fitted with ground glass stoppers) A and B. Into each tube add 1,0 ml of the eluent (4.3) and 1 ml of chromotropic acid (5 %). Add 10 ml sulphuric acid (86 %) (reference) to tube A and 10 ml (96 %) sulphuric acid to tube B (sample). Stopper and shake to homogenize, taking care not to wet the glass stopper. Immerse the tubes in a boiling water bath for exactly 10 minutes. Cool the tubes in the dark at 20 °C for exactly 90 minutes. Immediately measure the absorbence relative to the control at 420 nm in a 10 mm cell.

4.5.   Plotting the calibration curve

Pipette 5,0, 10,0, 15,0 and 20 ml aliquots respectively into 4 × 50 ml volumetric flasks. Make up to the mark with sodium sulphate solution (10 %).

These solutions correspond to eluates obtained from the wine containing 0,5, 1,0, 1,5 and 2,0 g/l of malic acid.

Continue as in 4.4.

The graph of the absorbences of these solutions is a function of their malic acid concentration represented as a straight line passing through the origin.

The intensity of the colour produced depends to a large extent on the strength of the sulphuric acid used, it is necessary to check the calibration curve with at least one point per series of readings to check if the concentration of the sulphuric acid has changed.

5.   EXPRESSION OF RESULTS

The concentration of the eluent is found using the calibration graph by extrapolation of the measured absorbence value to give the corresponding malic acid concentration in g/l. The result is expressed to one decimal place.

Repeatability:

Contents < 2 g/l: r

=

0,1 g/l.

Contents> 2 g/l: r

=

0,2 g/l.

Reproducibility:

R = 0,3 g/l.

22.   SORBIC ACID

1.   PRINCIPLE OF METHODS

1.1.   Determination by ultraviolet absorption spectrophotometry

Sorbic acid (trans, trans, 2,4-hexadienoic acid) extracted by steam distillation is determined in the wine distillate by ultraviolet absorption spectrophotometry. Substances that interfere in the ultraviolet are removed by evaporation to dryness using a lightly alkali, calcium hydroxide. Thin layer chromatography is used for confirmation of levels (1 mg/l) less than 20 mg/l.

1.2.   Determination by gas chromatography

Sorbic acid extracted in ethyl ether is determined by gas chromatography with an internal standard.

1.3.   Identification of traces by thin-layer chromatography

Sorbic acid extracted in ethyl ether is separated by thin layer chromatography and its concentration is evaluated semi-quantitatively.

2.   DETERMINATION BY ULTRAVIOLET ABSORPTION SPECTROPHOTOMETRY

2.1.   Reagents

2.1.1.

Crystalline tartaric acid, C4H6O6.

2.1.2.

Calcium hydroxide, Ca(OH)2, solution, approximately 0,02 M.

2.1.3.

Reference sorbic acid solution, 20 mg/l: Dissolve 20 mg of sorbic acid, C6H8O2, in approximately 2 ml of 0,1 M sodium hydroxide solution. Pour into a 1 000 ml volumetric flask, and make up to the mark with water. It is also possible to dissolve 26,8 mg of potassium sorbate, C6H7KO2, in water and make up to 1 000 ml with water.

2.2.   Apparatus

2.2.1.

Steam distillation apparatus (see chapter ‘Volatile acidity’).

2.2.2.

Water bath at 100 °C.

2.2.3.

Spectrophotometer enabling absorbence measurements to be made at a wavelength of 256 nm and having a quartz cell with a 1 cm optical path.

2.3.   Procedure

2.3.1.   Distillation

Place in the flask of the steam distillation apparatus 10 ml of wine and add 1 to 2 g tartaric acid (2.1.1). Collect 250 ml of the distillate.

2.3.2.   Preparation of the calibration curve

Prepare, by dilution of the reference solution (2.1.3), four dilute reference solutions with 0,5, 1,0, 2,5 and 5 mg of sorbic acid per litre. Measure their absorbences with the spectrophotometer at 256 nm using that of distilled water as a blank. Plot a curve showing the variation of absorbence as a function of concentration. The variation is linear.

2.3.3.   Determination

Place 5 ml of the distillate in an evaporating dish of 55 mm diameter, add 1 ml of calcium hydroxide solution (2.1.2). Evaporate to dryness on a water bath.

Dissolve the residue in several ml of distilled water, transfer completely to a 20 ml volumetric flask and make up to the mark with rinsing water. Measure the absorbence at 256 nm using the spectrophotometer against a blank consisting of a solution obtained by diluting 1 ml of calcium hydroxide solution (2.1.2) to 20 ml with water.

Plot the value of the measured absorbence on the calibration curve and from this find the concentration C of sorbic acid in the solution.

Note: In this method the loss due to evaporation can be neglected and the absorbence measured on the treated distillate diluted ¼ with distilled water.

2.4.   Expression of results

2.4.1.   Calculation

The sorbic acid concentration in the wine expressed in mg per litre is given by 100 × C

where

C

=

concentration of sorbic acid in the solution analysed by spectrophotometry expressed in mg per litre.

3.   DETERMINATION BY GAS CHROMATOGRAPHY

3.1.   Reagents

3.1.1.

Ethyl ether, (C2H5)2O, distilled just before use.

3.1.2.

Internal reference solution: solution of undecanoic acid, C11H22O2, in 95 % vol ethanol at a strength of 1 g/l.

3.1.3.

Aqueous solution of sulphuric acid, H2SO4 (ρ20 = 1,84 g/ml) diluted 1:3 (v/v).

3.2.   Apparatus

3.2.1.

Gas chromatograph fitted with a flame ionization detector and a stainless steel column (4 m × ⅛ inch) previously treated with dimethyldichlorosilane and packed with a stationary phase consisting of a mixture of diethyleneglycol succinate (5 %) and phosphoric acid (1 %) (DEGS — H3PO4) or of a mixture of diethyleneglycol adipate (7 %) and phosphoric acid (1 %) (DEGA — H3PO4) bonded on Gaschrom Q 80 — 100 mesh. Treatment of column with DMDCS — pass through the column a solution containing 2 to 3 g of DMDCS in toluene. Immediately wash with methanol, followed by nitrogen and then wash with hexane followed by more nitrogen. It is now ready to be packed. Operating conditions: Oven temperature: 175 °C. Temperature of the injector and detector: 230 °C. Carrier gas: nitrogen (flow rate = 200 ml/min).

3.2.2.

Microsyringe, 10 µl capacity graduated in 0,1 µl. Note: Other types of columns that give a good separation can be used, particularly capillary columns (e.g. FFAP). The working method described is given as an example.

3.3.   Procedure

3.3.1.   Preparation of sample to be analysed

Into a glass test tube of approximately 40 ml capacity and fitted with a ground glass stopper, introduce 20 ml of wine, add 2 ml of the internal reference solution (3.1.2) and 1 ml of dilute sulphuric acid (3.1.3).

After mixing the solution by repeatedly turning the tube over, add to its contents 10 ml of ethyl ether (3.1.1). Extract the sorbic acid in the organic phase by shaking the tube for five minutes. Leave to settle.

3.3.2.   Preparation of the reference solution

Select a wine for which the chromatogram of the ether extract shows no peak corresponding to the elution of sorbic acid. Overload this wine with sorbic acid at a concentration of 100 mg per litre. Treat 20 ml of the sample prepared in this way according to the procedure described in 3.3.1.

3.3.3.   Chromatography

Using a microsyringe, inject into the chromatograph in turn 2 µl of the ether-extract phase obtained in 3.3.2 and 2 µl of the ether-extracted phase obtained in 3.3.1.

Record the respective chromatograms: check the identity of the respective retention times of the sorbic acid and the internal standard. Measure the height (or area) of each of the recorded peaks.

3.4.   Expression of results

3.4.1.   Calculation

The concentration of sorbic acid in the analysed wine, expressed in mg per litre, is given by:

where

H

=

height of the sorbic acid peak in the reference solution

h

=

height of the sorbic acid peak in the sample for analysis

I

=

height of the internal standard peak in the reference solution

i

=

height of the internal standard peak in the sample for analysis

Note: The sorbic acid concentration may be determined in the same way from measurements of the areas under the respective peaks.

4.   IDENTIFICATION OF TRACES OF SORBIC ACID BY THIN LAYER CHROMATOGRAPHY

4.1.   Reagents

4.1.1.

Ethyl ether, (C2H5)2O.

4.1.2.

Aqueous sulphuric acid solution, H2SO4 (ρ20 = 1,84 g/ml), diluted 1:3 (v/v).

4.1.3.

Reference solution of sorbic acid in an approximately 10 % vol ethanol/water mixture containing 20 mg per litre.

4.1.4.

Mobile phase: hexane-pentane-acetic acid (20:20:3) (C6H14/C5H12/CH3COOH, ρ20 = 1,05 g/ml).

4.2.   Apparatus

4.2.1.

Precoated 20 × 20 cm plates for thin layer chromatography coated with polyamide gel (0,15 mm thick) with the addition of a fluorescent indicator.

4.2.2.

Cell for thin layer chromatography.

4.2.3.

Micropipette or microsyringe for delivering volumes of 5 µl to within ± 0,1 µl.

4.2.4.

Ultraviolet lamp (254 nm).

4.3.   Procedure

4.3.1.   Preparation of sample to be analysed

Into a glass test tube of approximately 25 ml capacity and fitted with a ground glass stopper, place 10 ml of wine, add 1 ml of dilute sulphuric acid (4.1.2) and 5 ml of ethyl ether (4.1.2). Mix by repeatedly turning the tube over. Leave to settle.

4.3.2.   Preparation of dilute reference solutions

Prepare five dilute reference solutions from the solution in 4.1.3 containing 2, 4, 6, 8 and 10 mg sorbic acid per litre.

4.3.3.   Chromatography

Using a microsyringe or micropipette, deposit 5 µl of the ether-extracted phase obtained in 4.3.1 and 5 µl of each of the dilute reference solutions (4.3.2) at points 2 cm from the lower edge of the plate and 2 cm apart from each other.

Place the mobile phase (4.1.4) in the chromatograph tank to a height of about 0,5 cm and allow the atmosphere in the tank to become saturated with solvent vapours. Place the plate in the tank. Allow the chromatogram to develop over 12 to 15 cm (development time approximately 30 minutes). Dry the plate in a current of cool air. Examine the chromatogram under a 254 nm ultraviolet lamp.

The spots indicating the presence of sorbic acid will appear to be dark violet against the yellow fluorescent background of the plate.

4.4.   Expression of results

A comparison of the intensities of the spots produced by the sample to be analysed and by the reference solutions will enable a semi-quantitative assessment to be made of the sorbic acid concentration between 2 and 10 mg per litre. A concentration of 1 mg per litre could be determined with the deposition of 10 µl of the sample solution to be analysed.

Concentrations above 10 mg per litre could be determined with the deposition of less than 5 µl of the solution to be analysed (measured out using a microsyringe).

23.   L-ASCORBIC ACID

1.   PRINCIPLE OF METHODS

The methods proposed enable the L-ascorbic acid and dehydroascorbic acid present in wines or musts to be determined.

1.1.   Reference method (fluorimetry)

The L-ascorbic acid is oxidized on activated carbon into dehydroascorbic acid. The latter forms a fluorescent compound by reacting with orthophenylenediamine (OPDA). A control test in the presence of boric acid enables spurious fluorescence to be determined (by the formation of a boric acid/dehydroascorbic acid complex) and the fluorimetric determination to be deduced.

1.2.   Usual method (colorimetry)

The L-ascorbic acid is oxidized by iodine to dehydroascorbic acid which is then precipitated using 2,4-dinitrophenylhydrazine to produce bis (2,4-dinitrophenylhydrazone). After separation by thin layer chromatography and dissolution in acetic acid medium the red-coloured derivative is determined by spectrophotometry at 500 nm.

2.   REFERENCE METHOD (fluorimetric method)

2.1.   Reagents

2.1.1.

Orthophenylenediamine dihydrochloride solution, C6H10Cl2N2, 0,02 g per 100 ml; prepared just before use.

2.1.2.

Sodium acetate trihydrate solution, CH3COONa · 3H2O, 500 g/litre.

2.1.3.

Mixed solution of boric acid and sodium acetate: dissolve 3 g of boric acid, H3BO3, in 100 ml of sodium acetate solution (2.1.2). This solution must be prepared just before use.

2.1.4.

Glacial acetic acid solution, CH3COOH (ρ20 = 1,05 g/ml), diluted to 56 % (v/v) with pH near to 1,2.

2.1.5.

Reference solution of L-ascorbic acid, 1 g/litre: Just before use, dissolve 50 mg of L-ascorbic acid, C6H8O6, previously dehydrated in a desiccator protected against light, in 50 ml of acetic acid solution (2.1.4).

2.1.6.

Very pure analytical grade activated carbon ( 12 ) Into a 2-litre conical flask, place 100 g of activated carbon and add 500 ml of 10 % (v/v) hydrochloric acid (HCl) solution (ρ20 = 1,19 g/ml). Bring to the boil, filter using a sintered glass filter of porosity 3. Collect the carbon treated in this way in a 2-litre conical flask, add 1 litre of water, shake and filter using a sintered glass filter of porosity 3. Repeat this operation two more times. Place the residue in an oven controlled to 115 ± 5 °C for 12 hours (overnight).

2.2.   Apparatus

2.2.1.

Fluorimeter. Use a spectrofluorimeter equipped with a lamp giving a continuous spectrum by using it at minimum power. The optimum excitation and emission wavelengths for the test will be determined beforehand and depend on the equipment used. As a guide, the excitation wavelength will be approximately 350 nm and the emission wavelength approximately 430 nm. Cells of 1 cm path length.

2.2.2.

Sintered glass filter of porosity 3.

2.2.3.

Test tubes (diameter approximately 10 mm).

2.2.4.

Stirring rods for test tubes.

2.3.   Procedure

2.3.1.   Preparation of the sample of wine or must

Take a volume of the wine or must and dilute to 100 ml in a graduated flask with the 56 % acetic acid solution (2.1.4) in order to obtain a solution with an L-ascorbic acid concentration between 0 and 60 mg/litre. Homogenize the contents of the flask by stirring. Add 2 g of activated carbon (2.1.6) and allow to stand for 15 minutes, stirring occasionally. Filter using ordinary filter paper, discarding the first few millilitres of filtrate.

Into two 100 ml graduated flasks, introduce 5 ml of the filtrate and, in the first, 5 ml of the mixed solution of boric acid and sodium acetate solution (2.1.3) (sample blank) and, in the second, 5 ml of the sodium acetate solution (2.1.2) (sample). Allow to stand for 15 minutes, stirring occasionally. Make up to 100 ml with distilled water.

Take 2 ml from the contents of each flask and add 5 ml of orthophenylenediamine solution (2.1.1), stir; leave the reaction to proceed for 30 minutes until the solution darkens and then make the spectrofluorimetric measurements.

2.3.2.   Preparation of the calibration curve

Into three 100 ml graduated flask place 2, 4 and 6 ml respectively of the reference L-ascorbic acid solution (2.1.5), make up to 100 ml with acetic acid solution (2.1.4) and homogenize by stirring. The reference solutions prepared in this way contain 2, 4 and 6 mg per 100 ml.

Add 2 g of activated carbon (2.1.6) to each of the flasks and allow to stand for 15 minutes, stirring occasionally. Filter through ordinary filter paper, discarding the first few millilitres. Introduce 5 ml of each filtrate collected into three 100-ml graduated flasks (first series). Repeat the operation and obtain a second series of three graduated flasks. To each of the flasks in the first series (corresponding to the blank test) add 5 ml of the mixed solution of boric acid and sodium acetate (2.1.5), and to each of the flasks in the second series add 5 ml of the sodium acetate solution (2.1.2).

Allow to stand for 15 minutes, stirring occasionally. Make up to 100 ml with distilled water. Take 2 ml of the contents of each flask, add 5 ml of orthophenylenediamine solution (2.1.1), stir, leave the reaction to proceed for 30 minutes until the solution darkens and then make the spectrofluorimetric measurements.

2.3.3.   Fluorimetric determination

For each solution contributing to the calibration curve and for the solution to be determined set the zero on the scale of measurements using the corresponding control test sample. Then measure the intensity of the fluorescence for each solution over the calibration range and for the solution to be determined.

Plot the calibration curve, which should be a straight line passing through the origin. On this line, find the value relative to the determination and thus deduce the concentration C L-ascorbic acid + dehydroascorbic acid in the solution to be analysed.

2.3.4.   Expression of results

The concentration of L-ascorbic acid and dehydroascorbic acid in the wine in milligrams per litre is given by C × F, where F is the dilution factor.

▼M8 —————

▼B

24.   pH

1.   PRINCIPLE

The difference in potential between two electrodes immersed in the liquid under test is measured. One of these two electrodes has a potential which is a function of the pH of the liquid, while the other has a fixed and known potential and constitutes the reference electrode.

2.   APPARATUS

2.1.	pH meter with a scale calibrated in pH units and enabling measurements to be made to at least ±0,05 pH unit.

2.2.	Electrodes: 2.2.1. Glass electrode, kept in distilled water. 2.2.2. Calomel-saturated potassium chloride reference electrode, kept in a saturated solution of potassium chloride. 2.2.3. Or a combined electrode, kept in distilled water.

3.   REAGENTS

3.1.   Buffer solutions

3.1.1.

Saturated solution of potassium hydrogen tartrate, containing at least 5,7 g of potassium hydrogen tartrate per litre (C4H5KO6) at 20 °C. (This solution may be kept for up to two months by adding 0,1 g of thymol per 200 ml.) pH 3,57 at 20 °C 3,56 at 25 °C 3,55 at 30 °C

3.1.2.

Solution of potassium hydrogen phthalate, 0,05 M, containing 10,211 g of potassium hydrogen phthalate (C8H5KO4) per litre at 20 °C. (Maximum keeping period, two months.) pH 3,999 at 15 °C 4,003 at 20 °C 4,008 at 25 °C 4,015 at 30 °C

3.1.3.

Solution containing: monopotassium phosphate, KH2PO4 … 3,402 g dipotassium phosphate, K2HPO4 … 4,354 g water to … 1 000 ml (maximum keeping period, two months) pH 6,90 at 15 °C 6,88 at 20°C 6,86 at 25 °C 6,85 at 30 °C Note: Commerical reference buffer solutions may also be used.

4.   PROCEDURE

4.1.   Preparation of the sample for analysis

4.1.1.

For must and wine: use the must or wine directly.

4.1.2.

For rectified concentrated must: dilute the rectified concentrated must with water to produce a concentration of 25 ± 0,5 % (m/m) of total sugars (25 ° Brix). If P is the percentage concentration (m/m) of total sugars in the rectified concentrated must, weigh a mass of and make up to 100 g with water. The water used must have a conductivity below 2 microsiemens per cm.

4.2.   Zeroing of the apparatus

Zeroing is carried out before any measurement is made, according to the instructions provided with the apparatus used.

4.3.   Calibration of the pH meter

Calibrate the pH meter at 20 °C using buffer solutions of pH 6,88 and 3,57 at 20 °C.

Use the buffer solution of pH 4,00 at 20 °C to check the calibration of the scale.

4.4.   Determination

Dip the electrode into the sample to be analysed, the temperature of which should be between 20 and 25 °C and as close as possible to 20 °C. Read the pH value directly off the scale.

Carry out at least two determinations on the same sample.

The final result is taken to be the arithmetic mean of two determinations.

5.   EXPRESSION OF RESULTS

The pH of the must, the wine or the 25 % (m/m) (25 ° Brix) solution of rectified concentrated must is quoted to two decimal places.

25.   SULPHUR DIOXIDE

1.   DEFINITIONS

Free sulphur dioxide is defined as the sulphur dioxide present in the must or wine in the following forms: H2SO3, HSO3−

The equilibrium between these forms is a function of pH and temperature:

H2SO3 H+ + HSO3−

H2SO3 represents molecular sulphur dioxide.

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

2.   FREE AND TOTAL SULPHUR DIOXIDE

2.1.   Principle of the methods

2.1.1.   Reference method

2.1.1.1.   For wines and musts

The sulphur dioxide is carried over by a current of air or nitrogen; it is fixed and oxidized by being bubbled through a dilute and neutral hydrogen peroxide solution. The sulphuric acid formed is determined by titration with a standard solution of sodium hydroxide. Free sulphur dioxide is purged from the wine by entrainment at low temperature (10 °C).

Total sulphur dioxide is purged from the wine by entrainment at high temperature (approximately 100 °C).

2.1.1.2.   For rectified concentrated musts

Total sulphur dioxide is extracted from the previously diluted rectified concentrated must by entrainment at high temperature (approximately 100 °C).

2.1.2.   Rapid method of determination (for wines and musts)

Free sulphur dioxide is determined by direct iodometric titration.

Combined sulphur dioxide is subsequently determined by iodometric titration after alkaline hydrolysis. When added to the free sulphur dioxide, it gives the total sulphur dioxide.

2.2.   Reference method

2.2.1.   Apparatus

2.2.1.1.

The apparatus used should conform to the diagram shown below, particularly with regard to the condenser. The dimensions given are in millimetres. The internal diameters of the four concentric tubes forming the condenser are 45, 34, 27 and 10 mm. The gas feed tube to the bubbler B ends in a small sphere of 1 cm diameter with 20 0,2-mm diameter holes around its largest horizontal circumference. Alternatively, this tube may end in a frit glass plate which produces a large number of very small bubbles and thus ensures good contact between the liquid and gaseous phases. The gas flow through the apparatus should be approximately 40 litres per hour. The bottle on the right of the diagram is intended to restrict the pressure reduction produced by the water pump to 20 to 30 cm of water. To regulate the vacuum to its correct value, a flow-meter with a semi-capillary tube should be installed between the bubbler and the bottle.

2.2.1.2.

A microburette.

2.2.2.   Reagents

2.2.2.1.

Phosphoric acid, 85 % (H3PO4, ρ20 = 1,71 g/ml).

2.2.2.2.

Hydrogen peroxide solution, 9,1 g H2O2/litre (three volumes).

2.2.2.3.

Indicator reagent: methyl red … 100 mg methylene blue … 50 mg alcohol, 50 % vol … 100 ml

2.2.2.4.

Sodium hydroxide solution, NaOH, 0,01 M

2.2.3.   Procedure

2.2.3.1.   Determination of free sulphur dioxide

The wine must be kept in a full and stoppered bottle at 20 °C for two days before the determination.

Place 2 to 3 ml of hydrogen peroxide solution (2.2.2.2) and two drops of the indicator reagent in the bubbler B and neutralize the hydrogen peroxide solution with the 0,01 M sodium hydroxide solution (2.2.2.4). Connect the bubbler to the apparatus.

Introduce 50 ml of the sample and 15 ml of phosphoric acid (2.2.2.1) into the flask A of the entrainment apparatus. Connect the flask into the apparatus.

Bubble air (or nitrogen) through it for 15 minutes. The free sulphur dioxide carried over is oxidized to sulphuric acid. Remove the bubbler from the apparatus and titrate the acid which has formed against the 0,01 M sodium hydroxide solution (2.2.2.4). Let n ml be the volume used.

2.2.3.2.   Expression of results

The liberated sulphur dioxide is expressed in mg/l to the nearest whole number.

2.2.3.2.1.

Calculation The free sulphur dioxide in milligrams per litre is 6,4 n.

2.2.3.3.   Determination of total sulphur dioxide

2.2.3.3.1.

For rectified concentrated musts, use the solution obtained by diluting the sample to be analysed to 40 % (m/v) as indicated in the chapter ‘Total acidity’, section 5.1.2. Introduce 50 ml of this solution and 5 ml of phosphoric acid (2.2.2.1) into the 250 ml flask A of the entrainment apparatus. Connect the flask into the apparatus.

2.2.3.3.2.

Wines and musts If the estimated concentration in the sample is no greater than 50 mg of total SO2 per litre, place 50 ml of the sample and 15 ml of phosphoric acid (2.2.2.1) in the 250-ml flask A of the entrainment apparatus. Connect the flask to the apparatus. However, until ►M5  31 August 1996 ◄ at the latest, to analyse the sulphur dioxide content of grape juice, 5 ml of a 25 % solution (m/v) of phosphoric acid (2.2.2.1) shall be used. If the estimated concentration in the sample is greater than 50 mg of total SO2 per litre, place 20 ml of the sample and 5 ml of phosphoric acid (2.2.2.1) in the 100 ml flask A of the entrainment apparatus. Connect the flask to the apparatus. Place 2 to 3 ml of hydrogen peroxide solution (2.2.2.2) in the bubbler B, neutralized as before, and bring the wine in the flask A to the boil using a small flame of 4 to 5 cm height which should directly touch the bottom of the flask. Do not put the flask on a metal plate but on a disc with a hole of approximately 30 mm diameter in it. This is to avoid overheating substances extracted from the wine that are deposited on the walls of the flask. Maintain boiling while passing a current of air (or nitrogen). Within 15 minutes the total sulphur dioxide has been carried over and oxidized. Determine the sulphuric acid which has formed by titration with the 0,01 M sodium hydroxide solution (2.2.2.4). Let n ml be the volume used.

2.2.3.4.   Expression of results

Musts and wines: Total sulphur dioxide is expressed in mg/l.

Rectified concentrated must: Total sulphur dioxide is expressed in mg/kg of total sugar.

2.2.3.4.1.

Calculation For wines: Total sulphur dioxide in milligrams per litre: — samples low in sulphur dioxide (50 ml test sample): 6,4 n — other samples (20 ml test sample): 16 n For rectified concentrated musts: Total sulphur dioxide in milligrams per kilogram of total sugars (50 ml prepared test sample (2.2.3.3.1)): where P = percentage concentration (m/m) of total sugars

2.2.3.4.2.

Repeatability (r) 50 ml test sample < 50 mg/l; r = 1 mg/l. 20ml test sample > 50 mg/l; r = 6 mg/l.

2.2.3.4.3.

Reproducibility (R) 50ml test sample < 50 mg/l; R = 9 mg/l. 20ml test sample > 50 mg/l; R = 15 mg/l.

▼M8 —————

▼B

3.   MOLECULAR SULPHUR DIOXIDE

3.1.   Principle of the method

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

For a given temperature and alcoholic strength:

H2SO3 H+ + HSO3− ,

(1)

with

pKM

=

L

=

where

I

=

ionic strength,

A and B

=

coefficients varying with temperature and alcoholic strength,

KT

=

thermodynamic dissociation constant: values of pKT are given in Table 1 for various alcoholic strengths and temperatures,

KM

=

mixed dissociation constant.

Taking a mean value of 0,038 for the ionic strength I, Table 2 gives values of pKM for various temperatures and alcoholic strengths.

The molecular sulphur dioxide content calculated using expression (1) is given in Table 3 for various values of pH, temperature and alcoholic strength.

3.2.   Calculation

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

The molecular sulphur dioxide content in mg/l is:

X × C

where

C

=

the free sulphur dioxide content in mg/l.

26.   SODIUM

1.   PRINCIPLE OF THE METHODS

1.1.   Reference method: atomic absorption spectrophotometry

Sodium is determined directly in the wine by atomic absorption spectrophotometry after the addition of an ionization suppression agent (caesium chloride) to prevent ionization of sodium.

1.2.   Usual method: flame photometry

Sodium is determined directly in diluted wine (at least 1:10) by flame photometry.

2.   REFERENCE METHOD

2.1.   Reagents

2.1.1.   Solution containing 1 g of sodium per litre:

Use a standard commercial solution containing 1 g of sodium per litre. This solution may be prepared by dissolving 2,542 g of anhydrous sodium chloride, NaCl, in distilled water and making up to a volume of 1 litre.

Keep this solution in a polyethylene bottle.

2.1.2.   Matrix (model) solution:

2.1.3.   Caesium chloride solution containing 5 % caesium:

dissolve 6,330 g of caesium chloride, CsCl, in 100 ml of distilled water.

2.2.   Apparatus

2.2.1.

Atomic absorption spectrophotometer equipped with an air-acetylene burner.

2.2.2.

Sodium hollow cathode lamp.

2.3.   Procedure

2.3.1.   Preparation of sample

Pipette 2,5 ml of wine into a 50 ml volumetric flask, add 1 ml of the caesium chloride solution (2.1.3) and make up to the mark with distilled water.

2.3.2.   Calibration

Place 5,0 ml of the matrix solution in each one of a set of 100 ml volumetric flasks and add 0, 2,5, 5,0, 7,5 and 10 ml respectively of the 1 g/l sodium solution (2.1.1) previously diluted by 1:100. Add 2 ml of the caesium chloride solution (2.1.3) to each flask and make up to 100 ml with distilled water.

The standard solutions prepared in this way contain 0, 0,25, 0,50, 0,75 and 1,00 mg of sodium per litre respectively and each contains 1 g of caesium per litre. Keep these solutions in polyethylene bottles.

2.3.3.   Determination

Set the wavelength to 589,0 nm. Zero the absorbence scale using the matrix solution containing 1 g of caesium per litre (2.3.2). Aspirate the diluted wine directly into the burner of the spectrophotometer, followed in succession by the standard solutions (2.3.2). Read off the absorbences. Repeat each measurement.

2.4.   Expression of results

2.4.1.   Method of calculation

Plot a graph giving the absorbence as a function of the sodium concentration in the standard solutions.

Record the absorbence obtained with the diluted wine on this graph and determine its sodium concentration C in milligrams per litre.

The sodium concentration in milligrams per litre of the wine will then be 20C, expressed to the nearest whole number.

2.4.2.   Repeatability (r)

r

=

1 + 0,024 xi mg/l.

xi

=

concentration of sodium in the sample in mg/l.

2.4.3.   Reproducibility (R)

R

=

2,5 + 0,05 xi mg/l.

xi

=

concentration of sodium in the sample in mg/l.

▼M8 —————

▼B

27.   POTASSIUM

1.   PRINCIPLE OF THE METHODS

1.1.   Reference method

Potassium is determined directly in the diluted wine by atomic absorption spectrophotometry after the addition of an ionization suppression agent (caesium chloride) to prevent ionization of potassium.

1.2.   Usual method

Potassium is determined directly in the diluted wine by flame photometry.

2.   REFERENCE METHOD

2.1.   Reagents

2.1.1.   Solution containing 1 g of potassium per litre:

Use a standard commercial solution containing 1 g of potassium per litre. This solution may be prepared by dissolving 4,813 g of potassium hydrogen tartrate (C4H5KO6) in distilled water and making up the volume to 1 litre.

2.1.2.   Matrix (model) solution:

2.1.3.   Caesium chloride solution containing 5 % caesium:

dissolve 6,33 g of caesium chloride, CsCl, in 100 ml of distilled water.

2.2.   Apparatus

2.2.1.

Atomic absorption spectrophotometer, equipped with an air-acetylene burner.

2.2.2.

Potassium hollow cathode lamp.

2.3.   Procedure

2.3.1.   Preparation of sample

Pipette 2,5 ml of wine (previously diluted by 1:10) into a 50-ml volumetric flask, add 1 ml of the caesium chloride solution (2.1.3) and make up to the mark with distilled water.

2.3.2.   Calibration

Introduce 5,0 ml of the matrix solution (2.1.2) into each one of a set of 100-ml volumetric flasks and add 0, 2,0, 4,0, 6,0 and 8,0 ml respectively of the 1 g/l potassium solution (2.1.1)(previously diluted by 1:10). Add 2 ml of the caesium chloride solution (2.1.3) to each flask and make up to 100 ml with distilled water.

The standard solutions prepared in this way contain 0, 2, 4, 6 and 8 mg of potassium per litre respectively and each contains 1 g of caesium per litre. Keep these solutions in polyethylene bottles.

2.3.3.   Determination

Set the wavelength to 769,9 nm. Zero the absorbence scale using the matrix solution containing 1 g of caesium per litre (2.3.2). Aspirate the diluted wine (2.3.1) directly into the burner of the spectrophotometer, followed in succession by the standard solutions (2.3.2). Read off the absorbences. Repeat each measurement.

2.4.   Expression of results

2.4.1.   Method of calculation

Plot a graph giving the variation in absorbence as a function of the potassium concentration in the standard solutions.

Record the mean value of the absorbence obtained with the sample of diluted wine on this graph and determine its potassium concentration C in milligrams per litre.

The potassium concentration expressed in milligrams per litre of the wine to the nearest whole number will then be F × C, where F is the dilution factor (here 200).

2.4.2.   Repeatability (r)

r = 35 mg/l.

2.4.3.   Reproducibility (R)

R = 66 mg/l.

2.4.4.   Other ways of expressing results

In milliequivalents per litre: 0,0256 × F × C.

In mg potassium hydrogen tartrate per litre: 4,813 × F × C.

▼M8 —————

▼B

28.   MAGNESIUM

1.   PRINCIPLE OF THE METHOD

Magnesium is determined directly on wine, suitably diluted, by atomic absorption spectrophotometry.

2.   REAGENTS

2.1.   Concentrated standard solution containing 1 g magnesium per litre

Use a standard commercial magnesium solution (1 g/l). This solution may be prepared by dissolving 8,3646 g of magnesium chloride (MgCl2.6H2O) in distilled water and making up to 1 litre.

2.2.   Dilute standard solution containing 5 mg magnesium per litre.

Note: Keep the standard magnesium solutions in polyethylene bottles.

3.   APPARATUS

3.1.	Atomic absorption spectrophotometer fitted with an air-acetylene burner.

3.2.	Magnesium hollow cathode lamp.

4.   PROCEDURE

4.1.   Preparation of sample

Dilute the wine by 1:100 with distilled water.

4.2.   Calibration

Place 5, 10, 15 and 20 ml of the dilute standard magnesium solution (2.2) into each one of a set of 100 ml volumetric flasks and make up to 100 ml with distilled water. The standard solutions prepared in this way contain 0,25, 0,50, 0,75 and 1,0 mg of magnesium per litre respectively. These solutions should be kept in polyethylene bottles.

4.3.   Determination

Set the wavelength to 285 nm. Zero the absorbence scale using distilled water. Aspirate the diluted wine directly into the burner of the spectrophotometer, followed in succession by the standard solutions (4.2).

Read off the absorbences. Repeat each measurement.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Plot a graph of the variation in absorbence as a function of the magnesium concentration in the standard solutions. Record the mean value of the absorbence obtained with the diluted sample of wine on this graph and determine its magnesium concentration C in milligrams per litre.

The magnesium concentration in milligrams per litre of the wine to the nearest whole number will be 100 C.

5.2.   Repeatability (r)

r = 3 mg/l.

5.3.   Reproducibility (R)

R = 8 mg/l.

29.   CALCIUM

1.   PRINCIPLE OF THE METHOD

Calcium is determined directly on wine, suitably diluted, by atomic absorption spectrophotometry, after addition of an ionization suppression agent.

2.   REAGENTS

2.1.   Standard solution containing 1 g calcium per litre

Use a standard commercial calcium solution 1 g/l. This solution may be prepared by dissolving 2,5 g of calcium carbonate, CaCO3, in a quantity of 1:10 (v/v) HCl sufficient to dissolve it completely and making up to one litre with distilled water.

2.2.   Dilute standard solution containing 50 mg calcium per litre

Note: Keep the standard calcium solutions in polyethylene bottles.

2.3.   Lanthanum chloride solution containing 50 g lanthanum per litre

Dissolve 13,369 g of lanthanum chloride, LaCl3 · 7H2O, in distilled water; add 1 ml of HCl diluted 1:10 (v/v) and make up to 100 ml.

3.   APPARATUS

3.1.	Atomic absorption spectrophotometer fitted with an air-acetylene burner.

3.2.	Calcium hollow cathode lamp.

4.   PROCEDURE

4.1.   Preparation of sample

Place 1 ml of the wine, 2 ml of the lanthanum chloride solution (2.3) in a 20 ml volumetric flask and make up to the mark with distilled water. The wine, diluted by 1:20, contains 5 g lanthanum per litre.

Note: For sweet wines, the concentration of 5 g lanthanum per litre is sufficient provided the dilution does not bring the sugar content to below 2,5 g per litre. For wines with higher concentrations of sugar, the lanthanum concentration should be increased to 10 g per litre.

4.2.   Calibration

Place 0, 5, 10, 15 and 20 ml of the dilute standard calcium solution (2.2) respectively into a set of 100 ml volumetric flasks, add to each flask 10 ml of the lanthanum chloride solution (2.3) and make up to 100 ml with distilled water. The solutions prepared in this way contain 0, 2,5, 5,0, 7,5 and 10 mg of calcium per litre respectively and each contains 5 g of lanthanum per litre. These solutions should be kept in polyethylene bottles.

4.3.   Determination

Set the wavelength to 422,7 nm. Zero the absorbence scale using the solution containing 5 g of lanthanum per litre (4.2). Aspirate the diluted wine directly into the burner of the spectrophotometer, followed in succession by the five standard solutions (4.2). Read the absorbences. Repeat each measurement.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Plot a graph giving the variation in absorbence as a function of the calcium concentration in the standard solutions.

Record the mean value of the absorbence obtained with the sample of diluted wine on this graph and determine its calcium concentration C. The calcium concentration in milligrams per litre of the wine to the nearest whole number will be 20 C.

5.2.   Repeatability (r)

Concentration < 60 mg/l: r

=

2,7 mg/l.

Concentration > 60 mg/l: r

=

4 mg/l.

5.3.   Reproducibility (R)

R

=

0,114xi − 0,5.

xi

=

concentration in the sample in mg/l.

30.   IRON

1.   PRINCIPLE OF THE METHODS

Reference method

After suitable dilution of the wine and removal of alcohol, iron is determined directly by atomic absorption spectrophotometry.

Usual method

After digestion in 30 % hydrogen peroxide solution, the total iron, now in the Fe(III) state, is reduced to the Fe(II) state and is determined using the coloration produced by orthophenanthroline.

2.   REFERENCE METHOD

2.1.   Reagents

2.1.1.

Concentrated standard iron solution containing 1 g Fe(III) per litre. Use a standard commercial solution (1 g/l). This solution may be prepared by dissolving 8,6341 g of ferric ammonium sulphate (FeNH4(SO4)2 · 12H2O) in distilled water slightly acidified with 1 M hydrochloric acid and making up to one litre.

2.1.2

Dilute standard iron solution containing 100 mg iron per litre.

2.2.   Apparatus

2.2.1

Rotary evaporator with thermostatically controlled waterbath.

2.2.2.

Atomic absorption spectrophotometer equipped with an air-acetylene burner.

2.2.3.

Iron hollow cathode lamp.

2.3.   Procedure

2.3.1.   Preparation of sample

Remove the alcohol from the wine by reducing the volume of the sample to half its original volume using a rotary evaporator (50 to 60 °C). Make up to the original volume with distilled water.

If necessary, dilute prior to the determination.

2.3.2.   Calibration

Place 1, 2, 3, 4 and 5 ml of the solution containing 100 mg iron per litre (2.1.2) respectively into a set of 100 ml volumetric flasks and make up to 100 ml with distilled water. The solutions prepared in this way contain 1, 2, 3, 4 and 5 mg of iron per litre respectively.

These solutions should be kept in polyethylene bottles.

2.3.3.   Determination

Set the wavelength to 248,3 nm. Zero the absorbence scale using distilled water. Aspirate the diluted sample directly into the burner of the spectrophotometer, followed in succession by the five standard solutions (2.3.2). Read off the absorbences. Repeat each measurement.

2.4.   Expression of results

2.4.1.   Method of calculation

Plot a graph giving the variation in absorbence as a function of the iron concentration in the standard solutions. Record the mean value of the absorbence obtained with the diluted wine sample on this this graph and determine its iron concentration C.

The iron concentration in milligrams per litre of the wine to one decimal place will be F.C, where F is the dilution factor.

▼M8 —————

▼B

31.   COPPER

1.   PRINCIPLE OF THE METHOD

The method is based on the use of atomic absorption spectrophotometry.

2.   APPARATUS

2.1.	Platinum dish.

2.2.	Atomic absorption spectrophotometer.

2.3.	Copper hollow cathode lamp.

2.4.	Gas supplies: air-acetylene or nitrous oxide/acetylene.

3.   REAGENTS

3.1.	Metallic copper.

3.2.	Nitric acid, HNO3, concentrated 65 %, ρ20 = 1,38 g/ml.

3.3.	Dilute nitric acid, 1:2 (v/v).

3.4.

Solution containing copper at 1 g/l. Use a standard commercial copper solution. This solution may be prepared by weighing 1,000 g of metallic copper and transferring it without loss to a 1 000 ml volumetric flask. Add 1:2 (v/v) dilute nitric acid (3.3) in just sufficient quantity to dissolve the metal, add 10 ml of concentrated nitric acid (3.2) and make up to the mark with doubly distilled water.

3.5.	Solution containing copper at 100 mg/l. Introduce 10 ml of the solution prepared as in 3.4 into a 100 ml volumetric flask and make up to the mark with doubly distilled water.

4.   PROCEDURE

4.1.   Preparation of sample and determination of copper

If required, prepare a suitably dilute solution with doubly distilled water.

4.2.   Calibration

Pipette 0,5, 1 and 2 ml of solution 3.5 (100 mg of copper per litre) into 100 ml volumetric flasks and make up to the volume with doubly distilled water: the solutions so obtained contain 0,5, 1 and 2 mg of copper per litre respectively.

4.3.	Measure the absorbence at 324,8 nm. Set the zero with doubly distilled water. Measure directly the absorbence of successive standard solutions prepared in 4.2, Carry out in duplicate.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Plot a graph giving the variations in absorbence as a function of the copper concentrations in the standard solutions.

Using the measured absorbence of the samples, read off the concentration C in mg/l from the calibration curve.

If F is the dilution factor, the concentration of the copper present is given in milligrams per litre by F × C. It is quoted to two decimal places.

Notes:

32.   CADMIUM

1.   Principle

The cadmium is determined directly in the wine by non-flame atomic absorption spectrophotometry.

2.   APPARATUS

All the glassware must be washed prior to use in concentrated nitric acid heated to 70 to 80 °C and rinsed in double distilled water.

2.1.	Atomic absorption spectrophotometer equipped with a graphite oven, background correction and a multipotentiometer.

2.2.	Cadmium hollow cathode lamp.

2.3.	5 µl micropipettes with special tips for atomic absorption measurements.

3.   REAGENTS

The water used must be double distilled using borosilicate glass apparatus, or water of a similar purity. All reagents must be of recognized analytical reagent grade and, in particular, free of cadmium.

3.1.	85 % phosphoric acid (ρ20 = 1,71 g/ml).

3.2.	Phosphoric acid solution obtained by diluting 8 ml of phosphoric acid with water to 100 ml.

3.3.	A 0,02 M solution of di-sodium salt of ethylene diamine tetra-acetic acid (EDTA).

3.4.	pH 9 buffer solution: dissolve 5,4 g of ammonium chloride in a few millilitres of water in a 100 ml volumetric flask, add 35 ml of ammonium hydroxide solution (ρ20 = 0,92 g/ml) diluted to 25 % (v/v) and make up to 100 ml with water.

3.5.	Eriochrome black T: 1 % (w/w) solid solution in sodium chloride.

3.6.

Cadmium sulphate (CdSO4·8H2O). The titre of the cadmium sulphate must be verified using the following method: Weigh exactly 102,6 mg of the cadmium sulphate sample into a cylindrical vessel with some water and shake until dissolved; add 5 ml of the pH 9 buffer solution and approximately 20 mg of eriochrome black T. Titrate with the EDTA solution until the indicator begins to turn blue. The volume of EDTA added must be equal to 20 ml. If the volume is slightly different, correct the weighed test portion of cadmium sulphate used in the preparation of the reference solution accordingly.

3.7.	Cadmium reference solution at 1 g per litre. Use a standard commercial grade solution. This solution may be obtained by dissolving 2,2820 g of cadmium sulphate in water and making up to one litre. Keep the solution in a borosilicate glass bottle with a ground glass stopper.

4.   PROCEDURE

4.1.   Preparation of the sample

Dilute the wine to 1:2 (v/v) with the phosphoric acid solution.

4.2.   Preparation of the calibration range of solutions

Using the cadmium reference solution, prepare successive dilutions with titres of 2,5, 5, 10 and 15 µg of cadmium per litre respectively.

4.3.   Determination

4.3.1.   Programming of oven (for guidance only):

Drying at 100 °C for 30 seconds

Mineralization at 900 °C for 20 seconds

Atomization at 2 250 °C for 2 to 3 seconds

Nitrogen flow (flushing gas): 6 litres/minute

Note: At the end of the procedure, increase the temperature to 2 700 °C to clean the oven.

4.3.2.   Atomic absorption measurements:

Select wavelength 228,8 nm. Set the zero on the absorbence scale with double distilled water. Using a micropipette, introduce into the oven three 5-µl portions of each of the solutions in the calibration range and of the sample solution to be analysed. Record the absorbences measured. Calculate the mean absorbence value from the results for the three portions.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Draw the absorbence variation curve as a function of the concentrations of cadmium in the solutions in the calibration range. Variation is linear. Enter the mean absorbence value of the sample solution on the calibration curve, derive from it the cadmium concentration C. The cadmium concentration expressed in micrograms per litre of wine is equal to:

2 C

33.   SILVER

1.   PRINCIPLE OF THE METHOD

The method is based on the use of atomic absorption spectrophotometry after ashing of the sample.

2.   APPARATUS

2.1.	Platinum dish.

2.2.	Waterbath, thermostatically controlled to 100 °C.

2.3.	Furnace controlled to 500 to 525 °C.

2.4.	Atomic absorption spectrophotometer.

2.5.	Silver hollow cathode lamp.

2.6.	Gas supplies: air, acetylene.

3.   REAGENTS

3.1.	Silver nitrate, AgNO3.

3.2.	Nitric acid, HNO3, concentrated 65 %, ρ20 = 1,38 g/ml.

3.3.	Dilute nitric acid, 1:10 (v/v).

3.4.	Solution containing silver at 1 g/l. Use a standard commercial silver solution. This solution may be prepared by dissolving 1,575 g of silver nitrate in dilute nitric acid and making up to a volume of 1 000 ml with dilute nitric acid (3.3).

3.5.	Solution containing silver at 10 mg/l. 10 ml of the solution prepared as in 3.4 are diluted to 1 000 ml with dilute nitric acid.

4.   PROCEDURE

4.1.   Preparation of sample

Place 20 ml of the sample in a platinum dish and evaporate to dryness over a boiling waterbath. Ash in the furnace at 500 to 525 °C. Moisten the white ash with 1 ml of concentrated nitric acid (3.2). Evaporate over a boiling waterbath, repeat the addition of 1 ml nitric acid (3.2) and evaporate a second time. Add 5 ml of dilute nitric acid (3.3) and heat slightly until dissolved.

4.2.   Calibration

Pipette 2, 4, 6, 8, 10 and 20 ml of solution 3.5 (10 mg of silver per litre) respectively into a set of 100 ml volumetric flasks and make up to the mark with dilute nitric acid (3.3): the solutions so obtained contain 0,20, 0,40, 0,60, 0,80, 1,0 and 2,0 mg of silver per litre respectively.

4.3.	Set the wavelength to 328,1 nm. Set zero using doubly distilled water. Measure the absorbence directly of successive standard solutions prepared in 4.2. Carry out in duplicate.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Plot a graph giving the variations in absorbence as a function of the silver concentrations in the standard solutions.

Using the measured absorbence of the sample, read off the concentration C in mg/l from the calibration curve.

The concentration of silver in the wine is given in milligrams per litre by 0,25 C. It is quoted to two decimal places.

Note:

Select the concentration of the solutions for the preparation of the calibration curve, the volume of the sample taken and the final volume of the liquid to be appropriate to the sensitivity of the apparatus to be used.

34.   ZINC

1.   PRINCIPLE OF THE METHOD

After removal of alcohol, zinc is determined directly in the wine by atomic absorption spectrophotometry.

2.   REAGENTS

The water used in borosilicate glass apparatus must be doubly distilled or of an equivalent degree of purity.

2.1.	Standard solution containing 1 g of zinc per litre: use a commercial standard zinc solution. This solution may be prepared by dissolving 4,3975 g of zinc sulphate. (ZnSO4 × 7H2O) in water and making up the volume to one litre.

2.2.	Dilute standard solution containing 100 mg of zinc per litre.

3.   APPARATUS

3.1.	Rotary evaporator with thermostatically controlled waterbath.

3.2.	Atomic absorption spectrophotometer equipped with an air-acetylene burner.

3.3.	Zinc hollow cathode lamp.

4.   PROCEDURE

4.1.   Preparation of sample

Remove the alcohol from 100 ml of the wine by reducing the volume of the sample to half its original volume using a rotary evaporator (50 to 60 °C). Make up to the original volume of 100 ml with doubly distilled water.

4.2.   Calibration

Place 0,5, 1, 1,5 and 2 ml of the solution containing 100 mg zinc per litre (2.2) into each one of a set of 100-ml volumetric flasks and make up to the mark with doubly distilled water. The solutions prepared in this way contain 0,5, 1, 1,5 and 2 mg of zinc per litre respectively.

4.3.   Determination

Set the wavelength to 213,9 nm. Zero the absorbence scale using doubly distilled water. Aspirate the wine directly into the burner of the spectrophotometer, followed in succession by the four standard solutions. Read the absorbences. Repeat each measurement.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Plot a graph giving the variation in absorbence as a function of zinc concentration in the standard solutions. Record the mean value of the absorbence obtained with the diluted wine sample on this graph and determine its zinc concentration to one decimal place.

35.   LEAD

1.   PRINCIPLE

The lead is determined directly in the wine by non-flame atomic absorption spectrophotometry.

2.   APPARATUS

All the glassware must be washed prior to use in concentrated nitric acid heated to 70 to 80 °C and rinsed in double distilled water.

2.1.	Atomic absorption spectrophotometer equipped with a graphite oven, non-specific absorption correction and a multipotentiometer.

2.2.	Lead hollow cathode lamp.

2.3.	5 µl micropipettes with special tips for atomic absorption measurements.

3.   REAGENTS

All reagents must be of recognized analytical reagent grade, and in particular, free of lead. The water used must be doubly distilled using borosilicate glass apparatus, or water of a similar purity.

3.1.	85 % phosphoric acid (ρ20 = 1,71 g/ml).

3.2.	Phosphoric acid solution obtained by diluting 8 ml of phosphoric acid with water to 100 ml.

3.3.	Nitric acid (ρ20 = 1,38 g/ml).

3.4.	Lead solution at 1 g per litre. Use a standard commercial grade solution. This solution may be obtained by dissolving 1,600 g of lead (II) nitrate, Pb(NO3)2 in nitric acid diluted to 1 % (v/v) and made up to one litre. Keep the solution in a borosilicate glass bottle with a ground glass stopper.

4.   PROCEDURE

4.1.   Preparation of the sample

Dilute the wine to 1:2 or 1:3 with the phosporic acid solution, depending on the presumed lead concentration.

4.2.   Preparation of the calibration range of solutions

Using the lead reference solution, prepare successive solutions with titres of 2,5, 5, 10 and 15 µg of lead per litre respectively, by diluting with double distilled water.

4.3.   Determination

4.3.1   Programming of oven (for guidance only):

Drying at 100 °C for 30 seconds

Mineralization at 900 °C for 20 seconds

Atomization at 2 250 °C for 2 to 3 seconds

Nitrogen flow (flushing gas): 6 litres/minute.

Note: At the end of the procedure, increase the temperature to 2 700 °C to cleanse the oven.

4.3.2.   Measurements

Select wavelength 217 nm. Set the zero on the absorbence scale with doubly distilled water. Using a micropipette, introduce into the programmed oven three 5-µl portions of each of the solutions in the calibration range and of the sample solution to be analysed. Record the absorbences measured. Calculate the mean absorbence value from the results for the three portions.

5.   EXPRESSION OF RESULTS

5.1.   Method of calculation

Draw the absorbence variation curve as a function of the concentrations of lead in the calibration range. The variation is linear. Carry over the mean absorbence value of the sample solution on the calibration curve, derive from it the lead concentration C. The lead concentration expressed in micrograms per litre of wine is equal to:

C × F

Where

F

=

the dilution factor.

36.   FLUORIDES

1.   PRINCIPLE

The fluoride content of the wine, added to a buffer solution, is determined using a solid membrane selective electrode. The measured potential is proportionate to the logarithm of the activity of the fluoride ions in the medium being analysed, in accordance with the following equation:

Where

E

=

potential of the ion-selective electrode measured against the reference electrode in the medium being analysed;

Eo

=

standard potential of the sensor;

S

=

slope of the ion-selective electrode (Nernst factor). At 25 °C the theoretical slope is equal to 59,2 mV;

a F

=

activity of the fluoride ions in the solution being analysed.

2.   APPARATUS

2.1.	Fluoride-ion-selective crystal membrane electrode.

2.2.	Reference electrode (calomel or Ag/AgCl).

2.3.	Millivoltmeter (pH meter with extended scale in millivolts), accurate to 0,1 mV.

2.4.	Magnetic stirrer with an insulating plate to protect the analysis solution from the heat of the motor. Stirring vessel covered with plastic (polythene or equivalent material).

2.5.	Plastic beakers with a capacity of 30 or 50 ml, and plastic bottles (polythene or equivalent material).

2.6.	Precision pipettes (pipettes graduated in microlitres or any other equivalent pipettes).

3.   REAGENTS

3.1.

Stock fluoride solution of 1 g/l. Use standard commercial quality solution of 1 g/l. This solution can be prepared by dissolving 2,210 g of sodium fluoride (dried for three to four hours at 105 °C) in distilled water. Make up to one litre with distilled water. The solution is kept in a plastic bottle.

3.2.	Standard fluoride solutions of appropriate concentration are prepared by diluting the stock solution with distilled water and kept in plastic bottles. Solutions the fluoride content of which is in mg/l must not be prepared in advance.

3.3.

Buffer solution, pH 5,5 10 g of trans-1,2-dicmiaocyclohexane tetra-acetic acid (CDTA) are added to water (about 50 ml); add a solution containing 58 g of sodium chloride and 29,4 g of trisodium citrate in 700 ml of distilled water. The CDTA is dissolved by adding approximately 6 ml of 32 % (m/v) sodium hydroxide solution. Lastly, add 57 ml of acetic acid (ρ20 = 1,05 g/ml) and bring the pH to 5,5 with 32 % sodium hydroxide solution (about 45 ml). Leave to cool and make up to one litre with distilled water.

4.   PROCEDURE

Preliminary comment:

Care should be taken to ensure that all the solutions remain at a temperature of 25 °C (± 1 °C) during measurement. (A deviation of more than 1 °C causes a modification of about 0,2 mV.)

4.1.   Direct method

Place a defined volume of wine in a plastic beaker with an equal volume of buffer solution.

The solution is stirred in an even and moderate manner. When the indicator is stable (stability is reached when the potential varies by not more than 0,2 to 0,3 mV/three minutes), read the value of the potential in mV.

4.2.   The known additions method

Stirring continuously, add a known volume of standard fluoride solution to the analysis medium using a precision pipette. When the indicator is stable, read the value of the potential in mV.

The concentration of the standard solution to be added is selected as follows:

The approximate concentration of the analysis medium is read on a calibration line drawn on a semi-logarithmic scale with the standard fluoride solutions with titres of 0,1, 0,2, 0,5, 1,0, 2,0 mg/l.

Note: If the approximate concentration of the analysis medium lies outside the concentration range of the standard solutions, dilute the sample.

Example:

If the approximate fluoride content of the analysis medium (20 ml volume) is 0,25 mg/l F−; the concentration must be increased by 0,25 mg/l. To do this, use the appropriate delivery pipette to add, for example, 0,20 ml (= 1 %) of a standard solution containing 25 mg/l F− or 0,050 ml of a standard solution with 100 mg/l F−.

5.   CALCULATIONS

The fluoride content of the analysis medium expressed in mg/l is obtained by applying the following formula:

C F

=

fluoride concentration of the analysis medium (mg/l);

C a

=

concentration of fluoride added (mg/l) to analysis medium (V a );

V o

=

initial volume of the analysis medium before overloading (ml);

V a

=

volume of the overloaded solution (ml);

ΔE

=

difference between potentials E1 and E2 obtained in 4.1 and 4.2 (mV);

S

=

slope of the electrode in the analysis solution.

If V a is very close to V o (see 4.2), the following simplified formula is applied:

The value obtained must be multiplied by the dilution factor arising from addition of the buffer solution.

37.   CARBON DIOXIDE

1.   PRINCIPLE OF METHODS

1.1.   Reference method

1.1.1.   Still wines (CO2 over pressure ≤ 0,5 × 105 Pa) ( 13 )

The volume of wine taken from the sample is cooled to around 0 °C and mixed with a sufficient quantity of sodium hydroxide to give a pH of 10 to 11. Titration is carried out with an acid solution in the presence of carbonic anhydrase. The carbon dioxide content is calculated from the volume of acid needed to change the pH from 8,6 (bicarbonate form) to 4,0 (carbonic acid). A blank titration is carried out in the same conditions on decarbonated wine in order to take account of the volume of sodium hydroxide solution taken up by the wine acids.

1.1.2.   Sparkling and semi-sparkling wines

The sample of wine to be analysed is cooled near to freezing point. After removal of a quantity to be used as a blank after decarbonation, the remainder of the bottle is made alkaline to fix all the carbon dioxide in the form of Na2CO3. Titration is carried out with an acid solution in the presence of carbonic anhydrase. The carbon dioxide content is calculated from the volume of acid solution needed to change the pH from 8,6 (bicarbonate form) to 4,0 (carbonic acid). A blank titration is carried out in the same conditions in decarbonated wine in order to take account of the volume of sodium hydroxide taken up by the wine acids.

1.2.   Usual method: sparkling and semi-sparkling wines

Manometric method: the excess pressure of the carbon dioxide is measured directly in the bottle using an aphrometer.

2.   REFERENCE METHOD

2.1.

Still wines (CO2 over pressure ≤ 0,5 × 105 Pa) 2.1.1.   Apparatus 2.1.1.1. Magnetic stirrer. 2.1.1.2. pH meter. 2.1.2.   Reagents 2.1.2.1. Sodium hydroxide solution, NaOH, 0,1 M. 2.1.2.2. Sulphuric acid solution, H2SO4, 0,05 M. 2.1.2.3. Carbonic anhydrase solution, 1 g/l. 2.1.3.   Procedure Cool the wine sample to approximately 0 °C together with the 10 ml pipette used for sampling. Place 25 ml of sodium hydroxide solution (2.1.2.1) in a 100 ml beaker; add two drops of aqueous solution of carbonic anhydrase (2.1.2.3). Introduce 10 ml of wine using the pipette cooled to 0 °C. Place the beaker on the magnetic stirrer, set up the pH electrode and stir moderately. When the liquid has reached room temperature, titrate slowly with the sulphuric acid solution (2.1.2.2) until the pH reaches 8,6. Note the burette reading. Continue titrating with the sulphuric acid (2.1.2.2) until the pH reaches 4,0. Let n ml be the volume used between pH 8,6 and 4,0. Remove CO2 from approximately 50 ml of the wine sample by agitation under vacuum for three minutes, the flask being heated in a waterbath to about 25 °C. Carry out the above procedure on 10 ml of the decarbonated wine. Let n′ ml be the volume used. 2.1.4.   Expression of results 1 ml of the titrated 0,1 M sodium hydroxide solution corresponds to 4,4 mg of CO2. The quantity of CO2 in grams per litre of wine is given by the formula: 0,44 (n − n′) It is quoted to two decimal places. Note: Where wines contain little CO2 (CO2< 1 g/l), the addition of carbonic anhydrase to catalyse the hydration of CO2 is unnecessary.

2.2.

Sparkling and semi-sparkling wines 2.2.1.   Apparatus 2.2.1.1. Magnetic stirrer. 2.2.1.2. pH meter. 2.2.2.   Reagents 2.2.2.1. Sodium hydroxide, NaOH, 50 % (m/m). 2.2.2.2. Sulphuric acid solution, H2SO4, 0,05 M. 2.2.2.3. Carbonic anhydrase solution, 1 g/l. 2.2.3.   Procedure Mark the level of wine in the bottle and then cool until freezing begins. Allow the bottle to warm up slightly, while shaking, until ice crystals disappear. Remove the stopper rapidly and place 45 to 50 ml of wine in a measuring cylinder for blank titration. The exact volume removed, ν ml, is determined by reading on the cylinder after it has returned to room temperature. Immediately after the blank sample has been removed, add 20 ml of the sodium hydroxide solution (2.2.2.1) in the bottle with a capacity of 750 ml. Wait until the wine has reached room temperature. Place 30 ml of boiled distilled water and two drops of the carbonic anhydrase solution (2.2.2.3) into a 100 ml beaker. Add 10 ml of wine which has been made alkaline. Place the beaker on the magnetic stirrer, set up the electrode and magnetic rod and stir moderately. Titrate with the sulphuric acid solution (2.2.2.2) slowly until the pH reaches 8,6. Note the burette reading. Continue titrating slowly with the sulphuric acid (2.2.2.2) until the pH reaches 4,0. Let n ml be the volume used between pH 8,6 and 4,0. Remove CO2 from the ν ml of wine placed on one side for the blank titration by agitating under vacuum for three minutes, the flask being heated in a waterbath at about 25 °C. Remove 10 ml of decarbonated wine and add to 30 ml of boiled distilled water, add two to three drops of sodium hydroxide solution (2.2.2.1) to bring the pH to 10 to 11. Then follow the above procedure. Let n′ ml be the volume of 0,5 M sulphuric acid added. 2.2.4.   Expression of results 1 ml of 0,05 M sulphuric acid corresponds to 4,4 mg of CO2. Empty the bottle of wine which has been made alkaline and determine to within 1 ml the initial volume of wine by making up to the mark with water, say V ml. The quantity of CO2 in grams per litre of wine is given by the following formula: The result is quoted to two decimal places.

2.3.	Expression of results The excess pressure at 20 °C (Paph20) expressed in pascals is given by the formula: where: Q = CO2 content in g/l of wine, A = the alcoholic strength of wine at 20 °C, S = the sugar content of the wine in g/l, Patm = the atmospheric pressure, expressed in pascals.

▼M8 —————

▼B

4.   RELATIONSHIP BETWEEN THE PRESSURE AND THE QUANTITY OF CARBON DIOXIDE CONTAINED IN A SEMI-SPARKLING WINE ( 14 )

From the excess pressure at 20 °C (Paph20), the absolute pressure at 20 °C (Pabs20) is calculated using the formula:

Pabs20 = Patm + Paph20

where Patm is the atmospheric pressure expressed in bars.

The quantity of carbon dioxide contained in a wine is given by the following relationships:

▼M7

38.   CYANIDE DERIVATIVES

(Caution: comply with safety measures for handling chemicals chloramine T, pyridine, potassium cyanide, hydrochloric acid, and phosphoric acid. Dispose of used products in the proper way, in compliance with environmental rules in force. Caution with hydrocyanic acid released during the distillation of acidified wine.)

1.   PRINCIPLE

The total free hydrocyanic acid in the wine is released by acid hydrolysis and separated by distillation. After reacting with chloramine-T and pyridine, the glutaconic dialdehyde formed is determined by colorimetry on the basis of the blue coloration it gives with 1,3-dimethyl-barbituric acid.

2.   APPARATUS

2.1.   Distillation apparatus

Use the distillation apparatus described for determining the alcohol content of wine

2.2.	500-ml round-bottomed flask with standardised ground joints

2.3.	Water bath thermostatically controlled at 20 °C

2.4.	Spectrophotometer allowing absorbance to be measured at wavelength of 590 nm

2.5.	Glass cells or single-use cells with optical paths of 20 mm

3.   REAGENTS

3.1.	Phosphoric acid (H3PO4) at 25 % (m/v)

3.2.	Chloramine-T solution (C7H7ClNNa O2S, 3H2O) 3 % (m/v)

3.3.	Solution of 1,3-dimethyl-barbituric acid: dissolve 3,658 g 1,3-dimethyl-barbituric acid (C6H8N2O3) in 15 ml pyridine and 3 ml hydrochloric acid (ρ20=1,19 g/ml) and add 50 ml distilled water

3.4.	Potassium cyanide (KCN)

3.5.	Potassium iodide (KI) solution at 10 % (m/v)

3.6.	Silver nitrate solution (AgNO3), 0,1 M

4.   PROCEDURE

4.1.   Distillation

Put 25 ml wine, 50 ml distilled water, 1 ml phosphoric acid (3.1) and a few glass beads into the 500-ml flask (2.2). Place flask immediately on the distillation apparatus. Use a tapering tube to conduct the distillate into a 50-ml calibrated flask containing 10 ml water. Immerse calibrated flask in iced water. Collect 30 to 35 ml of distillate (or around 45 ml liquid in total) in the calibrated flask.

Flush the tapering tube of the condenser with a few millilitres of distilled water, bring distillate to 20 °C and fill to the calibration line with distilled water.

4.2.   Measurement

Put 25 ml distillate in a 50-ml conical flask with a ground glass stopper, add 1 ml chloramine-T solution (3.2) and seal with the stopper. After exactly 60 seconds add 3 ml of 1,3-dimethyl-barbituric acid solution (3.3), seal with stopper and leave for 10 minutes. Then measure absorbance against a control (25 ml distilled water instead of 25 ml distillate) at wavelength of 590 nm in cells with optical paths of 20 mm.

5.   DETERMINING THE CALIBRATION CURVE

5.1.   Argentometric titration of the potassium cyanide

Dissolve around 0,2 g KCN (3.4), carefully measured, in 100 ml distilled water in a 300-ml calibrated flask. Add 0,2 ml of potassium iodide solution (3.5) and titrate with the 0,1 M silver nitrate solution (3.6) until a stable yellowish colouring is obtained.

Taking 1 ml of 0,1 M silver nitrate solution as corresponding to 13,2 mg KCN, calculate the concentration of the KCN sample.

5.2.   Standard curve

5.2.1.   Preparation of standard solutions

Having established the concentration of the KCN according to the procedure set out in 5.1, prepare a standard solution containing 30 ml/l hydrocyanic acid (30 ml HCN ≅ 72,3 ml KCN). Dilute the solution to 1/10.

Introduce 1,0 ml, 2,0 ml, 3,0 ml, 4,0 ml and 5,0 ml of the diluted sample solution into the 100-ml calibrated flasks and fill to the calibration line with distilled water. The solutions prepared contain 30 μg, 60 μg, 90 μg, 120 μg and 150 μg of hydrocyanic acid per litre respectively.

5.2.2.   Titration

Take 25-ml samples of the solutions thus obtained and continue as indicated above at 4.1 and 4.2.

The values obtained for absorbance with regard to the standard solutions as a function of the corresponding hydrocyanic acid content should produce a straight line passing through the origin.

6.   EXPRESSION OF RESULTS

The hydrocyanic acid is expressed in micrograms per litre (μg/l) with no decimal places.

6.1.   Method of calculation

Read off the hydrocyanic acid content on the calibration curve. If the sample has been diluted, multiply the result by the dilution factor.

Repeatability (r) and reproducibility (R)

White wine

=

r = 3,1 μg/l or approximately 6 %· xi R = 12 μg/l or approximately 25 %· xi

Red wine

=

r = 6,4 μg/l or approximately 8 %· xi R = 23 μg/l or approximately 29 %· xi

xi

=

average concentration of HCN in the wine

xi

=

48,4 μg/l for white wine

xi

=

80,5 μg/l for red wine.

▼B

39.   ALLYL ISOTHIOCYANATE

1.   PRINCIPLE OF THE METHOD

Any allyl isothiocyanate present in the wine is collected by distillation and identified by gas chromatography.

2.   REAGENTS

2.1.	Ethanol, absolute.

2.2.	Standard solution: solution of allyl isothiocyanate in absolute alcohol containing 15 mg of allyl isothiocyanate per litre.

2.3.	Freezing mixture consisting of ethanol and dry ice (temperature −60 °C).

3.   APPARATUS

3.1.	Distillation apparatus as shown in the figure overleaf. A stream of nitrogen is passed continuously through the apparatus.

3.2.	Heating mantle, thermostatically controlled.

3.3.	Flowmeter.

3.4.	Gas chromatograph fitted with a flame spectrophotometer detector equipped with a selective filter for sulphur compounds (wavelength = 394 nm) or any other suitable detector.

3.5.	Stainless steel chromatograph column of internal diameter 3 mm and length 3 m filled with Carbowax 20M at 10 % on Chromosorb WHP, 80 to 100 mesh.

3.6.	Microsyringe, 10µl.

4.   PROCEDURE

Put two litres of wine into the distillation flask, introduce a few millilitres of ethanol (2.1) into the two collecting tubes so that the porous parts of the gas dispersion rods are completely immersed. Cool the two tubes externally with the freezing mixture. Connect the flask to the collecting tubes and begin to flush the apparatus with nitrogen at a rate of three litres per hour. Heat the wine to 80 °C with the heating mantle, distil and collect 45 to 50 ml of the distillate.

Stabilize the chromatograph. It is recommended that the following conditions are used:

With the microsyringe, introduce a volume of the standard solution such that the peak corresponding to the allyl isothiocyanate can easily be identified on the gas chromatogram.

Similarly introduce an aliquot of the distillate into the chromatograph. Check that the retention time of the peak obtained corresponds with that of the peak of allyl isothiocyanate.

Under the conditions described above, compounds naturally present in the wine will not produce interfering peaks on the chromatogram of the sample solution.

Apparatus for distillation under a current of nitrogen

40.   CHROMATIC PROPERTIES

1.   WINES AND MUSTS

1.1.   Definitions

The chromatic properties of a wine are defined as its luminosity and its chromaticity.

The luminosity is represented by the transmittance and it varies inversely with the colour intensity of the wine.

The chromaticity is represented by the dominant wavelength (which characterizes the tint) an the purity of the colour.

By convention, and for reasons of convenience, the chromatic properties of red and rosé wines are given as the colour intensity and the tint, in keeping with a procedure adopted as the usual method.

1.2.   Principle of the methods

1.2.1.   Reference method

This is a spectrophotometric method which makes it possible to determine the tristimulus values and three chromaticity coordinates necessary for the specification of the colour as laid down by the International Commission on Illumination (CIE).

1.2.2.   Usual method (applicable to red and rosé wines)

This is a spectrophotometric method by which the chromatic properties are expressed by convention as follows:

The colour intensity is given by the sum of the absorbences at wavelengths af 420, 520 and 620 nm for radiation traversing a 1 cm optical path in the sample.

The tint is expressed by the ratio of the absorbences at 420 nm and 520 nm.

1.3.   Reference method

1.3.1.   Apparatus

1.3.1.1.

Spectrophotometer enabling measurements to be made between 300 and 700 nm.

1.3.1.2.

Glass cells in pairs, with optical paths, b, equal to 0,1, 0,2, 0,5, 1,2 and 4 cm.

1.3.2.   Procedure

1.3.2.1.   Preparation of the sample

Cloudy wine must be clarified by centrifugation. The bulk of the carbon dioxide in young and sparkling wines must be removed by shaking under vacuum.

1.3.2.2.   Measurements

The optical path, b, in the glass cell should be so chosen that the measured absorbence lies between 0,3 and 0,7.

The following guidance is given for the appropriate choice of the optical path: use cells of 2 (or 4) cm optical path for white wines, with 1 cm for rosé wines and with 0,1 cm (or 0,2 cm) for red wines.

The spectrophotometric measurements should be made using distilled water, in a cell with the same optical path, b, as reference liquid to establish the zero of the absorbence scale at wavelengths 445, 495, 550 and 625 nm.

The four corresponding absorbences for the wine should then be measured to three decimal places for the optical path, b. Let these be A 445, A 495, A 550, A 625.

1.3.3.   Calculations

Together with Table I, use these values of the absorbences for the optical path, b cm to obtain the corresponding transmittances (T%). Let these be T 445, T 495, T 550 and T 625.

1.3.4.   Expression of results

1.3.4.1.

The relative luminosity is given by the value of Y expressed as a percentage. (For complete darkness, Y = 0 %; for colourless liquids, Y = 100 %.)

1.3.4.2.

The chromaticity is expressed by the dominant wavelength and the purity. The determination of these two quantities makes use of the chromaticity diagram bounded by the spectral locus as given in Figure 1. The point O plotted in this diagram represents the white light source used and has the coordinates of a standard source, C, xo = 0,3101 and yo = 0,3163, representing daylight of average brightness. — Dominant wavelength — Plot the point C with coordinates x, y on the chromaticity diagram. — If C is outside the triangle AOB, draw the straight line joining O to C and extend it to cut the spectral locus at the point S, which corresponds to the dominant wavelength. — If C is inside the triangle AOB, draw the straight line from C to O and extend it to intersect the spectral locus at a point corresponding to the wavelength of the colour complementary to that of the wine. This wavelength is denoted by its value followed by the letter C. — Purity — If the point C is outside the triangle AOB, the purity is given as a percentage by the ratio: —  — If the point C is inside the triangle AOB, the purity is given as a percentage by the ratio: —   ( 15 ) — where P is the point where the straight line OC cuts the line of purples (line AB). — Purity is also given directly by chromaticity diagrams from the known values of x and y (Figures 2, 3, 4, 5 and 6).

1.3.4.3.

Results The colour of a wine is completely defined by its luminosity, its chromaticity (expressed by the dominant wavelength) and its purity. These should be indicated in the analysis report with the value of the optical path in which the measurements were made.

▼M8 —————

▼B

TABLE 1

Transformation of absorbences to transmittances (T%)

Method of use

Find the first decimal figure of the absorbence in the first vertical column, and call its row R. Find the second decimal figure of the absorbence in the top horizontal row and call its column C. Read the figure in the box at the intersection of the row R and the column C. To calculate the transmittance, divide this figure by 10 if the absorbence is less than 1, by 100 if it lies between 1 and 2, and by 1 000 if it lies between 2 and 3.

Note:

The figure in the top right-hand corner of each box enables the third decimal figure of the absorbence to be taken into account by interpolation.

Example:

Transmittances T % are to be expressed to the nearest 0,1 %.

FIGURE 1

Chromaticity diagram including all colours in the spectrum

FIGURE 2

Chromaticity diagram for unadulterated red (bright red) wines and red-brown (brick red) wines

FIGURE 3

Chromaticity diagram for unadulterated red (bright red) wines and red-brown (brick red) wines

FIGURE 4

Chromaticity diagram for unadulterated red (bright red) wines and purple wines

FIGURE 5

Chromaticity diagram for unadulterated red (bright red) wines and purple wines

FIGURE 6

Chromaticity diagram for red-brown (brick red) wines and purple wines

2.   RECTIFIED CONCENTRATED MUSTS

2.1.   Principle of the method

The absorbence of the rectified concentrated must is measured at 425 nm through a thickness of 1 cm after dilution to bring the sugar concentration to 25 % (m/m) (25° Brix).

2.2.   Apparatus

2.2.1.

Spectrophotometer enabling measurements to be made between 300 and 700 nm.

2.2.2.

Glass cells with optical paths of 1 cm.

2.2.3.

Membrane filter of pore diameter 0,45 µm.

2.3.   Procedure

2.3.1.   Preparation of the sample

Use the solution with a sugar concentration of 25 % (m/m) (25° Brix) prepared as described in the chapter ‘pH’, section 4.1.2. Filter is through a membrane filter of pore diameter 0,45 µm.

2.3.2.   Determination of absorbence

Zero the absorbence scale at a wavelength of 425 nm using a cell with an optical path of 1 cm containing distilled water.

Measure the absorbence A at the same wavelength of the solution containing 25 % sugar (25° Brix) prepared as in 2.3.1 and placed in a cell with an optical path of 1 cm.

2.4.   Expression of results

The absorbence at 425 nm of the rectified concentrated must in a solution with 25 % sugar (25° Brix) is quoted to two decimal places.

41.   FOLIN-CIOCALTEU INDEX

1.   DEFINITION

The Folin-Ciocalteu index is the result obtained from the application of the method described below.

2.   PRINCIPLE OF THE METHOD

All the phenolic compounds contained in the wine are oxidized by the Folin-Ciocalteu reagent. This reagent is formed from a mixture of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40) which, after oxidation of the phenols, is reduced to a mixture of the blue oxides of tungsten (W8O23) and molybdenum (Mo8O23).

The blue coloration produced has a maximum absorption in the region of 750 nm, and it is proportional to the total quantity of phenolic compounds originally present.

3.   REAGENTS

These must be of analytical reagent quality. The water used must be distilled or water of equivalent purity.

3.1.   Folin-Ciocalteu reagent

This reagent is available commercially in a form ready for use. It may be prepared as follows: dissolve 100 g of sodium tungstate (Na2WO4 · 2H2O) and 25 g of sodium molybdate (Na2MoO4 · 2H2O) in 700 ml of distilled water. Add 50 ml of 85 % phosphoric acid (ρ20 = 1,71 g/ml) and 100 ml of concentrated hydrochloric acid (ρ20 = 1,19 g/ml). Bring to the boil and boil for 10 hours under reflux conditions. Then add 150 g of lithium sulphate (Li2SO4 · H2O) and a few drops of bromine and boil once more for 15 minutes. Allow to cool and make up to one litre with distilled water.

3.2.	Anhydrous sodium carbonate, Na2CO3, made up into a 20 % m/v solution.

4.   APPARATUS

Normal laboratory apparatus, particularly:

4.1.	100 ml volumetric flasks.

4.2.	Spectrophotometer capable of operating at 750 nm.

5.   PROCEDURE

5.1.   Red wine

Introduce the following into a 100 ml volumetric flask (4.1) strictly in the order given:

Make up to 100 ml with distilled water.

Stir to homogenize. Wait 30 minutes for the reaction to stabilize. Determine the absorbence at 750 nm through a path length of 1 cm with respect to a blank prepared with distilled water in place of the wine.

If the absorbence is not around 0,3 an appropriate dilution should be made.

5.2.   White wine

Carry out the same procedure with 1 ml of undiluted wine.

5.3.   Rectified concentrated must

5.3.1.   Preparation of sample

Use the solution with a sugar concentration of 25 % (m/m) (25° Brix) prepared as described in the chapter ‘pH’, section 4.1.2.

5.3.2.   Measurement

Proceed as described for the case of red wine (5.1) using a 5 ml sample prepared as described in 5.3.1 and measuring the absorbence with respect to a control prepared with 5 ml of a 25 % (m/m) invert sugar solution.

6.   EXPRESSION OF RESULTS

6.1.   Method of calculation

The result is expressed in the form of an index obtained by multiplying the absorbence by 100 for red wines diluted 1:5 (or by the corresponding factor for other dilutions) and by 20 for white wines. For rectified concentrated musts, multiply by 16.

6.2.   Repeatability

The difference between the results of two determinations carried out simultaneously or very quickly one after the other by the same analyst must not be greater than 1.

Good repeatability of results is achieved by using scrupulously clean apparatus (volumetric flasks and spectrophotometer cells).

42.   SPECIAL METHODS OF ANALYSIS FOR RECTIFIED CONCENTRATED GRAPE MUST

(a)   TOTAL CATIONS

1.   PRINCIPLE OF THE METHOD

The test sample is treated by a strongly acid cation exchanger. The cations are exchanged with H+. Total cations are expressed by the difference between the total acidity of the effluent and that of the test sample.

2.   APPARATUS

2.1.	Glass column of internal diameter 10 to 11 mm and length approximately 300 mm, fitted with a drain tap.

2.2.	pH meter with a scale graduated at least in 0,1 pH units.

2.3.	Electrodes: — glass electrode, kept in distilled water, — calomel/saturated potassium chloride reference electrode, kept in a saturated solution of potassium chloride, — or a combined electrode, kept in distilled water.

3.   REAGENTS

3.1.	Strongly acid cation exchange resin in H+ form pre-swollen by soaking in water overnight.

3.2.	Sodium hydroxide solution, 0,1 M.

3.3.	Paper pH indicator.

4.   PROCEDURE

4.1.   Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v) as described in the chapter ‘Total acidity’, section 5.1.2.

4.2.   Preparation of the ion exchange column

Introduce into the column approximately 10 ml pre-swollen ion exchanger in H+ form. Rinse the column with distilled water until all acidity has been removed, using the paper indicator to monitor this.

4.3.   Ion exchange

Pass 100 ml of the rectified concentrated must solution prepared as in 4.1 through the column at the rate of one drop every second. Collect the effluent in a beaker. Rinse the column with 50 ml of distilled water. Titrate the acidity in the effluent (including the rinse water) with the 0,1 M sodium hydroxide solution until the pH is 7 at 20 °C. The alkaline solution should be added slowly and the solution continuously shaken. Let n ml be the volume of 0,1 M sodium hydroxide solution used.

5.   EXPRESSION OF RESULTS

The total cations are expressed in milliequivalents per kilogram of total sugar to one decimal place.

5.1.

Calculations — Acidity of the effluent expressed in milliequivalents per kilogram of rectified concentrated must: —  E = 2,5n — Total acidity of the rectified concentrated must in milliequivalents per kilogram (see ‘Total acidity’, section 6.1.2): a — Total cations in milliequivalents per kilogram of total sugars: —  —   Where P = percentage concentration (m/m) of total sugars.

(b)   CONDUCTIVITY

1.   PRINCIPLE OF THE METHOD

The electrical conductivity of a column of liquid defined by two parallel platinum electrodes at its ends is measured by incorporating it in one arm of a Wheatstone bridge.

The conductivity varies with temperature and it is therefore expressed at 20 °C.

2.   APPARATUS

2.1.	Conductivity meter enabling measurements of conductivity to be made over a range from 1 to 1 000 microsiemens per cm (µS cm−1).

2.2.	Waterbath for bringing the temperature of samples to be analysed to approximately 20 °C (20 ± 2 °C).

3.   REAGENTS

3.1.	Demineralized water with specific conductivity below 2 µS cm−1 at 20 °C.

3.2.

Reference solution of potassium chloride. Dissolve 0,581 g of potassium chloride, KCl, previously dried to constant mass at a temperature of 105 °C, in demineralized water (3.1). Make up to one litre with demineralized water (3.1). This solution has a conductivity of 1 000 µS cm−1 at 20 °C. It should not be kept for more than three months.

4.   PROCEDURE

4.1.   Preparation of the sample to be analysed

Use the solution with a total sugar concentration of 25 % (m/m) (25° Brix) as described in the chapter ‘pH’, section 4.1.2.

4.2.   Determination of conductivity

Bring the sample to be analysed to 20 °C by immersion in a waterbath. Maintain the temperature to within ± 0,1 °C.

Rinse the conductivity cell twice with the solution to be examined.

Measure the conductivity and express the result in µS cm−1.

5.   EXPRESSION OF RESULTS

The result is expressed in microsiemens per cm (µS cm−1) at 20 °C to the nearest whole number for the 25 % (m/m) (25° Brix) solution of rectified concentrated must.

5.1.   Calculations

If the apparatus is not provided with means for controlling the temperature, correct the measured conductivity using Table I. If the temperature is below 20 °C, add the correction; if the temperature is above 20 °C, subtract the correction.

(c)   HYDROXYMETHYLFURFURAL (HMF)

1.   PRINCIPLE OF THE METHODS

1.1.   Colorimetric method

Aldehydes derived from furan, the main one being hydroxymethylfurfural, react with barbituric acid and paratoluidine to give a red compound which is determined by colorimetry at 550 nm.

1.2.   High-performance liquid chromatography (HPLC)

Separation through a column by reversed-phase chromatography and determination at 280 nm.

2.   COLORIMETRIC METHOD

2.1.   Apparatus

2.1.1.

Spectrophotometer for making measurements between 300 and 700 nm.

2.1.2.

Glass cells with optical paths of 1 cm.

2.2.   Reagents

2.2.1.   Barbituric acid, 0,5 % solution (m/v).

Dissolve 500 mg of barbituric acid, C4O3N2H4, in distilled water and heat slightly over a waterbath at 100 °C. Make up to 100 ml with distilled water. The solution keeps for about a week.

2.2.2.   Paratoluidine solution, 10 % (m/v).

Place 10 g of paratoluidine, C6H4(CH3)NH2, in a 100 ml volumetric flask; add 50 ml of isopropanol, CH3CH(OH)CH3, and 10 ml of glacial acetic acid, CH3COOH (ρ20 = 1,05 g/ml). Make up to 100 ml with isopropanol. This solution should be renewed daily.

2.2.3.   Ethanal (acetaldehyde), CH3CHO, 1 % (m/v) aqueous solution.

Prepare just before use.

2.2.4.   Hydroxymethylfurfural, C6O3H6, 1 g/l aqueous solution.

Prepare successive dilutions containing 5, 10, 20, 30 and 40 mg/l. The 1 g/l and the diluted solutions must be freshly prepared.

2.3.   Procedure

2.3.1.   Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v) as described in the chapter ‘Total acidity’, section 5.1.2. Carry out the determination on 2 ml of this solution.

2.3.2.   Colorimetric determination

Into each of two 25 ml flasks a and b fitted with ground glass stoppers place 2 ml of the sample prepared as in 2.3.1. Place in each flask 5 ml of paratoluidine solution (2.2.2); mix. Add 1 ml of distilled water to flask b (control) and 1 ml barbituric acid (2.2.1) to flask a. Shake to homogenize. Transfer the contents of the flasks into spectrophotometer cells with optical paths of 1 cm. Zero the absorbence scale using the contents of flask b for a wavelength of 550 nm. Follow the variation in the absorbence of the contents of flask a; record the maximum value A, which is reached after two to five minutes.

Samples with hydroxymethylfurfural concentrations above 30 mg/l must be diluted before the analysis.

2.3.3.   Preparation of the calibration curve

Place 2 ml of each of the hydroxymethylfurfural solutions with 5, 10, 20, 30 and 40 mg/l (2.2.4) into two sets of 25 ml flasks a and b and treat them as described in 2.3.2.

The graph representing the variation of absorbence with the hydroxymethylfurfural concentration in mg/l is a straight line passing through the origin.

2.4.   Expression of results

The hydroxymethylfurfural concentration in rectified concentrated musts is expressed in milligrams per kilogram of total sugars.

2.4.1.   Method of calculation

The hydroxymethylfurfural concentration C mg/l in the sample to be analysed is that concentration on the calibration curve corresponding to the absorbence A measured on the sample.

The hydroxymethylfurfural concentration in milligrams per kilogram of total sugars is given by:

where

P

=

percentage (m/m) concentration of total sugars in the rectified concentrated must.

3.   HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

3.1.   Apparatus

3.1.1.   High-performance liquid chromatograph equipped with:

Flow rate of mobile phase: 1,5 ml/minute.

3.1.2.

Membrane filtration apparatus, pore diameter 0,45 µm.

3.2.   Reagents

3.2.1.

Doubly distilled water.

3.2.2.

Methanol, CH3OH, distilled or HPLC quality.

3.2.3.

Acetic acid CH3COOH, (ρ = 1,05 g/ml).

3.2.4.

Mobile phase: water-methanol (3.2.2)-acetic acid (3.2.3) previously filtered through a membrane filter (0,45 µm), (40:9:1 v/v). This mobile phase must be prepared daily and outgassed befor use.

3.2.5.

Reference solution of hydroxymethylfurfural, 25 mg/l (v/v). Into a 100-ml volumetric flask, place 25 mg of hydroxymethylfurfural, C6H3O6, accurately weighed, and make up to the mark with methanol (3.2.2). Dilute this solution 1:10 with methanol (3.2.2) and filter through a membrane filter (0,45 µm). If kept in a brown glass bottle in a refrigerator, this solution will keep for two to three months.

3.3.   Procedure

3.3.1.   Preparation of sample

Use the solution obtained by diluting the rectified concentrated must to 40 % (m/v) as described in the chapter ‘Total acidity’, section 5.1.2, and filter it through a 0,45 µm membrane filter.

3.3.2.   Chromatographic determination

Inject 5 (or 10) µl of the sample prepared as described in 3.3.1. and 5 (or 10) µl of the reference hydroxymethylfurfural solution (3.2.5) into the chromatograph. Record the chromatogram.

The retention time of hydroxymethylfurfural is approximately six to seven minutes.

3.4.   EXPRESSION OF RESULTS

The hydroxymethylfurfural concentration in rectified concentrated musts is expressed in milligrams per kilogram of total sugars.

3.4.1.   Method of calculation

Let the hydroxymethylfurfural concentration in the 40 % (m/v) solution of the rectified concentrated must be C mg/l.

The hydroxymethylfurfural concentration in milligrams per kilogram of total sugars is given by:

where

P

=

percentage (m/m) concentration of total sugars in the rectified concentrated must.

(d)   HEAVY METALS

1.   PRINCIPLE OF THE METHODS

I.   Rapid method for evaluation of heavy metals

Heavy metals are revealed in the suitably diluted rectified concentrated must by the coloration produced by the formation of sulphides. They are assessed by comparison with a standard lead solution corresponding to the maximum admissible concentration.

II.   Determination of lead content by atomic absorption spectrophotometry

The chelate given by lead with ammonium pyrrolidinedithiocarbamate is extracted with methylisobutylketone and the absorbence measured at 283,3 nm. The lead content is determined by using known additional amounts of lead in a set of reference solutions.

2.   RAPID METHOD FOR EVALUATION OF HEAVY METALS

2.1.   Reagents

2.1.1.   Dilute hydrochloric acid, 70 % (m/v).

Take 70 g of hydrochloric acid, HCl (ρ20 = 1,16 to 1,19 g/ml), and make up to 100 ml with water.

2.1.2.   Dilute hydrochloric acid, 20 % (m/v).

Take 20 g of hydrochloric acid, HCl (ρ20 = 1,16 to 1,19 g/ml), and make up to 100 ml with water.

2.1.3.

Dilute ammonia. Take 14 g of ammonia, NH3 (ρ20 = 0,931 to 0,934 g/ml) and make up to 100 ml with water.

2.1.4.

pH 3,5 buffer solution. Dissolve 25 g of ammonium acetate CH3COONH4, in 25 ml of water and add 38 ml of dilute hydrochloric acid (2.1.1). Adjust the pH if necessary with the dilute hydrochloric acid (2.1.2) or the dilute ammonia (2.1.3) and make up to 100 ml with water.

2.1.5.

Thioacetamide solution C2H5 SN, 4 % (m/v).

2.1.6.

Glycerol solution, C3H8O3, 85 % (m/v), ( = 1,449 to 1,455).

2.1.7.

Thioacetamide reagent. To 0,2 ml of thioacetamide solution (2.1.5) add 1 ml of a mixture of 5 ml of water, 15 ml of 1 M sodium hydroxide solution and 20 ml of glycerol (2.1.6). Heat on a waterbath at 100 °C for 20 seconds. Prepare just before use.

2.1.8.

Solution containing 0,002 g/l of lead. Prepare a 1 g/l lead solution by dissolving 0,400 g of lead nitrate, Pb(NO3)2, in water and making up to 250 ml with water. At the time of use, dilute this solution with water to two parts in 1 000 (v/v) in order to obtain a 0,002 g/l solution.

2.2.   Procedure

Dissolve a test sample of 10 g of the rectified concentrated must in 10 ml of water. Add 2 ml of the pH 3,5 buffer solution (2.1.4); mix. Add 1,2 ml of the thioacetamide reagent (2.1.7). Mix at once. Prepare the control under the same conditions by using 10 ml of the 0,002 g/l lead solution (2.1.8).

After two minutes, any brown coloration of the rectified concentrated must solution should not be more intense than that of the control.

2.3.   Calculations

Under the conditions of the above procedure, the control sample corresponds to a maximum admissible heavy metal concentration expressed as lead of 2 mg/kg of rectified concentrated must.

3.   DETERMINATION OF LEAD CONTENT BY ATOMIC ABSORPTION SPECTROPHOTOMETRY

3.1.   Apparatus

3.1.1.

Atomic absorption spectrophotometer equipped with an air-acetylene burner.

3.1.2.

Lead hollow cathode lamp.

3.2.   Reagents

3.2.1.   Dilute acetic acid.

Take 12 g of glacial acetic acid (ρ = 1,05 g/ml) and make up to 100 ml with water.

3.2.2.

Solution of ammonium pyrrolidinedithiocarbamate, C5H12N2S2, 1 % (m/v).

3.2.3.

Methylisobutylketone, (CH3)2CHCH2COCH3.

3.2.4.

Solution containing 0,010 g/l of lead. Dilute the 1 g/l lead solution (of 2.1.8) to 1 % (v/v).

3.3.   Procedure

3.3.1.   Preparation of solution to be examined

Dissolve 10 g of rectified concentrated must in a mixture of equal volumes of dilute acetic acid (3.2.1) and water, and make up to 100 ml with this mixture.

Add 2 ml of ammonium pyrrolidinedithiocarbamate solution (3.2.2) and 10 ml of methylisobutylketone (3.2.3). Shake for 30 seconds while protected from bright light. Leave the two layers to separate. Use the methylisobutylketone layer.

3.3.2.   Preparation of reference solutions

Prepare three reference solutions containing, in addition to 10 g of rectified concentrated must, 1, 2 and 3 ml respectively of the solution containing 0,010 g/l of lead (3.2.4). Treat these in the same way as the solution to be examined.

3.3.3.   Control

Prepare a control by proceeding under the same conditions as in 3.3.1, but without the addition of the rectified concentrated must.

3.3.4.   Determination

Set the wavelength to 283,3 nm.

Atomize the methylisobutylketone from the control sample in the flame and zero the absorbence scale.

By operating with their respective solvent extracts, determine the absorbences of the solution to be examined and the reference solutions.

3.4.   Expression of results

Express the lead content in milligrams per kilogram of rectified concentrated must to one decimal place.

3.4.1.   Calculations

Plot the curve giving the variation in absorbence as a function of the lead concentration added to the reference solutions, zero concentration corresponding to the solution to be examined.

Extrapolate the straight line joining the points until it cuts the negative part of the concentration axis. The distance of the point of intersection from the origin gives the lead concentration in the solution to be examined.

(e)   CHEMICAL DETERMINATION OF ETHANOL

This method is used for the determination of the alcoholic strength of low-alcohol liquids such as musts, concentrated musts and rectified concentrated musts.

1.   PRINCIPLE OF THE METHOD

Simple distillation of the liquid. Oxidation of the ethanol in the distillate by potassium dichromate. Titration of the excess dichromate with an iron (II) solution.

2.   APPARATUS

2.1.	Use the distillation apparatus described in the chapter ‘Alcoholic strength by volume’, section 3.2.

3.   REAGENTS

3.1.   Potassium dichromate solution.

Dissolve 33,600 g of potassium dichromate, K2Cr2O7, in sufficient quantity of water to make one litre of solution at 20 °C.

One millilitre of this solution oxidizes 7,8924 mg of alcohol.

3.2.   Iron (II) ammonium sulphate solution.

Dissolve 135 g of iron (II) ammonium sulphate, FeSO4 · (NH4)2SO4 · 6 H2O, in sufficient quantity of water to make one litre of solution and add 20 ml of concentrated sulphuric acid, H2SO4 (ρ20 = 1,84 g/ml). This solution more or less corresponds to half its volume of dichromate solution when just prepared. Subsequently, it oxidizes slowly.

3.3.   Potassium permanganate solution.

Dissolve 1,088 g of potassium permanganate, KMnO4, in a sufficient quantity of water to make one litre of solution.

3.4.   Dilute sulphuric acid, 1:2 (v/v).

A little at a time and stirring continuously, add 500 ml of sulphuric acid, H2SO4 (ρ20 = 1,84 g/ml) to 500 ml of water.

3.5.   Ferrous orthophenanthroline reagent.

Dissolve 0,695 g of ferrous sulphate, FeSO4 · 7 H2O, in 100 ml of water, and add 1,485 g of orthophenanthroline monohydrate, C12H8N2 · H2O. Heat to help the dissolution. This bright red solution keeps well.

4.   PROCEDURE

4.1.   Distillation

Place 100 g of rectified concentrated must and 100 ml of water in the distillation flask. Collect the distillate in a 100 ml volumetric flask and make up to the mark with water.

4.2.   Oxidation

Take a flask with a ground glass stopper and with a widened neck enabling the neck to be rinsed without loss. In the flask, place 20 ml of the titrant potassium dichromate solution (3.1) and 20 ml of the 1:2 (v/v) dilute sulphuric acid (3.4) and shake. Add 20 ml of the distillate. Stopper the flask, shake, and wait at least 30 minutes, shaking occasionally. (This is the ‘measurement’ flask.)

Carry out the titration of the iron (II) ammonium sulphate solution (3.2) with respect to the potassium dichromate solution by placing in an identical flask the same quantities of reagents but replacing the 20 ml of distillate by 20 ml of distilled water. (This is the ‘control’ flask.)

4.3.   Titration

Add four drops of the orthophenanthroline reagent (3.5) to the contents of the ‘measurement’ flask. Titrate the excess dichromate by adding to it the iron (II) ammonium sulphate solution (3.2). Stop adding the ferrous solution when the mixture changes from green-blue to brown.

To judge the end-point more precisely, change the colour of the mixture back from brown to green-blue with the potassium permanganate solution (3.3). Subtract a tenth of the volume of this solution used from the volume of the iron (II) solution added. Let the difference be n ml.

Proceed in the same way with the ‘control’ flask. Let n′ ml be the difference here.

5.   EXPRESSION OF RESULTS

The ethanol is expressed in grams per kilogram of sugar and is quoted to one decimal place.

5.1.   Method of calculation

n′ ml of ferrous solution reduces 20 ml of dichromate solution which oxidizes 157,85 mg of pure ethanol.

One millilitre of iron (II) solution has the same reducing power as

n − n′ ml of iron (II) solution have the same reducing power as

Ethanol concentration in g/kg of rectified concentrated must is given by:

Ethanol concentration in g/kg of total sugars is given by:

where

P

=

percentage concentration (m/m) of total sugars.

(f)   MESO-INOSITOL, SCYLLO-INOSITOL AND SUCROSE

1.   PRINCIPLE

Gas chromatography of silylated derivatives.

2.   REAGENTS

2.1.	Internal standard: xylitol (aqueous solution of about 10 g/l to which a spatula tip of sodium azide is added)

2.2.	Bis(trimethylsilyl)trifluoroacetamide — BSTFA — (C8H18F3NOSi2)

2.3.	Trimethylchlorosilane (C3H9ClSi)

2.4.	Pyridine p.A. (C5H5N)

2.5	Meso-inositol (C6H12O6)

3.   APPARATUS

3.1.	Gas chromatograph equipped with:

3.2.

Capillary column (e.g. in fused silica, coated with OV 1, film thickness of 0,15 µm, length 25 m and internal diameter of 0,3 mm) Operating conditions: — carrier gas: hydrogen or helium, — carrier gas flow rate: about 2 ml/minute, — injector and detector temperature: 300 °C, — programming of temperature: 1 minute at 160 °C, 4 °C per minute to 260 °C, constant temperature of 260 °C for 15 minutes, — splitter ratio: about 1:20.

3.3.	Integrator.

3.4.	Microsyringe, 10 µl.

3.5.	Micropipettes, 50, 100 and 200 µl.

3.6.	2 ml flask with Teflon stopper.

3.7.

Oven.

4.   METHOD OF OPERATION

An accurately weighed sample of about 5 g of rectified concentrated must is placed in a 50 ml flask. 1 µl of standard solution of xylitol (2.1) is added and water added to capacity. After mixing, 100 µl of solution is taken and placed in a flask (3.6) where it is dried under a gentle stream of air. 100 µl of absolute ethyl alcohol may be added if necessary to facilitate evaporation.

The residue is carefully dissolved in 100 µl of pyridine (2.4) and 100 µl of bis(trimethylsilyl)trifluoroacetamide (2.2) and 10 µl of trimethylchlorosilane (2.3) are added. The flask is closed with the Teflon stopper and heated at 60 °C for one hour.

Draw off 0,5 µl of clear fluid and inject using a heated hollow needle in accordance with the stated splitter ratio.

5.   CALCULATION OF RESULTS

5.1.

A solution is prepared containing: 60 g/l of glucose, 60 g/l of fructose, 1 g/l of meso-inositol and 1 g/l of sucrose. 5 g of the solution is weighed and the procedure at 4 followed. The results for meso-inositol and sucrose with respect to xylitol are calculated from the chromatogram. In the case of scyllo-inositol, which is not commercially available and has a retention time lying between the last peak of the anomeric form of glucose and the peak for meso-inositol (see diagram overleaf), the same result as for meso-inositol is taken.

6.   EXPRESSION OF RESULTS

6.1.	Meso-inositol and scyllo-inositol are expressed in milligrams per kilogram of sugar. Sucrose is expressed in grams per kilogram of must. Chromatogram in gaseous phase of meso-inositol, scyllo-inositol and sucrose

▼M6

43.   DETERMINATION OF THE ISOTOPIC RATIO 18O/16O OF THE WATER CONTENT IN WINES

I.   DESCRIPTION OF THE METHOD

1.   Method objective

The objective of the present method is to measure the isotopic ratio 18O/16O of waters of different origins. The isotopic ratio 18O/16O can be expressed in deviation δ ‰ in ratio to the value of isotopic ratio of the international reference V.SMOW:

2.   Principle

The isotopic ratio 18O/16O is determined by mass spectrometry of isotopic ratios (MSIR) from ionic currents m/z 46 (12C16O18O) and m/z 44 (12C16O2) produced by carbon dioxide obtained after an exchange with the water in wine according to the reaction:

C16O2 + H2 18O ↔ C16O18O + H2 16O

The carbon dioxide in the gaseous phase is used for analysis.

3.   Reagents

4.   Laboratory equipment

5.   Experimental determinations

5.1.   Manual method

Introduction of the sample

The two ramps are cooled down with liquid nitrogen, then the whole system is purged up to 0,1 mm Hg by opening the valves.

Then, shut the valves off and let it all heat up. The degasing cycle is repeated until there is no more pressure variation.

Cool the work ramps to − 70 °C (Liquid nitrogen and alcohol mix) to freeze the water and put it all in a vacuum. After stabilization of the vacuum, isolate the ramp by actioning the valve and purge the CO2 introduction system. Insert the gaseous CO2 into the work ramp and, after having isolated it from the rest of the system, introduce the ramp in a thermostated bath at 25 °C (± 0,5 °C) for 12 hours (one night). To optimize the necessary time for equilibration, it is advisable to prepare the samples at the end of the day and let the balance settle overnight.

A sample holder which supports as many measuring cells as balloon flasks containing exchanged CO2 is adapted on the empty line next to the work ramp. The empty cells are carefully purged and the exchanged gases contained in the ballons are transferred one after the other, into the measuring cells which have been cooled by liquid nitrogen. Then the measuring cells are allowed to heat up at room temperature.

5.2.   Use of an automatic exchange apparatus

In order to carry out the equilibration, sample phials are filled with either 2 ml of wine or 2 ml of water (laboratory work reference) and cooled down to − 18 °C. The sample slides containing the frozen products are adapted to the equilibration system, and after having created a vacuum in the system, carbon dioxide is introduced at a pressure of 800 hPa.

The balance is reached at a temperature of 22 ± 0,5 °C after a minimum period of five hours and with moderate agitation. Since the equilibration duration depends on the phial's geometry, the optimum duration should be determined first for the system used.

Carbon dioxide contained in the phials is then transferred to the introduction chamber of the mass spectrometer by a capillary tube and the measurement is carried out according to a specific protocol for each kind of equipment.

6.   Calculation and expression of the results

The relative difference δ′ of the ratio intensities of ions m/z 46 and 44 (I46/I44) between the sample and the reference is expressed in ‰ by means of the following equation:

The 18O content of the sample compared to the reference V.SMOW on the V.SMOW/SLAP scale, is given by the relation:

The value accepted for SLAP is equal to − 55,5 ‰ compared to V.SMOW. The isotopic ratio of reference must be determined after each series of 10 measurements on unknown samples.

7.   Fidelity

▼M7

44.   DETERMINATION OF ETHYL CARBAMATE IN WINE: SELECTIVE DETECTION METHOD USING GAS CHROMATOGRAPHY/MASS SPECTROMETRY

(Applicable to the determination of ethyl carbamate for concentrations between 10 and 200 μg/l)

(Caution: comply with safety measures for handling chemicals, ethanol, acetone and carcinogenic products (ethyl carbamate and dichloromethane). Dispose of used solvents in the proper way, in compliance with environmental rules in force).

A.   Principle

Propyl carbamate is added to a sample as an internal standard, the solution is diluted with water and placed in a 50 ml solid phase extraction column. Ethyl carbamate and propyl carbamate are eluted with dichloromethane.

The eluate is concentrated in a vacuum rotary evaporator. The concentrate is analysed by gas chromatography (GC). Detection is by mass spectrometry using fragmentometry in SIM (selected ion monitoring) mode.

B.   Apparatus and chromatographic conditions (example)

C.   Reagents

D.   Preparation of the test sample

Place the following volumes of test material in two separate 100 ml beakers:

To each beaker, add l ml of internal standard PC solution C(e)(3) and water, to obtain a total volume of 40 ml (or 40 g).

E.   Extraction

Extraction should be carried out under an extractor hood, with adequate ventilation.

Transfer the sample prepared under heading D to the extraction column.

Rinse the beaker with 10 ml of water and transfer the rinsing water to the column.

Leave the liquid to absorb for four minutes. Elute with 2 × 80 ml of dichloromethane. Collect the eluate in a 300 ml conical flask.

Evaporate the eluate from 2 to 3 ml in a water bath rotary evaporator at 30 °C. (

NB: do not allow to boil dry.

)

Transfer the concentrated residue to a 4 ml graduated tube with a Pasteur pipette.

Rinse the flask with 1 ml of dichloromethane and transfer the rinsing liquid to the tube. Concentrate the sample to 1 ml under a weak nitrogen stream.

If necessary, transfer the concentrate to auto sampler flask for GC/MS analysis.

F.   GC/MS analysis

G.   Collaborative analysis

The table shows individual results for the practical entrainment sample and for both types of wine.

The Cochran test led to the elimination of only one pair of results, for wine of alcoholic strength over 14 % vol and for wine of alcoholic strength of 14 % vol or less, from two different laboratories.

Relative reproducibility (RSDR) tends to decrease as the concentration of ethyl carbamate increases.

▼M10

45.   DETERMINATION BY ISOTOPE MASS SPECTROMETRY OF THE 13C/12C RATIO IN WINE ETHANOL OR ETHANOL OBTAINED BY THE FERMENTATION OF MUSTS, CONCENTRATED MUSTS OR RECTIFIED CONCENTRATED MUSTS

1.   FIELD OF APPLICATION

The method enables measurement of the 13C/12C isotope ratio in wine ethanol and ethanol obtained by fermentation of products of the vine (musts, concentrated musts, rectified concentrated musts).

2.   REFERENCE STANDARDS

ISO

:

5725:1994 ‘Accuracy (trueness and precision) of measurement methods and results: Basic method for the determination of repeatability and reproducibility of a standard measurement method’.

V-PDB

:

Vienna-Pee-Dee Belemnite (RPDB = 0,0112372).

Method 8 of the Annex to this Regulation

:

‘Detecting enrichment of grape musts, concentrated grape musts, rectified concentrated grape musts and wines by application of nuclear magnetic resonance of deuterium (SNIF-NMR).’

3.   TERMS AND DEFINITIONS

13C/12C

:

ratio of carbon 13 (13C) to carbon 12 (12C) isotopes for a given sample.

δ13C

:

carbon 13 content (13C) expressed in parts per 1 000 (‰).

SNIF-NMR

:

fractionating the particular natural isotope under study by nuclear magnetic resonance.

V-PDB

:

Vienna-Pee-Dee Belemnite. PDB is the primary reference material for measuring natural variations of carbon 13 isotope content, consisting of calcium carbonate from a Cretaceous belemnite guard from the Pee Dee Formation in South Carolina (USA). Its 13C/12C isotope ratio or RPDB is 0,0112372. PDB reserves have been exhausted for a long time, but it has remained the primary reference for expressing natural variations of carbon 13 isotope content and against which the reference material available at the International Atomic Energy Agency (IAEA) in Vienna (Austria) is calibrated. Isotopic indications of naturally occurring carbon 13 are conventionally expressed in relation to V-PDB.

m/z

:

mass-to-charge ratio.

4.   PRINCIPLE

During photosynthesis, the assimilation of carbon dioxide by plants occurs via two principle forms of metabolism, the C3 metabolism (Calvin cycle) and the C4 metabolism (Hatch and Slack). These two photosynthesis mechanisms present a different type of isotope fractionation. Products of C4 plants, such as sugars and alcohol derived from fermentation, have higher levels of carbon 13 than similar products of C3 plants. Most plants, including vines and sugar beets, belong to the C3 group. Sugar cane and maize belong to the C4 group. Measuring the carbon 13 content enables the detection and evaluation of sugars of C4 origin (sugar cane or maize isoglucose) added to grape products (grape musts, wines, etc.). The information on carbon 13 content combined with that obtained from SNIF-NMR enables the added quantities of mixtures of sugars or alcohols derived from C3 and C4 plants to be determined.

The carbon 13 content is determined on carbon dioxide produced during the complete combustion of the sample. The abundance of the principle isotopomers of masses 44 (12C16O2), 45 (13C16O2 and 12C17O16O) and 46 (12C16O18O), resulting from the different possible combinations of isotopes 18O, 17O, 16O, 13C and 12C, are determined from the ionic currents measured by three different collectors of a mass isotopic spectrometer. The contributions of isotopomers 13C17O16O and 12C17O2 may be disregarded given their low levels. The ionic current for m/z = 45 is corrected for the contribution of 12C17O16O, which is calculated according to the current intensity measured for m/z = 46, while taking the relative abundance of 18O and 17O into account (Craig correction). Comparison with a reference calibrated against the international reference V-PDB permits calculation of carbon 13 content on the δ13C relative scale.

5.   REAGENTS

The material and the consumables depend on the apparatus (point 6) used by the laboratory. The systems generally used are based on elemental analysers. These systems can be equipped to allow the introduction of samples placed in sealed metal capsules or the injection of liquid samples through a septum using a syringe.

Depending on the type of instrument used, the following reference materials, reagents, and consumables may be used:

6.   APPARATUS AND EQUIPMENT

6.1.   Isotope ratio mass spectrometer (IRMS)

Isotope ratio mass spectrometer (IRMS) capable of determining the relative 13C content of naturally occurring CO2 gas with an internal accuracy of 0,05 ‰ or better expressed as a relative value (point 9). Internal accuracy here is defined as the difference between two measurements of the same sample of CO2. The mass spectrometer used to measure isotope ratios is generally equipped with a triple collector to simultaneously measure intensities for m/z = 44, 45 and 46. The isotope ratio mass spectrometer must either be equipped with a dual inlet, to alternately measure the unknown sample and a reference sample, or use an integrated system that carries out the quantitative combustion of samples and separates the carbon dioxide from the other combustion products before measurement in the mass spectrometer.

6.2.   Combustion apparatus

Combustion apparatus able to quantitatively convert ethanol into carbon dioxide and eliminate all other combustion products including water, without any isotopic fractionation. The apparatus may be either a continuous-flow system integrated with the mass spectrometry apparatus (point 6.2.1) or a separate combustion system (point 6.2.2). The apparatus must permit an accuracy of at least that indicated in (point 11).

6.2.1.   Continuous-flow systems

These comprise either an elemental analyser or a gas chromatograph with an online combustion system.

The following laboratory equipment is needed for systems equipped for the introduction of samples contained in metal capsules:

Note: in order to reduce the risk of evaporation of ethanol samples, an absorbent material (for example Chromosorb W 45-60 mesh) may be placed in the capsules, it first having been verified by means of a measurement without a sample that is does not contain a significant quantify of carbon likely to affect the results.

The following laboratory equipment is needed when using an elemental analyser equipped with a liquid injector or in the case of a combustion chromatography preparation system:

The laboratory equipment indicated in the above lists are examples and may be replaced by other equipment of equivalent performance depending on the type of combustion and mass spectrometry apparatus used by the laboratory.

6.2.2.   Separate preparation system

The samples of carbon dioxide resulting from the combustion of the samples to be analysed and the reference sample are collected in bulbs which are then placed in the dual inlet of the spectrometer for isotopic analysis. Several combustion apparatuses described in the literature may be used:

7.   PREPARATION OF SAMPLES FOR TESTS

The ethanol must be extracted from the wine before isotopic testing. This is carried out by distilling wine as described in point 3.1 of method No 8 (SNIF-NMR).

In the case of grape musts, concentrated grape musts and rectified concentrated grape musts, the sugars must be fermented in ethanol first as described in point 3.2 of method No 8.

8.   PROCEDURE

All steps of the preparation must be carried out without any significant ethanol loss through evaporation that would change the isotopic composition of the sample.

The following description refers to the procedures generally used for ethanol sample combustion using commercial automated combustion systems. All other methods that ensure that all of the ethanol sample is converted into carbon dioxide without any loss of ethanol through evaporation may be used for the preparation of carbon dioxide for isotopic analysis.

Experimental procedure based on the use of an elemental analyser:

9.   CALCULATION

The purpose of the method is to measure the 13C/12C isotope ratio ofethanol extracted from wine or from products derived from grapes following fermentation. The 13C/12C isotope ratiocan be expressed by its deviation from a working reference. The isotopic deviation of carbon 13 (δ 13C) is then calculated on a delta scale per thousand (δ/1 000) by comparing the results obtained for the sample to be measured with those for a working reference previously calibrated on the basis of the primary international reference (V-PDB). The δ 13C values are expressed in relation to the working reference as follows:

δ13Csam/ref ‰ = 1 000 × (Rsam-Rref)/Rref

where Rsam and Rref are respectively the 13C/12C isotope ratios of the sample and of the carbon dioxide used as the reference gas.

The δ 13C values are expressed in relation to V-PDB as follows:

δ13Csam/V-PDB ‰ = δ13Csam/ref +δ13Cref/V-PDB + (δ13Csam/ref × δ13Cref/V-PDB)/1 000,

where δ13Cref/V-PDB is the previously determined isotopic deviation of the working reference from V-PDB.

Small variations may occur while measuring on line due to changes in the instrumental conditions. In this case the δ 13C values of the samples must be corrected according to the difference in the measured δ13C value of the standard working sample and its true value, previously calibrated against V-PDB by comparison with one of the international reference materials. Between two measurements of the standard working sample, the variation, and therefore the correction to be applied to the results obtained from the samples, may be assumed to be linear. The standard working sample must be measured at the beginning and at the end of all sample series. A correction can then be calculated for each sample using linear interpolation.

10.   QUALITY ASSURANCE AND CONTROL

Check that the 13C value for the working reference does not differ by more than 0,5 ‰ from the admissible value. If not, the spectrometry apparatus settings should be checked and, if necessary, adjusted.

For each sample, check that the difference in the results for two capsules measured successively is less than 0,3 ‰. The final result for a given sample is the average value for the two capsules. If the deviation is greater than 0,3 ‰, the measurement must be repeated.

Checks on correct measurement can be based on the ionic current where m/z = 44, which is proportional to the quantity of carbon injected into the elemental analyser. Under standard conditions, the ionic current should be almost constant for the samples analysed. A significant deviation could be indicative of ethanol evaporation (for example an imperfect seal on a capsule) or instability of the elemental analyser or mass spectrometer.

11.   PERFORMANCE CHARACTERISTICS OF THE METHOD (Accuracy)

An initial collaborative study (point 11.1) was carried out on distillates containing alcohol of vinous origin and cane and beet alcohol, as well as different mixtures of alcohol of those three origins. Since this study did not take into account the distillation procedure, further information from other interlaboratory studies on wine (point 11.2) and, in particular, series of proficiency tests (point 11.3) for isotopic measurements were also considered. The results show that under satisfactory conditions, and in particular those for measurement using SNIF-NMR, the different distillation systems do not produce significant variation in the determination of the δ13C value of wine ethanol. The accuracy parameters observed for wine are almost identical to those obtained in the joint study on distillates (point 11.1).

11.1.   Joint study on distillates

Year of interlaboratory tests

:

1996

Number of laboratories

:

20

Number of samples

:

Six samples in double-blind comparison

Analyte

:

δ 13C of ethanol

11.2.   Interlaboratory study on two wines and one alcohol

Year of interlaboratory tests

:

1996

Number of laboratories

:

14 for distillation of wine of which seven also measured δ 13C of wine ethanol, Eight for measuring δ 13C of alcohol sample,

Number of samples

:

Three (white wine of 9,3 % ASV, white wine of 9,6 % ASV and alcohol of strength 93 % m/m).

Analyte

:

δ 13C of ethanol

Different distillation systems were used by the participating laboratories. The isotopic determinations (δ 13C) carried out in a single laboratory on the whole number of distillates returned by the participants do not reveal any aberrant values or values that differ significantly from the average values. The variation in results (S2 = 0,0059) is comparable to the repeatability variances Sr2 in the joint study on distillates (point 11.1).

11.3.   Results of the exercises carried out to monitor proficiency in performing isotopic tests

Since December 1994, international proficiency tests for the determination of isotopic measurements for wine and alcohol (distillates of 96 % ASV) have been regularly organised. The results enable participating laboratories to check the quality of their analyses. Statistical results permit appreciation of the variation in measurements under conditions of reproducibility and therefore an estimation of the parameters of variance and the limit of reproducibility. The results obtained for the determination of δ

13

C for wine and distillate ethanol are summarised in the following table:

11.4.   Limits of repeatability and reproducibility

On the basis of the data from the different interlaboratory tests given in the above tables, the following limits of repeatability and reproducibility can be established for this method, including the distillation stage:

---

( 1 ) OJ No L 84, 27. 3. 1987, p. 1.

( 2 ) OJ No L 132, 23. 5. 1990, p. 19.

( 3 ) OJ No L 133, 14. 5. 1982, p. 1.

( 4 ) Any pycnometer with equivalent characteristics may be used.

( 5 ) A numeric example is given in section 6 of this chapter.

( 6 ) A numeric example is given in section 6 of this chapter.

( 7 ) The sugar concentration is expressed in terms of invert sugar.

( 8 ) The sugar concentration is expressed in terms of invert sugar.

( 9 ) For example, for an alcoholic strength of 12 % by weight, p = 0,12.

( 10 ) Before carrying out this calculation, the specific gravity (or the density) of the wine measured as specified above should be corrected for the effect of the volatile acidity using the formula:

d v = − 0,0000086a or ρ v = ρ 20 − 0,0000086a

where a is the volatile acidity expressed in milliequivalents per litre.

( 11 ) These values are given pending the creation of a Community data bank of such values.

( 12 ) One of the trade names is ‘Norite’.

( 13 ) 105 pascal (Pa) = 1 bar.

( 14 ) No account is taken of other gases present (O2, N2, etc.) in amounts that are too small to have any effect on the excess pressure.

( 15 ) This distance must be given in a direction going from O to C.

( 16 ) For certain particularly rich wines, a 50 m capillary column may be desirable.

( 17 ) For certain particularly rich wines, a temperature programme of 2 °C/minute may be desirable.