1990R2676 — EN — 18.03.2003 — 007.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSIONREGULATION(EEC)No000/90
of17September1990
determining Communitymethodsfortheanalysisofwines
(OJ L 272, 3.10.1990, p. 1)
Amended by:
Official Journal
No page date
►M1CommissionRegulation(EEC) No 2645/92 of 11 September 1992 L 266 10 12.9.1992
►M2CommissionRegulation(EC) No 60/95 of 16 Jan uary 1995 L 11 19 17.1.1995
►M3CommissionRegulation(EC) No 69/96 of 18 Jan uary 1996 L 14 13 19.1.1996
►M4CommissionRegulation(EC) No 822/97 of 6 May 1997 L 117 10 7.5.1997
►M5CommissionRegulation(EEC) No 2348/91 of 29 July 1991 L 214 39 2.8.1991
►M6amended by Commission Regulation (EC) No 1932/97 of 3 October
1997L 272 10 4.10.1997
►M7CommissionRegulation(EC) No 761/1999 of 12 April 1999 L 99 4 14.4.1999
►M8CommissionRegulation(EC) No 440/2003 of 10 March 2003 L 66 15 11.3.2003
▼B
COMMISSIONREGULATION(EEC)No000/90
of17September1990
determining Communitymethodsfortheanalysisofwines
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Regulation (EEC) No 822/87 of 16 March
1987 on the common organization of the market in wine (1), as
amended by Regulation (EEC) No 1325/90 (2), and in particular Article
74 thereof,
Whereas Article 74 (1) of Regulation(EEC) No 822/87 prescribes the
adoption of methods of analysis for establishing the composition of theproducts indicated in Article 1 of that Regulation and of rules forchecking whether these products have been subjected to treatments inviolationof authorized oen ological pratice;
Whereas, inasmuch as the Community has not yet laid down maximum
levels for substances whose presence indicates that certain oenologicalpractices have been used and has not yet adopted tables enablinganalysis data to be compared, there is good reason to authorizeMember States to determine such maximum levels;
Whereas Article 13 (1) of Regulation(EEC) No 822/87 provides for an
analytical test, including at the least an assessment of the characteris-tics, as listed in the Annex to that Regulation, of the quality wine psrinquestion ;
Whereas the verification of the particulars on documents concerning
the products in question calls for the introduction of uniform methodsof analysis to ensure that accurate and comparable information isobtained; whereas, consequently, these methods should be compulsoryfor all commercial transactions and all verification procedures;whereas, however, inview of the trade's limited facilities, a restricted
number of usual procedures should be admitted enabling the requisite
factors to be determined rapidly and with reasonable accuracy;
Whereas, as far as is possible, generally recognized methods such as
those developed under the 1954 International Convention for the Unifi-cation of Methods of Analysis and Appraisal of Wines, which are
published inthe Recueil des méthodes internationales d'analyse des
vins(Compendium of international methods for the analysis of wines)
by the International Office of Vine and Wine, may usefully be retained;
Whereas the Community methods of analysis applicable to wine were
laid downinCommissionRegulation(EEC) No 1108/82 (
3); whereas
scientific progress has made it necessary to replace certain of the
methods by some that are more suitable, to modify others, and to intro-duce new methods, particularly those approved since the aforesaidRegulation by the International Office of Vine and Wine; whereas,because of the profusion and complexity of these changes, all the
analyses should be reassembled in a new Regulation, and Regulation
(EEC) No 1108/82 should be repealed;
Whereas, in order to ensure the comparability of the results obtained by
applying the analytical methods referred to in Article 74 of Regulation(EEC) No 822/87, steps should be takento refer, inregard to the accu-
racy, repeatability and reproducibility of these results, to the definitions
laid down by the International Office of Vine and Wine;
Whereas, in order to recognize the scientific advances on the one hand
and the technical equipment of official laboratories on the other, andwith the aim of increasing the efficiency and profitability of these1990R2676 — EN — 18.03.2003 — 007.001 — 2
(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.
▼B
laboratories, there is good reasonto allow automated an alytical
methods to be applied under certain conditions; whereas it is importantto specify that, where a dispute arises, the automated methods may not
replace the reference methods and the usual methods;
Whereas the results of a density measurement using the automated
method based on the principle of the frequency oscillator are, in respectof their accuracy, repeatability and reproducibility, at least as good asthe results obtained by the methods listed in section 1 of the Annex to
the present Regulation for measuring the density or specific gravity;
whereas it is therefore indicated, by virtue of Article 74 (3) of Regula-tion(EEC) No 822/87, that this automated method shall be con sideredas equivalent to the said methods listed in the Annex to the presentRegulation;
Whereas the procedure described in Chapter 25 under point 2.2.3.3.2 in
the Annex hereto for analysing the total sulphur dioxide content of
wines and grape musts of a presumed content of less than 50 mg/lresults inbetter extractionof that substan ce compared to the methodsdescribed in Chapter 13 under point 13.4 of the Annex to Regulation(EEC) No 1108/82; whereas the result is higher total sulphur dioxide
contents of the products analysed, which may exceed, in particular, in
the case of certaingrape juices, the maximum limit laid down ;whereas, inorder to avoid difficulties inthe disposal of grape juicealready prepared at the time of entry into force of this Regulation anduntil such time as the production processes are adapted to result inmore complete de-sulphiting of grape musts with fermentation arrested
by the additionof alcohol, the procedure described inthe abovemen -
tioned Regulation should be allowed to be used during a transitionalperiod;
Whereas the measures provided for inthis Regulationare inaccor-
dance with the opinion of the Management Committee for Wine,
HAS ADOPTED THIS REGULATION:
Article 1
1. The Community methods for the analysis of wine making it
possible, in the context of commercial transactions and all controloperations, to:
— establish the compositionof the products listed inArticle 1 of
Regulation(EEC) No 822/87,
— check whether these products have beensubjected to treatmen ts in
violationof authorized oen ological practice,
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:
(a) the repeatability shall be the value below which the absolute differ-
ence between the two single test results obtained on identical testmaterial, under the same conditions (same operator, same appa-ratus, same laboratory and a short interval of time), may be
expected to lie with a specified probability;
(b) the reproducibility shall be the value below which the absolute
difference between two single test results obtained identical test
material, under different conditions (different operators, different
apparatus and/or different laboratories and/or different time), maybe expected to lie with a specified probability.
The term ‘single test result’ shall be the value obtained when the stan-
dardized test method is applied fully and once to a single sample.
Unless otherwise stated, the probability shall be 95 %.1990R2676 — EN — 18.03.2003 — 007.001 — 3
▼B
Article 3
1. Automated analytical methods shall be acceptable, under the
responsibility of the Director of a laboratory on condition that the accu-
racy, repeatability and reproducibility of the results are at least
equivalent to those of the results obtained by the analytical methodslisted in the Annex.
Where a dispute arises, the methods listed in the Annex may not be
replaced by automated methods.
2. The automated method for measuring density based on the prin-
ciple of the frequency oscilliator shall be considered as equivalent tothe methods listed in section 1 of the Annex to the present Regulation.
Article 4
Wherever mention is made of water for solution, dilution, or washing
purposes, this shall meandistilled water or demin eralized 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 Regulationshall apply un til 31 December
1990.
Article 6
This Regulation shall enter into force on the day of its publication in
theOfficial 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
inall Member States.1990R2676 — EN — 18.03.2003 — 007.001 — 4
▼B
ANNEX
1.DENSITYANDSPECIFICGRAVITYAT20ºC
1. DEFINITIONS
The density is the mass per unit volume of wine or must at 20 ºC. It
is expressed in grams per millilitre and denoted by the symbol ρ
20ºC.
The specific gravity at 20 ºC (or the 20 ºC/20 ºC relative density) is
the ratio, expressed as a decimal number, of the density of a certain
volume of the wine or must at 20 ºC to the density of same volume
water at the same temperature. It is denoted by the symbol d20 ºC20 ºC.
2. PRINCIPLE OF METHODS
The density and specific gravity at 20 ºC are measured on a test
sample:
— either by pigmentary: reference method,
— or by hydrometry or densimetry using a hydrostatic balance:
usual methods.
Note:For very accurate measurement, the density must be corrected for
the effect of sulphur dioxide by using the formula:
ρ
20 ºC=ρ′20 ºC− 0,0006 × S
where ρ20 ºC= corrected density
ρ′20 ºC= 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 filtrationun der reduced pressure through 2 g of cotton
wool placed inanexten siontube.
4. REFERENCE METHOD4.1.Apparatus:
Normal laboratory equipment, and in particular:
4.1.1. A Pyrex pycnometer (
1), of approximately 100 ml capacity, with a
removable ground-glass jointed thermometer calibrated in tenths of
a degree from 10 to 30 ºC (Figure 1). The thermometer must be
standardized.1990R2676 — EN — 18.03.2003 — 007.001 — 5
(1) Any pycnometer with equivalent characteristics may be used.
▼B
Figure 1
Thepycnometeranditstare
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 solution2,0 % m/v).
A thermally insulated container, exactly fitting the body of the pycn-
ometer.
4.1.3. A twin-pan balance with a range of at least 300 g and a sensitivity
of 0,1 mg,
ora single-pan balance with a range of at least 200 g and a sensitivity
of 0,1 mg.
4.2.Calibrationofpycnometer
Calibration of the pycnometer involves a determination of the
following quantities:
— tare of the empty pycnometer,
— volume of the pycnometer at 20 ºC,
— mass of the water-filled pycnometer at 20 ºC.
4.2.1.Methodusingatwin-panbalance
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 pgrams.
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 thermo-
meter 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 tºC carefully, possibly correcting it for the
inaccuracy in the temperature scale. Weigh the water-filled pycn-
ometer against the tare and record the weight p′ingrams required
to establish equilibrium.1990R2676 — EN — 18.03.2003 — 007.001 — 6
▼B
Calculation (1):
Taring of empty pycnometer:
tare of empty pycnometer = p+m,
wherem= mass of air contained in the pycnometer,
m = 0,0012 ( p−p′).
Volume at 20 ºC:V
20 ºC=(p+m−p′)·Ft,
whereFt= a factor takenfrom Table I for the temperature tºC.
V20 ºCshould be known to within ± 0,001 ml.
Mass of water at 20 ºC:
M20 ºC= 0,998203 V20 ºC,
where 0,998203 is the density of water at 20 ºC.
4.2.2. Method using a single-pan balance
Determine:
— the mass of the clean and dry pycnometer: let this be P,
— the mass of the pycnometer filled with water at tºC, following
the procedure described in4.2.1 above: let this be P1,
— the mass of the tare: T0.
Calculation(1):
Taring of empty pycnometer:tare of empty pycnometer = P−m,
wherem= mass of air contained in the pycnometer,
m = 0,0012 ( P
1−P).
Volume at 20 ºC:V
20 ºC=[P1−(P− m)] ×Ft
whereFt= a factor takenfrom Table I for the temperature tºC.
The volume at 20 ºC should be known to within ± 0,001 ml.
Mass of water at 20 ºC:M
20 ºC= 0,998203 V20 ºC,
where 0,998203 is the density of water at 20 ºC.
4.3.Methodofmeasurement (1)
4.3.1. Method using twin-pan balance
Fill the pycnometer with the prepared test sample, following the
procedure described in4.2.1 above.
Letp″be the weight ingrams required to establish equilibrium at t
ºC.
Mass of liquid contained in the pycnometer = p+m−p″.
Apparent density at tºC:
ρtº C¼pþm/C0p00
V20 ºC
Calculate the density at 20 ºC 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 ºC/20 ºC specific gravity of the wine is calculated by
dividing its density at 20 ºC by 0,998203.1990R2676 — EN — 18.03.2003 — 007.001 — 7
(1) A numeric example is given in section 6 of this chapter.
▼B
4.3.2. Method using a single-pan balance (1)
Weigh the tare bottle and let its mass be T.
Calculate d T=T1−T0.
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
ºC beP2.
Mass of liquid contained in pycnometer at tºC =P2−(P−m+dT)
Apparent density at tºC:
ρtº C¼P2/C0P/C0mþdT ðÞ
V20 ºC
Calculate the density at 20 ºC of the liquid under test (dry wine,
natural or concentrated must, or sweet wine) as indicated in 4.3.1
above.
The 20 ºC/20 ºC specific gravity is calculated by dividing the
density at 20 ºC 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.
5. USUAL METHODS5.1.Hydrometry
5.1.1. Apparatus
5.1.1.1. Hydrometer
Hydrometers shall conform to ISO Standards as regards their dimen-
sions and graduation.
They shall have a cylindrical bulb, and a stem of circular cross-
section with a minimum diameter of 3 mm. For dry wines, they shall
be graduated from 0,983 to 1,003 with graduationmarks every
0,0010 and 0,0002. Each mark at 0,0010 shall be separated from
the next corresponding mark by at least 5 mm. For measuring the
density of de-alcoholized wines, sweet wines and musts, a set of
five hydrometers graduated from 1,000 to 1,030, 1,030 to 1,060,
1,060 to 1,090, 1,090 to 1,120 and 1,120 to 1,150 shall be used.
These hydrometers shall be graduated inden sities at 20 ºC with
marks at least every 0,0010 and 0,0005, each mark at 0,0010 being
separated by at least 3 mm from the next corresponding mark.
These hydrometers shall be graduated so as to be read ‘at the top of
the meniscus’. An indication of the graduation in density or specific
gravity at 20 ºC and of the reading at the top of the meniscus shall
be carried either onthe graduated scale or ona slip of paper
enclosed in the bulb. These hydrometers shall be calibrated by agovernment department.
5.1.1.2. A calibrated thermometer graduated in minimum intervals of 0,5 ºC.5.1.1.3. A measuring cylinder of 36 mm internal diameter and 320 mm
height, held vertical by supporting levelling screws.
5.1.2. Procedure
5.1.2.1. Method of measurement
Pour 250 ml of the test sample, prepared as in5.1.1.3 above, in to the
measuring cylinder and introduce the thermometer and hydrometer1990R2676 — EN — 18.03.2003 — 007.001 — 8
(1) A numeric example is given in section 6 of this chapter.
▼B
into it. Read the temperature one minute after mixing the sample
well to produce a uniform temperature. Remove the thermometer
and read the apparent density at tºC from the hydrometer after a
further minute.
The apparent density at tºC is thencorrected to 20 ºC by usin g the
tables applicable to dry wines (Table V), musts (Table VI) or wines
containing sugars (Table VII).
The 20 ºC/20 ºC specific gravity is obtained by dividing the density
at 20 ºC by 0,998203.
5.2.Densimetryusingahydrostaticbalance
5.2.1. Apparatus
5.2.1.1. Hydrostatic balance with a maximum capacity of at least 100 g and
a sensitivity of 0,1 mg.
Identical Pyrex glass floats with a volume of at least20 ml are fixed
under both pans by suspending them with a thread of diameter not
exceeding 0,1 mm.
The float suspended under the right-hand pan must be capable of
being introduced into a measuring cylinder bearing a mark indicating
the level. The measuring cylinder must have an internal diameter at
least 6 mm larger thanthat of the float. The float must be capable of
being contained completely within the volume of the measuring
cylinder located below the mark; the surface of the liquid to be
measured must be penetrated only by the supporting thread. The
temperature of the liquid in the measuring cylinder is measured by
a thermometer graduated insteps of 0,2 ºC.
5.2.1.2. A single-pan hydrostatic balance may also be used.5.2.2. Procedure
5.2.2.1. Standardization of a hydrostatic balance
With the two floats in air, establish balance by placing weights in
the right-hand pan. Record the mass pof these added weights.
Fill the measuring cylinder with pure water up to the mark and read
the temperature tºC after shaking and allowing to stand for two or
three minutes.
Re-establish balance by placing weights in the right-hand pan. Let
the mass of these be p′.
The volume of the float at 20 ºC is givenby:
V
20 ºC=(p′−p)(F+ 0,0012)
whereFis the factor giveninTable I for the temperature tºC.
pandV20 ºCare the characteristics of the float.
5.2.2.2. Method of measurement
The right-hand float is immersed in the measuring cylinder filled
with wine (or must) up to the mark. The temperature tºC of the
wine (or must) is read and the mass p″needed to re-establish
balance is recorded.
The apparent density ρtis givenby
ρtº C¼p00/C0p ðÞ
Vþ0;0012
This density is corrected to 20 ºC by using one of the Tables II, III
or IV if the float is of Pyrex glass.1990R2676 — EN — 18.03.2003 — 007.001 — 9
▼B
6. EXAMPLE OF THE CALCULATION OF THE DENSITY AT 20 ºC
AND THE 20 ºC/20 ºC SPECIFIC GRAVITY (REFERENCE
METHOD)
6.1.Pycnometryusingatwin-panbalance
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 tºC:
Tare = pycnometer + water + p′
p′= 1,2396 g at t= 20,5 ºC
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 ºC = F(p+m−p′)tºC
F20,5 ºC= 1,001900
V20 ºC= (105,0698 − 1,2396) × 1,001900
V20 ºC= 104,0275 ml
Mass of water at 20 ºC =M
20 ºC=V20 ºC· 0,998203
M20 ºC= 103,8405 g
6.1.2. Determination of the density and specific gravity at 20 ºC/20 ºC of a
dry wine
ρ″= 1,2622 at 17,80 ºC
ρ17;80 ºC¼105 ;0698 /C01;2622
104 ;0275
ρ17,80 ºC= 0,99788 g/ml
Table II enables ρ20 ºCto be calculated from ρtºCusing the relation-
ship:
ρ20 ºC¼ρtº C±c
1000
Fort= 17,80 ºC and for an alcoholic strength of 11 % vol. c= 0,54.
ρ20 ºC¼0;99788 /C00;54
1000
ρ20 ºC= 0,99734 g/ml
d20 ºC
20 ºC¼0;99734
0;998203¼0;999131990R2676 — EN — 18.03.2003 — 007.001 — 10
▼B
6.2.Pycnometryusingasingle-panbalance
6.2.1. Standardization of the pycnometer:
1. Weight of cleandry pycn ometer:
P= 67,7913 g
2. Weight of pycnometer filled with water at tºC:
P1= 169,2715 at 21,65 ºC
3. Mass of air contained in the pycnometer:
m= 0,0012 ( P1−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 ºC = [ P1−(P−m)]FtºC
F21,65 ºC= 1,002140
V20 ºC= 1,002140 (169,2715 − 67,6695)
V20 ºC= 101,8194 ml
Mass of water at 20 ºC:
M20 ºC=V20 ºC× 0,998203
M20 ºC= 101,6364 g
Mass of tare bottle, To:
To= 171,9160 g
6.2.2. Determination of the density and specific gravity at 20º C of a dry
wine
T1= 171,9178 g
dT= 171,9178 − 171,9160 = 0,0018 g
P−m+dT= 67,6695 + 0,0018 = 67,6713 g
P2= 169,2799 at 18 ºC
ρ18 ºC¼169 ;2799 /C067;6713
101 ;8194
ρ18 ºC= 0,99793 g/ml
Table II enables ρ20 ºCto be calculated from ρtº Cusing the relation-
ship:
ρ20 ºC¼ρtº C±c
1000
Fort= 18 ºC and an alcoholic strength of 11 % vol. c= 0,49.
ρ20 ºC¼0;99793 /C00;49
1000
ρ20 ºC= 0,99744 g/ml
d20 ºC
20 ºC¼0;99744
0;998203¼0;999231990R2676 — EN — 18.03.2003 — 007.001 — 11
▼B
TABLE I
FfactorsbywhichthemassofwatercontainedinthePyrexpycnometerattºChastobemulti-
pliedinordertocalculate thepycnometervolumeat20ºC
tºC F tºC F tºC F tºC F tºC F tºC F tºC F
10,0 1,000398 13,0 1,000691 16,0 1,001097 19,0 1,001608 22,0 1,002215 25,0 1,002916 28,0 1,003704
,1 1,000406 ,1 1,000703 ,1 1,001113 ,1 1,001627 ,1 1,002238 ,1 1,002941 ,1 1,003731
,2 1,000414 ,2 1,000714 ,2 1,001128 ,2 1,001646 ,2 1,002260 ,2 1,002966 ,2 1,003759
,3 1,000422 ,3 1,000726 ,3 1,001144 ,3 1,001665 ,3 1,002282 ,3 1,002990 ,3 1,003787
,4 1,000430 ,4 1,000738 ,4 1,001159 ,4 1,001684 ,4 1,002304 ,4 1,003015 ,4 1,003815
10,5 1,000439 13,5 1,000752 16,5 1,001175 19,5 1,001703 22,5 1,002326 25,5 1,003041 28,5 1,003843
,6 1,000447 ,6 1,000764 ,6 1,001191 ,6 1,001722 ,6 1,002349 ,6 1,003066 ,6 1,003871
,7 1,000456 ,7 1,000777 ,7 1,001207 ,7 1,001741 ,7 1,002372 ,7 1,003092 ,7 1,003899
,8 1,000465 ,8 1,000789 ,8 1,001223 ,8 1,001761 ,8 1,002394 ,8 1,003117 ,8 1,003928
,9 1,000474 ,9 1,000803 ,9 1,001239 ,9 1,001780 ,9 1,002417 ,9 1,003143 ,9 1,003956
11,0 1,000483 14,0 1,000816 17,0 1,001257 20,0 1,001800 23,0 1,002439 26,0 1,003168 29,0 1,003984
,1 1,000492 ,1 1,000829 ,1 1,001273 ,1 1,001819 ,1 1,002462 ,1 1,003194 ,1 1,004013
,2 1,000501 ,2 1,000842 ,2 1,001290 ,2 1,001839 ,2 1,002485 ,2 1,003222 ,2 1,004042
,3 1,000511 ,3 1,000855 ,3 1,001306 ,3 1,001859 ,3 1,002508 ,3 1,003247 ,3 1,004071
,4 1,000520 ,4 1,000868 ,4 1,001323 ,4 1,001880 ,4 1,002531 ,4 1,003273 ,4 1,004099
11,5 1,000530 14,5 1,000882 17,5 1,001340 20,5 1,001900 23,5 1,002555 26,5 1,003299 29,5 1,004128
,6 1,000540 ,6 1,000895 ,6 1,001357 ,6 1,001920 ,6 1,002578 ,6 1,003326 ,6 1,004158
,7 1,000550 ,7 1,000909 ,7 1,001374 ,7 1,001941 ,7 1,002602 ,7 1,003352 ,7 1,004187
,8 1,000560 ,8 1,000923 ,8 1,001391 ,8 1,001961 ,8 1,002625 ,8 1,003379 ,8 1,004216
,9 1,000570 ,9 1,000937 ,9 1,001409 ,9 1,001982 ,9 1,002649 ,9 1,003405 ,9 1,004245
12,0 1,000580 15,0 1,000951 18,0 1,001427 21,0 1,002002 24,0 1,002672 27,0 1,003432 30,0 1,004275
,1 1,000591 ,1 1,000965 ,1 1,001445 ,1 1,002023 ,1 1,002696 ,1 1,003458
,2 1,000601 ,2 1,000979 ,2 1,001462 ,2 1,002044 ,2 1,002720 ,2 1,003485
,3 1,000612 ,3 1,000993 ,3 1,001480 ,3 1,002065 ,3 1,002745 ,3 1,003513
,4 1,000623 ,4 1,001008 ,4 1,001498 ,4 1,002086 ,4 1,002769 ,4 1,003540
12,5 1,000634 15,5 1,001022 18,5 1,001516 21,5 1,002107 24,5 1,002793 27,5 1,003567
,6 1,000645 ,6 1,001037 ,6 1,001534 ,6 1,002129 ,6 1,002817 ,6 1,003594
,7 1,000656 ,7 1,001052 ,7 1,001552 ,7 1,002151 ,7 1,002842 ,7 1,003621
,8 1,000668 ,8 1,001067 ,8 1,001570 ,8 1,002172 ,8 1,002866 ,8 1,003649
,9 1,000679 ,9 1,001082 ,9 1,001589 ,9 1,002194 ,9 1,002891 ,9 1,0036761990R2676 — EN — 18.03.2003 — 007.001 — 12
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TABLE II
Temperaturecorrections ctothedensityofalcohol-freedrywinesmeasuredwithaPyrexglasspycnometerat t
ºCtorelate theresultto20ºC
ρ20¼ρt±c
1000/C144/C147/C147/C147/C147/C145/C147/C147/C147/C147/C146−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Alcoholic strengths
0567891 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7Temperature (ºC)10 1,59 1,64 1,67 1,71 1,77 1,84 1,91 2,01 2,11 2,22 2,34 2,46 2,60 2,73 2,88 3,03 3,19 3,35 3,52 3,70 3,87 4,06 4,25 4,44
11 1,48 1,53 1,56 1,60 1,64 1,70 1,77 1,86 1,95 2,05 2,16 2,27 2,38 2,51 2,63 2,77 2,91 3,06 3,21 3,36 3,53 3,69 3,86 4,03
12 1,36 1,40 1,43 1,46 1,50 1,56 1,62 1,69 1,78 1,86 1,96 2,05 2,16 2,27 2,38 2,50 2,62 2,75 2,88 3,02 3,16 3,31 3,46 3,61
13 1,22 1,26 1,28 1,32 1,35 1,40 1,45 1,52 1,59 1,67 1,75 1,83 1,92 2,01 2,11 2,22 2,32 2,44 2,55 2,67 2,79 2,92 3,05 3,18
14 1,08 1,11 1,13 1,16 1,19 1,23 1,27 1,33 1,39 1,46 1,52 1,60 1,67 1,75 1,84 1,93 2,03 2,11 2,21 2,31 2,42 2,52 2,63 2,74
15 0,92 0,96 0,97 0,99 1,02 1,05 1,09 1,13 1,19 1,24 1,30 1,36 1,42 1,48 1,55 1,63 1,70 1,78 1,86 1,95 2,03 2,12 2,21 2,3016 0,76 0,79 0,80 0,81 0,84 0,86 0,89 0,93 0,97 1,01 1,06 1,10 1,16 1,21 1,26 1,32 1,38 1,44 1,51 1,57 1,64 1,71 1,78 1,85
17 0,59 0,61 0,62 0,63 0,65 0,67 0,69 0,72 0,75 0,78 0,81 0,85 0,88 0,95 0,96 1,01 1,05 1,11 1,15 1,20 1,25 1,30 1,35 1,40
18 0,40 0,42 0,42 0,43 0,44 0,46 0,47 0,49 0,51 0,53 0,55 0,57 0,60 0,63 0,65 0,68 0,71 0,74 0,77 0,81 0,84 0,87 0,91 0,94
19 0,21 0,21 0,22 0,22 0,23 0,23 0,24 0,25 0,26 0,27 0,28 0,29 0,30 0,32 0,33 0,34 0,36 0,37 0,39 0,41 0,42 0,44 0,46 0,47
20
21 0,21 0,22 0,22 0,23 0,23 0,24 0,25 0,26 0,27 0,28 0,29 0,30 0,31 0,32 0,34 0,36 0,37 0,38 0,40 0,41 0,43 0,44 0,46 0,48
22 0,44 0,45 0,46 0,47 0,48 0,49 0,51 0,52 0,54 0,56 0,59 0,61 0,63 0,66 0,69 0,71 0,74 0,77 0,80 0,83 0,87 0,90 0,93 0,97
23 0,68 0,70 0,71 0,72 0,74 0,76 0,78 0,80 0,83 0,86 0,90 0,93 0,96 1,00 1,03 1,08 1,13 1,17 1,22 1,26 1,31 1,37 1,41 1,46
24 0,93 0,96 0,97 0,99 1,01 1,03 1,06 1,10 1,13 1,18 1,22 1,26 1,31 1,36 1,41 1,47 1,52 1,58 1,64 1,71 1,77 1,84 1,90 1,97
25 1,19 1,23 1,25 1,27 1,29 1,32 1,36 1,40 1,45 1,50 1,55 1,61 1,67 1,73 1,80 1,86 1,93 2,00 2,08 2,16 2,24 2,32 2,40 2,4826 1,47 1,51 1,53 1,56 1,59 1,62 1,67 1,72 1,77 1,83 1,90 1,96 2,03 2,11 2,19 2,27 2,35 2,44 2,53 2,62 2,72 2,81 2,91 3,01
27 1,75 1,80 1,82 1,85 1,89 1,93 1,98 2,04 2,11 2,18 2,25 2,33 2,41 2,50 2,59 2,68 2,78 2,88 2,98 3,09 3,20 3,31 3,42 3,53
28 2,04 2,10 2,13 2,16 2,20 2,25 2,31 2,38 2,45 2,53 2,62 2,70 2,80 2,89 3,00 3,10 3,21 3,32 3,45 3,57 3,69 3,82 3,94 4,07
29 2,34 2,41 2,44 2,48 2,53 2,58 2,65 2,72 2,81 2,89 2,99 3,09 3,19 3,30 3,42 3,53 3,65 3,78 3,92 4,05 4,19 4,33 4,47 4,61
30 2,66 2,73 2,77 2,81 2,86 2,92 3,00 3,08 3,17 3,27 3,37 3,48 3,59 3,72 3,84 3,97 4,11 4,25 4,40 4,55 4,70 4,85 4,92 5,17
Note:This table may be used to convert the specific gravity d20tto the specific gravity d2020.
TABLE III
Temperature corrections cto the density of natural musts and of concentrated musts measured with a Pyrex
glasspycnometerat tºCtorelate theresultto20ºC
ρ20¼ρt±c
1000−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Densities
1,05 1,06 1,07 1,08 1,09 1,10 1,11 1,12 1,13 1,14 1,15 1,16 1,18 1,20 1,22 1,24 1,26 1,28 1,30 1,32 1,34 1,36Temperature (ºC)10º 2,31 2,48 2,66 2,82 2,99 3,13 3,30 3,44 3,59 3,73 3,88 4,01 4,28 4,52 4,76 4,98 5,18 5,42 5,56 5,73 5,90 6,05
11º 2,12 2,28 2,42 2,57 2,72 2,86 2,99 3,12 3,25 3,37 3,50 3,62 3,85 4,08 4,29 4,48 4,67 4,84 5,00 5,16 5,31 5,45
12º 1,92 2,06 2,19 2,32 2,45 2,58 2,70 2,82 2,94 3,04 3,15 3,26 3,47 3,67 3,85 4,03 4,20 4,36 4,51 4,65 4,78 4,91
13º 1,72 1,84 1,95 2,06 2,17 2,27 2,38 2,48 2,58 2,69 2,78 2,88 3,05 3,22 3,39 3,55 3,65 3,84 3,98 4,11 4,24 4,36
14º 1,52 1,62 1,72 1,81 1,90 2,00 2,09 2,17 2,26 2,34 2,43 2,51 2,66 2,82 2,96 3,09 3,22 3,34 3,45 3,56 3,67 3,76
15º 1,28 1,36 1,44 1,52 1,60 1,67 1,75 1,82 1,89 1,96 2,04 2,11 2,24 2,36 2,48 2,59 2,69 2,79 2,88 2,97 3,03 3,10
16º 1,05 1,12 1,18 1,25 1,31 1,37 1,43 1,49 1,55 1,60 1,66 1,71 1,81 1,90 2,00 2,08 2,16 2,24 2,30 2,37 2,43 2,49
17º 0,80 0,86 0,90 0,95 1,00 1,04 1,09 1,13 1,18 1,22 1,26 1,30 1,37 1,44 1,51 1,57 1,62 1,68 1,72 1,76 1,80 1,84
18º 0,56 0,59 0,62 0,66 0,68 0,72 0,75 0,77 0,80 0,83 0,85 0,88 0,93 0,98 1,02 1,05 1,09 1,12 1,16 1,19 1,21 1,24
19º 0,29 0,31 0,32 0,34 0,36 0,37 0,39 0,40 0,42 0,43 0,44 0,45 0,48 0,50 0,52 0,54 0,56 0,57 0,59 0,60 0,61 0,62
20º21º 0,29 0,30 0,32 0,34 0,35 0,37 0,38 0,40 0,41 0,42 0,44 0,46 0,48 0,50 0,53 0,56 0,58 0,59 0,60 0,61 0,62 0,62
22º 0,58 0,61 0,64 0,67 0,70 0,73 0,76 0,79 0,81 0,84 0,87 0,90 0,96 1,00 1,05 1,09 1,12 1,15 1,18 1,20 1,22 1,23
23º 0,89 0,94 0,99 1,03 1,08 1,12 1,16 1,20 1,25 1,29 1,33 1,37 1,44 1,51 1,57 1,63 1,67 1,73 1,77 1,80 1,82 1,84
24º 1,20 1,25 1,31 1,37 1,43 1,49 1,54 1,60 1,66 1,71 1,77 1,82 1,92 2,01 2,10 2,17 2,24 2,30 2,36 2,40 2,42 2,44
25º 1,51 1,59 1,66 1,74 1,81 1,88 1,95 2,02 2,09 2,16 2,23 2,30 2,42 2,53 2,63 2,72 2,82 2,89 2,95 2,99 3,01 3,0526º 1,84 1,92 2,01 2,10 2,18 2,26 2,34 2,42 2,50 2,58 2,65 2,73 2,87 3,00 3,13 3,25 3,36 3,47 3,57 3,65 3,72 3,79
27º 2,17 2,26 2,36 2,46 2,56 2,66 2,75 2,84 2,93 3,01 3,10 3,18 3,35 3,50 3,66 3,80 3,93 4,06 4,16 4,26 4,35 4,42
28º 2,50 2,62 2,74 2,85 2,96 3,07 3,18 3,28 3,40 3,50 3,60 3,69 3,87 4,04 4,21 4,36 4,50 4,64 4,75 4,86 4,94 5,00
29º 2,86 2,98 3,10 3,22 3,35 3,47 3,59 3,70 3,82 3,93 4,03 4,14 4,34 4,53 4,72 4,89 5,05 5,20 5,34 5,46 5,56 5,64
30º 3,20 3,35 3,49 3,64 3,77 3,91 4,05 4,17 4,30 4,43 4,55 4,67 4,90 5,12 5,39 5,51 5,68 5,84 5,96 6,08 6,16 6,22
Note:This table may be used to convert the specific gravity d20tto the specific gravity d2020.1990R2676 — EN — 18.03.2003 — 007.001 — 13
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TABLE IV
Temperature corrections cto the density of wines of 13 % vol and above containing residual sugar measured
withaPyrexglasspycnometerat tºCtorelate theresultto20ºC
ρ20¼ρt±c
1000/C144/C147/C147/C147/C147/C145/C147/C147/C147/C147/C146−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Wines of 13 % vol Wines of 15 % vol Wines of 17 % vol
Densities Densities Densities
1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120Temperature (ºC)10º 2,36 2,71 3,06 3,42 3,72 3,96 4,32 2,64 2,99 3,36 3,68 3,99 4,30 4,59 2,94 3,29 3,64 3,98 4,29 4,60 4,89
11º 2,17 2,49 2,80 2,99 3,39 3,65 3,90 2,42 2,73 3,05 3,34 3,63 3,89 4,15 2,69 3,00 3,32 3,61 3,90 4,16 4,41
12º 1,97 2,25 2,53 2,79 3,05 3,29 3,52 2,19 2,47 2,75 3,01 3,27 3,51 3,73 2,42 2,70 2,98 3,24 3,50 3,74 3,96
13º 1,78 2,02 2,25 2,47 2,69 2,89 3,09 1,97 2,21 2,44 2,66 2,87 3,08 3,29 2,18 2,42 2,64 2,87 3,08 3,29 3,49
14º 1,57 1,78 1,98 2,16 2,35 2,53 2,70 1,74 1,94 2,14 2,32 2,52 2,69 2,86 1,91 2,11 2,31 2,50 2,69 2,86 3,03
15º 1,32 1,49 1,66 1,82 1,97 2,12 2,26 1,46 1,63 1,79 1,95 2,10 2,25 2,39 1,60 1,77 1,93 2,09 2,24 2,39 2,5316º 1,08 1,22 1,36 1,48 1,61 1,73 1,84 1,18 1,32 1,46 1,59 1,71 1,83 1,94 1,30 1,44 1,58 1,71 1,83 1,95 2,06
17º 0,83 0,94 1,04 1,13 1,22 1,31 1,40 0,91 1,02 1,12 1,21 1,30 1,39 1,48 1,00 1,10 1,20 1,30 1,39 1,48 1,56
18º 0,58 0,64 0,71 0,78 0,84 0,89 0,95 0,63 0,69 0,76 0,83 0,89 0,94 1,00 0,69 0,75 0,82 0,89 0,95 1,00 1,06
19º 0,30 0,34 0,37 0,40 0,43 0,46 0,49 0,33 0,37 0,40 0,43 0,46 0,49 0,52 0,36 0,39 0,42 0,46 0,49 0,52 0,54
20º21º 0,30 0,33 0,36 0,40 0,43 0,46 0,49 0,33 0,36 0,39 0,43 0,46 0,49 0,51 0,35 0,39 0,42 0,45 0,48 0,51 0,54
22º 0,60 0,67 0,73 0,80 0,85 0,91 0,98 0,65 0,72 0,78 0,84 0,90 0,96 1,01 0,71 0,78 0,84 0,90 0,96 1,01 1,07
23º 0,93 1,02 1,12 1,22 1,30 1,39 1,49 1,01 1,10 1,20 1,29 1,38 1,46 1,55 1,10 1,19 1,29 1,38 1,46 1,55 1,63
24º 1,27 1,39 1,50 1,61 1,74 1,84 1,95 1,37 1,49 1,59 1,72 1,84 1,95 2,06 1,48 1,60 1,71 1,83 1,95 2,06 2,17
25º 1,61 1,75 1,90 2,05 2,19 2,33 2,47 1,73 1,87 2,02 2,17 2,31 2,45 2,59 1,87 2,01 2,16 2,31 2,45 2,59 2,73
26º 1,94 2,12 2,29 2,47 2,63 2,79 2,95 2,09 2,27 2,44 2,62 2,78 2,94 3,10 2,26 2,44 2,61 2,79 2,95 3,11 3,26
27º 2,30 2,51 2,70 2,90 3,09 3,27 3,44 2,48 2,68 2,87 3,07 3,27 3,45 3,62 2,67 2,88 3,07 3,27 3,46 3,64 3,81
28º 2,66 2,90 3,13 3,35 3,57 3,86 4,00 2,86 3,10 3,23 3,55 3,77 3,99 4,20 3,08 3,31 3,55 3,76 3,99 4,21 4,41
29º 3,05 3,31 3,56 3,79 4,04 4,27 4,49 3,28 3,53 3,77 4,02 4,26 4,49 4,71 3,52 3,77 4,01 4,26 4,50 4,73 4,95
30º 3,44 3,70 3,99 4,28 4,54 4,80 5,06 3,68 3,94 4,23 4,52 4,79 5,05 5,30 3,95 4,22 4,51 4,79 5,07 5,32 5,57
Wines of 19 % vol Wines of 21 % vol
Densities Densities
1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120Temperature (ºC)10º 3,27 3,62 3,97 4,30 4,62 4,92 5,21 3,62 3,97 4,32 4,66 4,97 5,27 5,56
11º 2,99 3,30 3,61 3,90 4,19 4,45 4,70 3,28 3,61 3,92 4,22 4,50 4,76 5,01
12º 2,68 2,96 3,24 3,50 3,76 4,00 4,21 2,96 3,24 3,52 3,78 4,03 4,27 4,49
13º 2,40 2,64 2,87 3,09 3,30 3,51 3,71 2,64 2,88 3,11 3,33 3,54 3,74 3,95
14º 2,11 2,31 2,51 2,69 2,88 3,05 3,22 2,31 2,51 2,71 2,89 3,08 3,25 3,43
15º 1,76 1,93 2,09 2,25 2,40 2,55 2,69 1,93 2,10 2,26 2,42 2,57 2,72 2,8616º 1,43 1,57 1,70 1,83 1,95 2,08 2,18 1,56 1,70 1,84 1,97 2,09 2,21 2,32
17º 1,09 1,20 1,30 1,39 1,48 1,57 1,65 1,20 1,31 1,41 1,50 1,59 1,68 1,77
18º 0,76 0,82 0,88 0,95 1,01 1,06 1,12 0,82 0,88 0,95 1,01 1,08 1,13 1,18
19º 0,39 0,42 0,45 0,49 0,52 0,55 0,57 0,42 0,46 0,49 0,52 0,55 0,58 0,61
20º
21º 0,38 0,42 0,45 0,48 0,51 0,54 0,57 0,41 0,45 0,48 0,51 0,54 0,57 0,60
22º 0,78 0,84 0,90 0,96 1,02 1,07 1,13 0,84 0,90 0,96 1,02 1,08 1,14 1,19
23º 1,19 1,28 1,38 1,47 1,55 1,64 1,72 1,29 1,39 1,48 1,57 1,65 1,74 1,82
24º 1,60 1,72 1,83 1,95 2,06 2,18 2,29 1,73 1,85 1,96 2,08 2,19 2,31 2,42
25º 2,02 2,16 2,31 2,46 2,60 2,74 2,88 2,18 2,32 2,47 2,62 2,76 2,90 3,0426º 2,44 2,62 2,79 2,96 3,12 3,28 3,43 2,53 2,81 2,97 3,15 3,31 3,47 3,62
27º 2,88 3,08 3,27 3,42 3,66 3,84 4,01 3,10 3,30 3,47 3,69 3,88 4,06 4,23
28º 3,31 3,54 3,78 4,00 4,22 4,44 4,64 3,56 3,79 4,03 4,25 4,47 4,69 4,89
29º 3,78 4,03 4,27 4,52 4,76 4,99 5,21 4,06 4,31 4,55 4,80 5,04 5,27 5,48
30º 4,24 4,51 4,80 5,08 5,36 5,61 5,86 4,54 4,82 5,11 5,39 5,66 5,91 6,161990R2676 — EN — 18.03.2003 — 007.001 — 14
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TABLE V
Temperature corrections cto the density of dry wines and alcohol-free dry wines measured with an ordinary
glasspycnometerorhydrometerat tºCtorelate theresultto20ºC
ρ20¼ρt±c
1000/C144/C147/C147/C147/C147/C145/C147/C147/C147/C147/C146−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Alcoholic strength
0 5 6 7 8 9 1 01 11 21 31 41 51 61 71 81 92 02 12 22 32 42 52 62 7Temperature (ºC)10º 1,45 1,51 1,55 1,58 1,64 1,70 1,78 1,88 1,98 2,09 2,21 2,34 2,47 2,60 2,75 2,90 3,06 3,22 3,39 3,57 3,75 3,93 4,12 4,31
11º 1,35 1,40 1,43 1,47 1,52 1,58 1,65 1,73 1,83 1,93 2,03 2,15 2,26 2,38 2,51 2,65 2,78 2,93 3,08 3,24 3,40 3,57 3,73 3,90
12º 1,24 1,28 1,31 1,34 1,39 1,44 1,50 1,58 1,66 1,75 1,84 1,94 2,04 2,15 2,26 2,38 2,51 2,63 2,77 2,91 3,05 3,19 3,34 3,49
13º 1,12 1,16 1,18 1,21 1,25 1,30 1,35 1,42 1,49 1,56 1,64 1,73 1,82 1,91 2,01 2,11 2,22 2,33 2,45 2,57 2,69 2,81 2,95 3,07
14º 0,99 1,03 1,05 1,07 1,11 1,14 1,19 1,24 1,31 1,37 1,44 1,52 1,59 1,67 1,75 1,84 1,93 2,03 2,13 2,23 2,33 2,44 2,55 2,66
15º 0,86 0,89 0,90 0,92 0,95 0,98 1,02 1,07 1,12 1,17 1,23 1,29 1,35 1,42 1,49 1,56 1,63 1,71 1,80 1,88 1,96 2,05 2,14 2,2316º 0,71 0,73 0,74 0,76 0,78 0,81 0,84 0,87 0,91 0,96 0,99 1,05 1,10 1,15 1,21 1,27 1,33 1,39 1,45 1,52 1,59 1,66 1,73 1,80
17º 0,55 0,57 0,57 0,59 0,60 0,62 0,65 0,67 0,70 0,74 0,77 0,81 0,84 0,88 0,92 0,96 1,01 1,05 1,11 1,15 1,20 1,26 1,31 1,36
18º 0,38 0,39 0,39 0,40 0,41 0,43 0,44 0,46 0,48 0,50 0,52 0,55 0,57 0,60 0,62 0,65 0,68 0,71 0,74 0,78 0,81 0,85 0,88 0,91
19º 0,19 0,20 0,20 0,21 0,21 0,22 0,23 0,24 0,25 0,26 0,27 0,28 0,29 0,30 0,32 0,33 0,35 0,36 0,38 0,39 0,41 0,43 0,44 0,46
20º
21º 0,21 0,22 0,22 0,23 0,23 0,24 0,25 0,25 0,26 0,27 0,29 0,29 0,31 0,32 0,34 0,35 0,36 0,38 0,39 0,41 0,43 0,44 0,46 0,48
22º 0,43 0,45 0,45 0,46 0,47 0,49 0,50 0,52 0,54 0,56 0,58 0,60 0,63 0,65 0,68 0,71 0,73 0,77 0,80 0,83 0,86 0,89 0,93 0,96
23º 0,67 0,69 0,70 0,71 0,72 0,74 0,77 0,79 0,82 0,85 0,88 0,91 0,95 0,99 1,03 1,07 1,12 1,16 1,21 1,25 1,30 1,35 1,40 1,45
24º 0,91 0,93 0,95 0,97 0,99 1,01 1,04 1,07 1,11 1,15 1,20 1,24 1,29 1,34 1,39 1,45 1,50 1,56 1,62 1,69 1,76 1,82 1,88 1,95
25º 1,16 1,19 1,21 1,23 1,26 1,29 1,33 1,37 1,42 1,47 1,52 1,57 1,63 1,70 1,76 1,83 1,90 1,97 2,05 2,13 2,21 2,29 2,37 2,4526º 1,42 1,46 1,49 1,51 1,54 1,58 1,62 1,67 1,73 1,79 1,85 1,92 1,99 2,07 2,14 2,22 2,31 2,40 2,49 2,58 2,67 2,77 2,86 2,96
27º 1,69 1,74 1,77 1,80 1,83 1,88 1,93 1,98 2,05 2,12 2,20 2,27 2,35 2,44 2,53 2,63 2,72 2,82 2,93 3,04 3,14 3,25 3,37 3,48
28º 1,97 2,03 2,06 2,09 2,14 2,19 2,24 2,31 2,38 2,46 2,55 2,63 2,73 2,83 2,93 3,03 3,14 3,26 3,38 3,50 3,62 3,75 3,85 4,00
29º 2,26 2,33 2,37 2,40 2,45 2,50 2,57 2,64 2,73 2,82 2,91 2,99 3,11 3,22 3,34 3,45 3,58 3,70 3,84 3,97 4,11 4,25 4,39 4,54
30º 2,56 2,64 2,67 2,72 2,77 2,83 2,90 2,98 3,08 3,18 3,28 3,38 3,50 3,62 3,75 3,88 4,02 4,16 4,30 4,46 4,61 4,76 4,92 5,07
Note:This table may be used to convert the specific gravity d20tto the specific gravity d2020.
TABLE VI
Temperature corrections cto the density of natural musts and of concentrated musts measured with a pycn-
ometerorhydrometerofordinaryglassat tºCtorelate theresultto20ºC
ρ20¼ρt±c
1000/C144/C147/C147/C147/C147/C145/C147/C147/C147/C147/C146−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Density
1,05 1,06 1,07 1,08 1,09 1,10 1,11 1,12 1,13 1,14 1,15 1,16 1,18 1,20 1,22 1,24 1,26 1,28 1,30 1,32 1,34 1,36Temperature (ºC)10º 2,17 2,34 2,52 2,68 2,85 2,99 3,16 3,29 3,44 3,58 3,73 3,86 4,13 4,36 4,60 4,82 5,02 5,25 5,39 5,56 5,73 5,87
11º 2,00 2,16 2,29 2,44 2,59 2,73 2,86 2,99 3,12 3,24 3,37 3,48 3,71 3,94 4,15 4,33 4,52 4,69 4,85 5,01 5,15 5,29
12º 1,81 1,95 2,08 2,21 2,34 2,47 2,58 2,70 2,82 2,92 3,03 3,14 3,35 3,55 3,72 3,90 4,07 4,23 4,37 4,52 4,64 4,77
13º 1,62 1,74 1,85 1,96 2,07 2,17 2,28 2,38 2,48 2,59 2,68 2,77 2,94 3,11 3,28 3,44 3,54 3,72 3,86 3,99 4,12 4,24
14º 1,44 1,54 1,64 1,73 1,82 1,92 2,00 2,08 2,17 2,25 2,34 2,42 2,57 2,73 2,86 2,99 3,12 3,24 3,35 3,46 3,57 3,65
15º 1,21 1,29 1,37 1,45 1,53 1,60 1,68 1,75 1,82 1,89 1,97 2,03 2,16 2,28 2,40 2,51 2,61 2,71 2,80 2,89 2,94 3,01
16º 1,00 1,06 1,12 1,19 1,25 1,31 1,37 1,43 1,49 1,54 1,60 1,65 1,75 1,84 1,94 2,02 2,09 2,17 2,23 2,30 2,36 2,42
17º 0,76 0,82 0,86 0,91 0,96 1,00 1,05 1,09 1,14 1,18 1,22 1,25 1,32 1,39 1,46 1,52 1,57 1,63 1,67 1,71 1,75 1,79
18º 0,53 0,56 0,59 0,63 0,65 0,69 0,72 0,74 0,77 0,80 0,82 0,85 0,90 0,95 0,99 1,02 1,06 1,09 1,13 1,16 1,18 1,20
19º 0,28 0,30 0,31 0,33 0,35 0,36 0,38 0,39 0,41 0,42 0,43 0,43 0,46 0,48 0,50 0,52 0,54 0,55 0,57 0,58 0,59 0,60
20º21º 0,28 0,29 0,31 0,33 0,34 0,36 0,37 0,39 0,40 0,41 0,43 0,44 0,46 0,48 0,51 0,54 0,56 0,57 0,58 0,59 0,60 0,60
22º 0,55 0,58 0,61 0,64 0,67 0,70 0,73 0,76 0,78 0,81 0,84 0,87 0,93 0,97 1,02 1,06 1,09 1,12 1,15 1,17 1,19 1,19
23º 0,85 0,90 0,95 0,99 1,04 1,08 1,12 1,16 1,21 1,25 1,29 1,32 1,39 1,46 1,52 1,58 1,62 1,68 1,72 1,75 1,77 1,79
24º 1,15 1,19 1,25 1,31 1,37 1,43 1,48 1,54 1,60 1,65 1,71 1,76 1,86 1,95 2,04 2,11 2,17 2,23 2,29 2,33 2,35 2,37
25º 1,44 1,52 1,59 1,67 1,74 1,81 1,88 1,95 2,02 2,09 2,16 2,22 2,34 2,45 2,55 2,64 2,74 2,81 2,87 2,90 2,92 2,9626º 1,76 1,84 1,93 2,02 2,10 2,18 2,25 2,33 2,41 2,49 2,56 2,64 2,78 2,91 3,03 3,15 3,26 3,37 3,47 3,55 3,62 3,60
27º 2,07 2,16 2,26 2,36 2,46 2,56 2,65 2,74 2,83 2,91 3,00 3,07 3,24 3,39 3,55 3,69 3,82 3,94 4,04 4,14 4,23 4,30
28º 2,39 2,51 2,63 2,74 2,85 2,96 3,06 3,16 3,28 3,38 3,48 3,57 3,75 3,92 4,08 4,23 4,37 4,51 4,62 4,73 4,80 4,86
29º 2,74 2,86 2,97 3,09 3,22 3,34 3,46 3,57 3,69 3,80 3,90 4,00 4,20 4,39 4,58 4,74 4,90 5,05 5,19 5,31 5,40 5,48
30º 3,06 3,21 3,35 3,50 3,63 3,77 3,91 4,02 4,15 4,28 4,40 4,52 4,75 4,96 5,16 5,35 5,52 5,67 5,79 5,91 5,99 6,04
Note:This table may be used to convert the specific gravity d20tto the specific gravity d2020.1990R2676 — EN — 18.03.2003 — 007.001 — 15
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TABLE VII
Temperature corrections cto the density of wines of 13 % vol and above containing residual sugar measured
withahydrometerorpycnometerofordinaryglassat tºCtorelate theresultto20ºC
ρ20¼ρt±c
1000/C144/C147/C147/C147/C147/C145/C147/C147/C147/C147/C146−i ftºC is lower than20 ºC
+i ftºC is higher than20 ºC
Wines of 13 % vol Wines of 15 % vol Wines of 17 % vol
Densities Densities Densities
1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120Temperature (ºC)10º 2,24 2,58 2,93 3,27 3,59 3,89 4,18 2,51 2,85 3,20 3,54 3,85 4,02 4,46 2,81 3,15 3,50 3,84 4,15 4,45 4,74
11º 2,06 2,37 2,69 2,97 3,26 3,53 3,78 2,31 2,61 2,93 3,21 3,51 3,64 4,02 2,57 2,89 3,20 3,49 3,77 4,03 4,28
12º 1,87 2,14 2,42 2,67 2,94 3,17 3,40 2,09 2,36 2,64 2,90 3,16 3,27 3,61 2,32 2,60 2,87 3,13 3,39 3,63 3,84
13º 1,69 1,93 2,14 2,37 2,59 2,80 3,00 1,88 2,12 2,34 2,56 2,78 2,88 3,19 2,09 2,33 2,55 2,77 2,98 3,19 3,39
14º 1,49 1,70 1,90 2,09 2,27 2,44 2,61 1,67 1,86 2,06 2,25 2,45 2,51 2,77 1,83 2,03 2,23 2,42 2,61 2,77 2,94
15º 1,25 1,42 1,59 1,75 1,90 2,05 2,19 1,39 1,56 1,72 1,88 2,03 2,11 2,32 1,54 1,71 1,87 2,03 2,18 2,32 2,4716º 1,03 1,17 1,30 1,43 1,55 1,67 1,78 1,06 1,27 1,40 1,53 1,65 1,77 1,88 1,25 1,39 1,52 1,65 1,77 1,89 2,00
17º 0,80 0,90 1,00 1,09 1,17 1,27 1,36 0,87 0,98 1,08 1,17 1,26 1,35 1,44 0,96 1,06 1,16 1,26 1,35 1,44 1,52
18º 0,54 0,61 0,68 0,75 0,81 0,86 0,92 0,60 0,66 0,73 0,80 0,85 0,91 0,97 0,66 0,72 0,79 0,86 0,92 0,97 1,03
19º 0,29 0,33 0,36 0,39 0,42 0,45 0,48 0,32 0,36 0,39 0,42 0,45 0,48 0,51 0,35 0,38 0,41 0,45 0,48 0,51 0,53
20º21º 0,29 0,32 0,35 0,39 0,42 0,45 0,47 0,32 0,35 0,38 0,42 0,45 0,48 0,50 0,34 0,38 0,41 0,44 0,47 0,50 0,53
22º 0,57 0,64 0,70 0,76 0,82 0,88 0,93 0,63 0,69 0,75 0,81 0,87 0,93 0,98 0,68 0,75 0,81 0,87 0,93 0,99 1,04
23º 0,89 0,98 1,08 1,17 1,26 1,34 1,43 0,97 1,06 1,16 1,25 1,34 1,42 1,51 1,06 1,15 1,25 1,34 1,42 1,51 1,59
24º 1,22 1,34 1,44 1,56 1,68 1,79 1,90 1,32 1,44 1,54 1,66 1,78 1,89 2,00 1,43 1,56 1,65 1,77 1,89 2,00 2,11
25º 1,61 1,68 1,83 1,98 2,12 2,26 2,40 1,66 1,81 1,96 2,11 2,25 2,39 2,52 1,80 1,94 2,09 2,24 2,39 2,52 2,66
26º 1,87 2,05 2,22 2,40 2,56 2,71 2,87 2,02 2,20 2,37 2,54 2,70 2,85 3,01 2,18 2,36 2,53 2,71 2,86 3,02 3,17
27º 2,21 2,42 2,60 2,80 3,00 3,18 3,35 2,39 2,59 2,78 2,98 3,17 3,35 3,52 2,58 2,78 2,97 3,17 3,36 3,54 3,71
28º 2,56 2,80 3,02 3,25 3,47 3,67 3,89 2,75 2,89 3,22 3,44 3,66 3,86 4,07 2,97 3,21 3,44 3,66 3,88 4,09 4,30
29º 2,93 3,19 3,43 3,66 3,91 4,14 4,37 3,16 3,41 3,65 3,89 4,13 4,36 4,59 3,40 3,66 3,89 4,13 4,38 4,61 4,82
30º 3,31 3,57 3,86 4,15 4,41 4,66 4,92 3,55 3,81 4,10 4,38 4,66 4,90 5,16 3,82 4,08 4,37 4,65 4,93 5,17 5,42
Wines of 19 % vol Wines of 21 % vol
Densities Densities
1,000 1,020 1,040 1,060 1,080 1,100 1,120 1,000 1,020 1,040 1,060 1,080 1,100 1,120Temperature (ºC)10º 3,14 3,48 3,83 4,17 4,48 4,78 5,07 3,50 3,84 4,19 4,52 4,83 5,12 5,41
11º 2,87 3,18 3,49 3,78 4,06 4,32 4,57 3,18 3,49 3,80 4,09 4,34 4,63 4,88
12º 2,58 2,86 3,13 3,39 3,65 3,88 4,10 2,86 3,13 3,41 3,67 3,92 4,15 4,37
13º 2,31 2,55 2,77 2,99 3,20 3,41 3,61 2,56 2,79 3,01 3,23 3,44 3,65 3,85
14º 2,03 2,23 2,43 2,61 2,80 2,96 3,13 2,23 2,43 2,63 2,81 3,00 3,16 3,33
15º 1,69 1,86 2,02 2,18 2,33 2,48 2,62 1,86 2,03 2,19 2,35 2,50 2,65 2,8016º 1,38 1,52 1,65 1,78 1,90 2,02 2,13 1,51 1,65 1,78 1,91 2,03 2,15 2,26
17º 1,06 1,16 1,26 1,35 1,44 1,53 1,62 1,15 1,25 1,35 1,45 1,54 1,63 1,71
18º 0,73 0,79 0,85 0,92 0,98 1,03 1,09 0,79 0,85 0,92 0,98 1,05 1,10 1,15
19º 0,38 0,41 0,44 0,48 0,51 0,52 0,56 0,41 0,44 0,47 0,51 0,54 0,57 0,59
20º
21º 0,37 0,41 0,44 0,47 0,50 0,53 0,56 0,41 0,44 0,47 0,51 0,54 0,57 0,59
22º 0,75 0,81 0,87 0,93 0,99 1,04 1,10 0,81 0,88 0,94 1,00 1,06 1,10 1,17
23º 1,15 1,30 1,34 1,43 1,51 1,60 1,68 1,25 1,34 1,44 1,63 1,61 1,70 1,78
24º 1,55 1,67 1,77 1,89 2,00 2,11 2,23 1,68 1,80 1,90 2,02 2,13 2,25 2,36
25º 1,95 2,09 2,24 2,39 2,53 2,67 2,71 2,11 2,25 2,40 2,55 2,69 2,83 2,9726º 2,36 2,54 2,71 2,89 3,04 3,20 3,35 2,55 2,73 2,90 3,07 3,22 3,38 3,54
27º 2,79 2,99 3,18 3,38 3,57 3,75 3,92 3,01 3,20 3,40 3,59 3,78 3,96 4,13
28º 3,20 3,44 3,66 3,89 4,11 4,32 4,53 3,46 3,69 3,93 4,15 4,36 4,58 4,77
29º 3,66 3,92 4,15 4,40 4,64 4,87 5,08 3,95 4,20 4,43 4,68 4,92 5,15 5,36
30º 4,11 4,37 4,66 4,94 5,22 5,46 5,71 4,42 4,68 4,97 5,25 5,53 5,77 6,021990R2676 — EN — 18.03.2003 — 007.001 — 16
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2.EVALUATION BY REFRACTOMETRY OF THE SUGAR CONCEN-
TRATION IN GRAPE MUSTS, CONCENTRATED GRAPE MUSTS
ANDRECTIFIEDCONCENTRATEDGRAPEMUSTS
1. PRINCIPLE OF THE METHOD
The refractive index at 20 ºC, 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, concen-
trated grape musts and rectified concentrated grape musts.
2. APPARATUS2.1.Abbérefractometer
The refractometer used must be fitted with a scale giving:
— either percentage by mass of sucrose to 0,1 %,
— or refractive indices to four decimal places.
The refractometer must be equipped with a thermometer having a
scale extending at least from + 15 ºC to + 25 ºC and with an
arrangement for circulating water enabling measurements to be
made at a temperature of 20 ± 5 ºC.
The operating instructions for this instrument must be strictly
adhered to, particularly with regard to calibrationan d the light
source.
3. PREPARATION OF THE SAMPLE3.1.Mustandconcentratedmust
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.Rectifiedconcentrated 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 ºC. Place a small test
sample onthe lower prism of the refractometer, takin g 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 tºC.
5. CALCULATION5.1.Temperaturecorrection
5.1.1. Instruments graduated in percentage by mass of sucrose: use Table I
to obtainthe temperature correction .
5.1.2. Instruments graduated in refractive index: find the index measured at
tºC in Table II to obtain (column 1) the corresponding value of the
percentage by mass of sucrose at tºC. This value is corrected for
temperature and expressed as a concentration at 20 ºC by means of
Table I.
5.2.Sugarconcentrationinmustandconcentratedmust
Find the percentage by mass of sucrose at 20 ºC in Table II and read
from the same row the sugar concentration in grams per litre and1990R2676 — EN — 18.03.2003 — 007.001 — 17
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grams per kilogram. The sugar concentration is expressed in terms of
invert sugar to one decimal place.
5.3.Sugarconcentrationinrectified concentratedmust
Find the percentage by mass of sucrose at 20 ºC 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 dilutionfactor.
5.4.Refractive index of must, concentrated must and rectified
concentratedmust
Find the percentage by mass of sucrose at 20 ºC in Table II and read
from the same row the refractive index at 20 ºC. 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
temperaturedifferentfrom20ºC
Temperature
ºCSucrose ingrams per 100 grams of product
51 01 52 03 04 05 06 07 07 5
Subtract
15 0,25 0,27 0,31 0,31 0,34 0,35 0,36 0,37 0,36 0,36
16 0,21 0,23 0,27 0,27 0,29 0,31 0,31 0,32 0,31 0,2317 0,16 0,18 0,20 0,20 0,22 0,23 0,23 0,23 0,20 0,17
18 0,11 0,12 0,14 0,15 0,16 0,16 0,15 0,12 0,12 0,09
19 0,06 0,07 0,08 0,08 0,08 0,09 0,09 0,08 0,07 0,05
Add
21 0,06 0,07 0,07 0,07 0,07 0,07 0,07 0,07 0,07 0,07
22 0,12 0,14 0,14 0,14 0,14 0,14 0,14 0,14 0,14 0,1423 0,18 0,20 0,20 0,21 0,21 0,21 0,21 0,22 0,22 0,2224 0,24 0,26 0,26 0,27 0,28 0,28 0,28 0,28 0,29 0,29
25 0,30 0,32 0,32 0,34 0,36 0,36 0,36 0,36 0,36 0,37
Temperatures must not differ from 20 ºC by more than ± 5 ºC.1990R2676 — EN — 18.03.2003 — 007.001 — 18
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TABLE II
Table giving the sugar concentration (1)in must and concentrated must in grams per litre and
gramsperkilogram,determinedbymeansofarefractometer graduatedeitherinpercentageby
massofsucroseat20ºCorinrefractive indexat20ºC.Thedensityat20ºCisalsogiven
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
10.0 1.34781 1.0390 82.3 79.2 4,89
10.1 1.34798 1.0394 83.4 80.2 4,95
10.2 1.34814 1.0398 84.5 81.3 5,02
10.3 1.34830 1.0402 85.6 82.2 5,09
10.4 1.34845 1.0406 86.6 83.2 5,14
10.5 1.34860 1.0410 87.6 84.1 5,20
10.6 1.34875 1.0414 88.6 85.1 5,26
10.7 1.34890 1.0419 89.7 86.1 5,33
10.8 1.34906 1.0423 90.8 87.1 5,39
10.9 1.34921 1.0427 91.8 88.1 5,45
11.0 1.34936 1.0431 92.9 89.1 5,52
11.1 1.34952 1.0435 94.0 90.0 5,58
11.2 1.34968 1.0439 95.0 91.0 5,64
11.3 1.34984 1.0443 96.1 92.0 5,71
11.4 1.34999 1.0447 97.1 92.9 5,77
11.5 1.35015 1.0452 98.2 94.0 5,83
11.6 1.35031 1.0456 99.3 95.0 5,90
11.7 1.35046 1.0460 100.3 95.9 5,96
11.8 1.35062 1.0464 101.4 96.9 6,02
11.9 1.35077 1.0468 102.5 97.9 6,09
12.0 1.35092 1.0473 103.6 98.9 6,15
12.1 1.35108 1.0477 104.7 99.9 6,22
12.2 1.35124 1.0481 105.7 100.8 6,28
12.3 1.35140 1.0485 106.8 101.9 6,35
12.4 1.35156 1.0489 107.9 102.9 6,41
12.5 1.35172 1.0494 109.0 103.8 6,47
12.6 1.35187 1.0498 110.0 104.8 6,53
12.7 1.35203 1.0502 111.1 105.8 6,60
12.8 1.35219 1.0506 112.2 106.8 6,66
12.9 1.35234 1.0510 113.2 107.8 6,73
13.0 1.35249 1.0514 114.3 108.7 6,79
13.1 1.35266 1.0519 115.4 109.7 6,86
13.2 1.35282 1.0523 116.5 110.7 6,92
13.3 1.35298 1.0527 117.6 111.7 6,99
13.4 1.35313 1.0531 118.6 112.6 7,05
13.5 1.35329 1.0536 119.7 113.6 7,11
13.6 1.35345 1.0540 120.8 114.6 7,18
13.7 1.35360 1.0544 121.8 115.6 7,24
13.8 1.35376 1.0548 122.9 116.5 7,30
13.9 1.35391 1.0552 124.0 117.5 7,37
14.0 1.35407 1.0557 125.1 118.5 7,43
14.1 1.35424 1.0561 126.2 119.5 7,50
14.2 1.35440 1.0565 127.3 120.5 7,56
14.3 1.35456 1.0569 128.4 121.5 7,63
14.4 1.35472 1.0574 129.5 122.5 7,69
14.5 1.35488 1.0578 130.6 123.4 7,76
14.6 1.35503 1.0582 131.6 124.4 7,82
14.7 1.35519 1.0586 132.7 125.4 7,88
14.8 1.35535 1.0591 133.8 126.3 7,95
14.9 1.35551 1.0595 134.9 127.3 8,011990R2676 — EN — 18.03.2003 — 007.001 — 19
(1) The sugar concentration is expressed in terms of invert sugar.
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Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
15.0 1.35567 1.0599 136.0 128.3 8,08
15.1 1.35583 1.0603 137.1 129.3 8,15
15.2 1.35599 1.0608 138.2 130.3 8,21
15.3 1.35615 1.0612 139.3 131.3 8,27
15.4 1.35631 1.0616 140.4 132.3 8,34
15.5 1.35648 1.0621 141.5 133.2 8,41
15.6 1.35664 1.0625 142.6 134.2 8,47
15.7 1.35680 1.0629 143.7 135.2 8,54
15.8 1.35696 1.0633 144.8 136.2 8,60
15.9 1.35712 1.0638 145.9 137.2 8,67
16.0 1.35728 1.0642 147.0 138.1 8,73
16.1 1.35744 1.0646 148.1 139.1 8,80
16.2 1.35760 1.0651 149.2 140.1 8,86
16.3 1.35776 1.0655 150.3 141.1 8,93
16.4 1.35793 1.0660 151.5 142.1 9,00
16.5 1.35809 1.0664 152.6 143.1 9,06
16.6 1.35825 1.0668 153.7 144.1 9,13
16.7 1.35842 1.0672 154.8 145.0 9,20
16.8 1.35858 1.0677 155.9 146.0 9,26
16.9 1.35874 1.0681 157.0 147.0 9,33
17.0 1.35890 1.0685 158.1 148.0 9,39
17.1 1.35907 1.0690 159.3 149.0 9,46
17.2 1.35923 1.0694 160.4 150.0 9,53
17.3 1.35939 1.0699 161.5 151.0 9,59
17.4 1.35955 1.0703 162.6 151.9 9,66
17.5 1.35972 1.0707 163.7 152.9 9,73
17.6 1.35988 1.0711 164.8 153.9 9,79
17.7 1.36004 1.0716 165.9 154.8 9,86
17.8 1.36020 1.0720 167.0 155.8 9,92
17.9 1.36036 1.0724 168.1 156.8 9,99
18.0 1.36053 1.0729 169.3 157.8 10,06
18.1 1.36070 1.0733 170.4 158.8 10,12
18.2 1.36086 1.0738 171.5 159.7 10,19
18.3 1.36102 1.0742 172.6 160.7 10,25
18.4 1.36119 1.0746 173.7 161.6 10,32
18.5 1.36136 1.0751 174.9 162.6 10,39
18.6 1.36152 1.0755 176.0 163.6 10,46
18.7 1.36169 1.0760 177.2 164.6 10,53
18.8 1.36185 1.0764 178.3 165.6 10,59
18.9 1.36201 1.0768 179.4 166.6 10,66
19.0 1.36217 1.0773 180.5 167.6 10,72
19.1 1.36234 1.0777 181.7 168.6 10,80
19.2 1.36251 1.0782 182.8 169.5 10,86
19.3 1.36267 1.0786 183.9 170.5 10,93
19.4 1.36284 1.0791 185.1 171.5 11,00
19.5 1.36301 1.0795 186.3 172.5 11,07
19.6 1.36318 1.0800 187.4 173.5 11,13
19.7 1.36335 1.0804 188.6 174.5 11,21
19.8 1.36351 1.0809 189.7 175.5 11,27
19.9 1.36367 1.0813 190.8 176.5 11,341990R2676 — EN — 18.03.2003 — 007.001 — 20
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Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
20.0 1.36383 1.0817 191.9 177.4 11,40
20.1 1.36400 1.0822 193.1 178.4 11,47
20.2 1.36417 1.0826 194.2 179.4 11,54
20.3 1.36434 1.0831 195.3 180.4 11,60
20.4 1.36451 1.0835 196.5 181.4 11,67
20.5 1.36468 1.0840 197.7 182.3 11,75
20.6 1.36484 1.0844 198.8 183.3 11,81
20.7 1.36501 1.0849 200.0 184.3 11,88
20.8 1.36518 1.0853 201.1 185.3 11,96
20.9 1.36534 1.0857 202.2 186.2 12,01
21.0 1.36550 1.0862 203.3 187.2 12,08
21.1 1.36568 1.0866 204.5 188.2 12,15
21.2 1.36585 1.0871 205.7 189.2 12,22
21.3 1.36601 1.0875 206.8 190.2 12,29
21.4 1.36618 1.0880 207.9 191.1 12,35
21.5 1.36635 1.0884 209.1 192.1 12,42
21.6 1.36652 1.0889 210.3 193.1 12,49
21.7 1.36669 1.0893 211.4 194.1 12,56
21.8 1.36685 1.0897 212.5 195.0 12,63
21.9 1.36702 1.0902 213.6 196.0 12,69
22.0 1.36719 1.0906 214.8 196.9 12,76
22.1 1.36736 1.0911 216.0 198.0 12,83
22.2 1.36753 1.0916 217.2 199.0 12,90
22.3 1.36770 1.0920 218.3 199.9 12,97
22.4 1.36787 1.0925 219.5 200.9 13,04
22.5 1.36804 1.0929 220.6 201.8 13,11
22.6 1.36820 1.0933 221.7 202.8 13,17
22.7 1.36837 1.0938 222.9 203.8 13,24
22.8 1.36854 1.0943 224.1 204.8 13,31
22.9 1.36871 1.0947 225.2 205.8 13,38
23.0 1.36888 1.0952 226.4 206.7 13,45
23.1 1.36905 1.0956 227.6 207.7 13,52
23.2 1.36922 1.0961 228.7 208.7 13,59
23.3 1.36939 1.0965 229.9 209.7 13,66
23.4 1.36956 1.0970 231.1 210.7 13,73
23.5 1.36973 1.0975 232.3 211.6 13,80
23.6 1.36991 1.0979 233.4 212.6 13,87
23.7 1.37008 1.0984 234.6 213.6 13,94
23.8 1.37025 1.0988 235.8 214.6 14,01
23.9 1.37042 1.0993 237.0 215.6 14,08
24.0 1.37059 1.0998 238.2 216.6 14,15
24.1 1.37076 1.1007 239.3 217.4 14,22
24.2 1.37093 1.1011 240.3 218.2 14,28
24.3 1.37110 1.1016 241.6 219.4 14,35
24.4 1.37128 1.1022 243.0 220.5 14,44
24.5 1.37145 1.1026 244.0 221.3 14,50
24.6 1.37162 1.1030 245.0 222.1 14,56
24.7 1.37180 1.1035 246.4 223.2 14,64
24.8 1.37197 1.1041 247.7 224.4 14,72
24.9 1.37214 1.1045 248.7 225.2 14,781990R2676 — EN — 18.03.2003 — 007.001 — 21
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
25.0 1.37232 1.1049 249.7 226.0 14,84
25.1 1.37249 1.1053 250.7 226.8 14,90
25.2 1.37266 1.1057 251.7 227.6 14,96
25.3 1.37283 1.1062 253.0 228.7 15,03
25.4 1.37300 1.1068 254.4 229.9 15,11
25.5 1.37317 1.1072 255.4 230.7 15,17
25.6 1.37335 1.1076 256.4 231.5 15,23
25.7 1.37353 1.1081 257.8 232.6 15,32
25.8 1.37370 1.1087 259.1 233.7 15,39
25.9 1.37387 1.1091 260.1 234.5 15,45
26.0 1.37405 1.1095 261.1 235.3 15,51
26.1 1.37423 1.1100 262.5 236.4 15,60
26.2 1.37440 1.1106 263.8 237.5 15,67
26.3 1.37457 1.1110 264.8 238.3 15,73
26.4 1.37475 1.1114 265.8 239.2 15,79
26.5 1.37493 1.1119 267.2 240.3 15,88
26.6 1.37510 1.1125 268.5 241.4 15,95
26.7 1.37528 1.1129 269.5 242.2 16,01
26.8 1.37545 1.1133 270.5 243.0 16,07
26.9 1.37562 1.1138 271.8 244.1 16,15
27.0 1.37580 1.1144 273.2 245.2 16,23
27.1 1.37598 1.1148 274.2 246.0 16,29
27.2 1.37615 1.1152 275.2 246.8 16,35
27.3 1.37632 1.1157 276.5 247.9 16,43
27.4 1.37650 1.1163 277.9 249.0 16,51
27.5 1.37667 1.1167 278.9 249.8 16,57
27.6 1.37685 1.1171 279.9 250.6 16,63
27.7 1.37703 1.1176 281.3 251.6 16,71
27.8 1.37721 1.1182 282.6 252.7 16,79
27.9 1.37739 1.1186 283.6 253.5 16,85
28.0 1.37757 1.1190 284.6 254.3 16,91
28.1 1.37775 1.1195 286.0 255.4 16,99
28.2 1.37793 1.1201 287.3 256.5 17,07
28.3 1.37810 1.1205 288.3 257.3 17,13
28.4 1.37828 1.1209 289.3 258.1 17,19
28.5 1.37846 1.1214 290.7 259.2 17,27
28.6 1.37863 1.1220 292.0 260.3 17,35
28.7 1.37881 1.1224 293.0 261.0 17,41
28.8 1.37899 1.1228 294.0 261.8 17,47
28.9 1.37917 1.1233 295.3 262.9 17,55
29.0 1.37935 1.1239 296.7 264.0 17,63
29.1 1.37953 1.1244 298.1 265.1 17,71
29.2 1.37971 1.1250 299.4 266.1 17,79
29.3 1.37988 1.1254 300.4 266.9 17,85
29.4 1.38006 1.1258 301.4 267.7 17,91
29.5 1.38024 1.1263 302.8 268.8 17,99
29.6 1.38042 1.1269 304.1 269.9 18,07
29.7 1.38060 1.1273 305.1 270.6 18,13
29.8 1.38078 1.1277 306.1 271.4 18,19
29.9 1.38096 1.1282 307.4 272.5 18,261990R2676 — EN — 18.03.2003 — 007.001 — 22
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
30.0 1.38114 1.1288 308.8 273.6 18,35
30.1 1.38132 1.1293 310.0 274.5 18,42
30.2 1.38150 1.1298 311.2 275.5 18,49
30.3 1.38168 1.1302 312.4 276.4 18,56
30.4 1.38186 1.1307 313.6 277.3 18,63
30.5 1.38204 1.1312 314.8 278.3 18,70
30.6 1.38222 1.1317 316.0 279.2 18,77
30.7 1.38240 1.1322 317.2 280.2 18,85
30.8 1.38258 1.1327 318.4 281.1 18,92
30.9 1.38276 1.1332 319.6 282.0 18,99
31.0 1.38294 1.1336 320.8 283.0 19,06
31.1 1.38312 1.1341 322.0 283.9 19,13
31.2 1.38330 1.1346 323.2 284.9 19,20
31.3 1.38349 1.1351 324.4 285.8 19,27
31.4 1.38367 1.1356 325.6 286.8 19,35
31.5 1.38385 1.1361 326.8 287.7 19,42
31.6 1.38403 1.1366 328.1 288.6 19,49
31.7 1.38421 1.1371 329.3 289.6 19,56
31.8 1.38440 1.1376 330.5 290.5 19,64
31.9 1.38458 1.1380 331.7 291.5 19,71
32.0 1.38476 1.1385 332.9 292.4 19,78
32.1 1.38494 1.1391 334.2 293.4 19,86
32.2 1.38513 1.1396 335.5 294.4 19,93
32.3 1.38531 1.1401 336.7 295.4 20,00
32.4 1.38550 1.1406 338.0 296.4 20,08
32.5 1.38568 1.1411 339.3 297.3 20,16
32.6 1.38586 1.1416 340.6 298.3 20,24
32.7 1.38605 1.1422 341.9 299.3 20,31
32.8 1.38623 1.1427 343.1 300.3 20,38
32.9 1.38642 1.1432 344.4 301.3 20,46
33.0 1.38660 1.1437 345.7 302.3 20,54
33.1 1.38678 1.1442 346.9 303.2 20,61
33.2 1.38697 1.1447 348.1 304.1 20,68
33.3 1.38715 1.1452 349.3 305.0 20,75
33.4 1.38734 1.1457 350.5 305.9 20,82
33.5 1.38753 1.1461 351.7 306.9 20,90
33.6 1.38771 1.1466 352.9 307.8 20,97
33.7 1.38790 1.1471 354.1 308.7 21,04
33.8 1.38808 1.1476 355.3 309.6 21,11
33.9 1.38827 1.1481 356.5 310.5 21,18
34.0 1.38845 1.1486 357.7 311.4 21,25
34.1 1.38864 1.1491 359.0 312.4 21,33
34.2 1.38882 1.1496 360.3 313.4 21,41
34.3 1.38901 1.1501 361.5 314.3 21,48
34.4 1.38919 1.1506 362.8 315.3 21,55
34.5 1.38938 1.1512 364.1 316.3 21,63
34.6 1.38957 1.1517 365.4 317.3 21,71
34.7 1.38975 1.1522 366.7 318.2 21,79
34.8 1.38994 1.1527 367.9 319.2 21,86
34.9 1.39012 1.1532 369.2 320.2 21,941990R2676 — EN — 18.03.2003 — 007.001 — 23
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
35.0 1.39031 1.1537 370.5 321.1 22,01
35.1 1.39050 1.1543 371.8 322.1 22,09
35.2 1.39069 1.1548 373.0 323.0 22,16
35.3 1.39087 1.1553 374.3 324.0 22,24
35.4 1.39106 1.1558 375.6 325.0 22,32
35.5 1.39125 1.1563 376,9 325.9 22,39
35.6 1.39144 1.1568 378.1 326.9 22,45
35.7 1.39163 1.1573 379.4 327.8 22,54
35.8 1.39181 1.1579 380.7 328.8 22,62
35.9 1.39200 1.1584 381.9 329.7 22,69
36.0 1.39219 1.1589 383.2 330.7 22,77
36.1 1.39238 1.1594 384.5 331.6 22,85
36.2 1.39257 1.1599 385.8 332.6 22,92
36.3 1.39276 1.1604 387.0 333.5 22,99
36.4 1.39295 1.1610 388.3 334.5 23,07
36.5 1.39314 1.1615 389.6 335.4 23,15
36.6 1.39332 1.1620 390.9 336.4 23,22
36.7 1.39351 1.1625 392.2 337.3 23,30
36.8 1.39370 1.1630 393.4 338.3 23,37
36.9 1.39389 1.1635 394.7 339.2 23,45
37.0 1.39408 1.1641 396.0 340.2 23,53
37.1 1.39427 1.1646 397.3 341.1 23,60
37.2 1.39446 1.1651 398.6 342.1 23,68
37.3 1.39465 1.1656 399.8 343.0 23,75
37.4 1.39484 1.1661 401.1 344.0 23,83
37.5 1.39504 1.1666 402.4 344.9 23,91
37.6 1.39523 1.1672 403.7 345.9 23,99
37.7 1.39542 1.1677 405.0 346.8 24,06
37.8 1.39561 1.1682 406.2 347.7 24,13
37.9 1.39580 1.1687 407.5 348.7 24,21
38.0 1.39599 1.1692 408.8 349.6 24,29
38.1 1.39618 1.1698 410.1 350.6 24,37
38.2 1.39637 1.1703 411.3 351.5 24,44
38.3 1.39657 1.1708 412.6 352.4 24,51
38.4 1.39676 1.1713 413.9 353.4 24,59
38.5 1.39695 1.1718 415.2 354.3 24,67
38.6 1.39714 1.1723 416.4 355.2 24,74
38.7 1.39733 1.1728 417.7 356.1 24,82
38.8 1.39753 1.1733 419.0 357.1 24,90
38.9 1.39772 1.1739 420.2 358.0 24,97
39.0 1.39791 1.1744 421.5 358.9 25,04
39.1 1.39810 1.1749 422.8 359.8 25,12
39.2 1.39830 1.1754 424.1 360.8 25,20
39.3 1.39849 1.1759 425.3 361.7 25,27
39.4 1.39869 1.1764 426.6 362.6 25,35
39.5 1.39888 1.1770 427.9 363.6 25,42
39.6 1.39907 1.1775 429.2 364.5 25,50
39.7 1.39927 1.1780 430.5 365.4 25,58
39.8 1.39946 1.1785 431.7 366.3 25,65
39.9 1.39966 1.1790 433.0 367.3 25,731990R2676 — EN — 18.03.2003 — 007.001 — 24
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
40.0 1.39985 1.1796 434.3 368.2 25,80
40.1 1.40004 1.1801 435.6 369.2 25,88
40.2 1.40024 1.1806 437.0 370.1 25,96
40.3 1.40043 1.1812 438.3 371.1 26,04
40.4 1.40063 1.1817 439.7 372.1 26,12
40.5 1.40083 1.1823 441.0 373.0 26,20
40.6 1.40102 1.1828 442.3 374.0 26,28
40.7 1.40122 1.1833 443.7 374.9 26,36
40.8 1.40141 1.1839 445.0 375.9 26,44
40.9 1.40161 1.1844 446.4 376.9 26,52
41.0 1.40180 1.1850 447.7 377.8 26,60
41.1 1.40200 1.1855 449.0 378.7 26,68
41.2 1.40219 1.1860 450.2 379.6 26,75
41.3 1.40239 1.1865 451.5 380.5 26,83
41.4 1.40259 1.1870 452.8 381.4 26,90
41.5 1.40279 1.1875 454.1 382.3 26,98
41.6 1.40298 1.1881 455.3 383.2 27,05
41.7 1.40318 1.1886 456.6 384.2 27,13
41.8 1.40338 1.1891 457.9 385.1 27,21
41.9 1.40357 1.1896 459.1 386.0 27,28
42.0 1.40377 1.1901 460.4 386.9 27,35
42.1 1.40397 1.1907 461.7 387.8 27,43
42.2 1.40417 1.1912 463.1 388.8 27,52
42.3 1.40436 1.1917 464.4 389.7 27,59
42.4 1.40456 1.1923 465.8 390.7 27,68
42.5 1.40476 1.1928 467.2 391.6 27,76
42.6 1.40496 1.1934 468.5 392.6 27,84
42.7 1.40516 1.1939 469.9 393.5 27,92
42.8 1.40535 1.1945 471.2 394.5 28,00
42.9 1.40555 1.1950 472.6 395.4 28,08
43.0 1.40575 1.1956 473.9 396.4 28,16
43.1 1.40595 1.1961 475.2 397.3 28,23
43.2 1.40615 1.1967 476.6 398.3 28,32
43.3 1.40635 1.1972 477.9 399.2 28,40
43.4 1.40655 1.1977 479.3 400.1 28,48
43.5 1.40675 1.1983 480.6 401.1 28,56
43.6 1.40695 1.1988 481.9 402.0 28,63
43.7 1.40715 1.1994 483.3 402.9 28,72
43.8 1.40735 1.1999 484.6 403.9 28,79
43.9 1.40755 1.2005 486.0 404.8 28,88
44.0 1.40775 1.2010 487.3 405.7 28,95
44.1 1.40795 1.2015 488.6 406.7 29,03
44.2 1.40815 1.2021 490.0 407.6 29,11
44.3 1.40836 1.2026 491.3 408.5 29,19
44.4 1.40856 1.2032 492.7 409.5 29,27
44.5 1.40876 1.2037 494.0 410.4 29,35
44.6 1.40896 1.2042 495.3 411.3 29,43
44.7 1.40916 1.2048 496.7 412.3 29,51
44.8 1.40937 1.2053 498.0 413.2 29,59
44.9 1.40957 1.2059 499.4 414.1 29,671990R2676 — EN — 18.03.2003 — 007.001 — 25
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
45.0 1.40977 1.2064 500.7 415.0 29,75
45.1 1.40997 1.2070 502.1 416.0 29,83
45.2 1.41018 1.2076 503.5 417.0 29,92
45.3 1.41038 1.2081 504.9 417.9 30,00
45.4 1.41058 1.2087 506.3 418.9 30,08
45.5 1.41079 1.2093 507.8 419.9 30,17
45.6 1.41099 1.2098 509.2 420.9 30,25
45.7 1.41119 1.2104 510.6 421.8 30,34
45.8 1.41139 1.2110 512.0 422.8 30,42
45.9 1.41160 1.2115 513.4 423.7 30,50
46.0 1.41180 1.2121 514.8 424.7 30,59
46.1 1.41200 1.2127 516.1 425.6 30,66
46.2 1.41221 1.2132 517.5 426.5 30,75
46.3 1.41241 1.2137 518.8 427.5 30,82
46.4 1.41262 1.2143 520.2 428.4 30,91
46.5 1.41282 1.2148 521.5 429.3 30,99
46.6 1.41302 1.2154 522.8 430.2 31,06
46.7 1.41323 1.2159 524.2 431.1 31,15
46.8 1.41343 1.2165 525.5 432.0 31,22
46.9 1.41364 1.2170 526.9 432.9 31,31
47.0 1.41384 1.2175 528.2 433.8 31,38
47.1 1.41405 1.2181 529.6 434.8 31,47
47.2 1.41425 1.2187 531.0 435.7 31,55
47.3 1.41446 1.2192 532.4 436.7 31,63
47.4 1.41466 1.2198 533.8 437.6 31,72
47.5 1.41487 1.2204 535.3 438.6 31,81
47.6 1.41508 1.2210 536.7 439.5 31,89
47.7 1.41528 1.2215 538.1 440.5 31,97
47.8 1.41549 1.2221 539.5 441.4 32,05
47.9 1.41569 1.2227 540.9 442.4 32,14
48.0 1.41590 1.2232 542.3 443.3 32,22
48.1 1.41611 1.2238 543.6 444.2 32,30
48.2 1.41632 1.2243 545.0 445.1 32,38
48.3 1.41652 1.2249 546.3 446.0 32,46
48.4 1.41673 1.2254 547.7 446.9 32,59
48.5 1.41694 1.2260 549.1 447.8 32,63
48.6 1.41715 1.2265 550.4 448.7 32,70
48.7 1.41736 1.2271 551.8 449.7 32,79
48.8 1.41756 1.2276 553.1 450.6 32,86
48.9 1.41777 1.2282 554.5 451.4 32,95
49.0 1.41798 1.2287 555.8 452.3 33,02
49.1 1.41819 1.2293 557.2 453.3 33,11
49.2 1.41840 1.2298 558.6 454.2 33,19
49.3 1.41861 1.2304 560.0 455.1 33,27
49.4 1.41882 1.2310 561.4 456.1 33,36
49.5 1.41903 1.2315 562.8 457.0 33,44
49.6 1.41924 1.2321 564.2 457.9 33,52
49.7 1.41945 1.2327 565.6 458.8 33,61
49.8 1.41966 1.2332 567.0 459.8 33,69
49.9 1.41987 1.2338 568.4 460.7 33,771990R2676 — EN — 18.03.2003 — 007.001 — 26
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
50.0 1.42008 1.2344 569.8 461.6 33,86
50.1 1.42029 1.2349 571.2 462.5 33,94
50.2 1.42050 1.2355 572.6 463.5 34,02
50.3 1.42071 1.2361 574.0 464.4 34,10
50.4 1.42092 1.2366 575.4 465.3 34,19
50.5 1.42114 1.2372 576.9 466.2 34,28
50.6 1.42135 1.2378 578.3 467.2 34,36
50.7 1.42156 1.2384 579.7 468.1 34,44
50.8 1.42177 1.2389 581.1 469.0 34,53
50.9 1.42198 1.2395 582.5 469.9 34,61
51.0 1.42219 1.2401 583.9 470.9 34,69
51.1 1.42240 1.2407 585.4 471.8 34,78
51.2 1.42261 1.2413 586.9 472.8 34,87
51.3 1.42283 1.2419 588.3 473.8 34,95
51.4 1.42304 1.2425 589.8 474.7 35,04
51.5 1.42325 1.2431 591.3 475.7 35,13
51.6 1.42346 1.2437 592.8 476.6 35,22
51.7 1.42367 1.2443 594.3 477.6 35,31
51.8 1.42389 1.2449 595.7 478.6 35,39
51.9 1.42410 1.2455 597.2 479.5 35,48
52.0 1.42431 1.2461 598.7 480.5 35,57
52.1 1.42452 1.2466 600.1 481.4 35,65
52.2 1.42474 1.2472 601.5 482.3 35,74
52.3 1.42495 1.2478 602.9 483.2 35,82
52.4 1.42517 1.2483 604.3 484.1 35,91
52.5 1.42538 1.2489 605.8 485.0 35,99
52.6 1.42559 1.2495 607.2 485.9 36,08
52.7 1.42581 1.2500 608.6 486.8 36,16
52.8 1.42602 1.2506 610.0 487.7 36,24
52.9 1.42624 1.2512 611.4 488.6 36,33
53.0 1.42645 1.2518 612.8 489.6 36,41
53.1 1.42666 1.2524 614.3 490.5 36,50
53.2 1.42686 1.2530 615.8 491.4 36,59
53.3 1.42707 1.2536 617.2 492.4 36,67
53.4 1.42727 1.2542 618.7 493.3 36,76
53.5 1.42748 1.2548 620.2 494.3 36,85
53.6 1.42769 1.2554 621.7 495.2 36,94
53.7 1.42789 1.2560 623.2 496.2 37,03
53.8 1.42810 1.2566 624.6 497.1 37,11
53.9 1.42830 1.2571 626.1 498.0 37,20
54.0 1.42851 1.2577 627.6 499.0 37,29
54.1 1.42874 1.2583 629.0 499.9 37,37
54.2 1.42897 1.2589 630.4 500.8 37,45
54.3 1.42919 1.2595 631.8 501.7 37,54
54.4 1.42942 1.2600 633.2 502.6 37,62
54.5 1.42965 1.2606 634.7 503.5 37,71
54.6 1.42988 1.2612 636.1 504.3 37,79
54.7 1.43011 1.2617 637.5 505.2 37,88
54.8 1.43033 1.2623 638.9 506.1 37,96
54.9 1.43056 1.2629 640.3 507.0 38,041990R2676 — EN — 18.03.2003 — 007.001 — 27
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
55.0 1.43079 1.2635 641.7 507.9 38,11
55.1 1.43101 1.2640 643.2 508.8 38,22
55.2 1.43123 1.2646 644.6 509.7 38,30
55.3 1.43145 1.2652 646.1 510.7 38,39
55.4 1.43167 1.2658 647.6 511.6 38,48
55.5 1.43189 1.2664 649.1 512.5 38,57
55.6 1.43210 1.2670 650.5 513.4 38,65
55.7 1.43232 1.2676 652.0 514.3 38,74
55.8 1.43254 1.2682 653.5 515.3 38,83
55.9 1.43276 1.2688 654.9 516.2 38,91
56.0 1.43298 1.2694 656.4 517.1 39,00
56.1 1.43320 1.2700 657.9 518.0 39,09
56.2 1.43342 1.2706 659.4 518.9 39,18
56.3 1.43364 1.2712 660.8 519.9 39,26
56.4 1.43386 1.2718 662.3 520.8 39,35
56.5 1.43409 1.2724 663.8 521.7 39,44
56.6 1.43431 1.2730 665.3 522.6 39,53
56.7 1.43453 1.2736 666.8 523.5 39,62
56.8 1.43475 1.2742 668.2 524.4 39,70
56.9 1.43497 1.2748 669.7 525.4 39,79
57.0 1.43519 1.2754 671.2 526.3 39,88
57.1 1.43541 1.2760 672.7 527.2 39,97
57.2 1.43563 1.2766 674.3 528.2 40,06
57.3 1.43586 1.2773 675.8 529.1 40,15
57.4 1.43608 1.2779 677.4 530.1 40,25
57.5 1.43630 1.2785 678.9 531.0 40,34
57.6 1.43652 1.2791 680.4 532.0 40,43
57.7 1.43674 1.2797 682.0 532.9 40,52
57.8 1.43697 1.2804 683.5 533.8 40,61
57.9 1.43719 1.2810 685.1 534.8 40,70
58.0 1.43741 1.2816 686.6 535.7 40,80
58.1 1.43763 1.2822 688.1 536.6 40,88
58.2 1.43786 1.2828 689.6 537.5 40,97
58.3 1.43808 1.2834 691.0 538.4 41,06
58.4 1.43831 1.2840 692.5 539.3 41,14
58.5 1.43854 1.2846 694.0 540.2 41,23
58.6 1.43876 1.2852 695.5 541.1 41,32
58.7 1.43899 1.2858 697.0 542.0 41,41
58.8 1.43921 1.2864 698.4 542.9 41,50
58.9 1.43944 1.2870 699.9 543.8 41,58
59.0 1.43966 1.2876 701.4 544.7 41,67
59.1 1.43989 1.2882 702.9 545.7 41,76
59.2 1.44011 1.2888 704.5 546.6 41,86
59.3 1.44034 1.2895 706.0 547.5 41,95
59.4 1.44056 1.2901 707.6 548.5 42,04
59.5 1.44079 1.2907 709.1 549.4 42,13
59.6 1.44102 1.2913 710.6 550.3 42,22
59.7 1.44124 1.2920 712.2 551.2 42,32
59.8 1.44147 1.2926 713.7 552.2 42,41
59.9 1.44169 1.2932 715.3 553.1 42,501990R2676 — EN — 18.03.2003 — 007.001 — 28
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
60.0 1.44192 1.2938 716.8 554.0 42,59
60.1 1.44215 1.2944 718.3 554.9 42,68
60.2 1.44237 1.2950 719.8 555.8 42,77
60.3 1.44260 1.2956 721.2 556.7 42,85
60.4 1.44283 1.2962 722.7 557.6 42,94
60.5 1.44306 1.2968 724.2 558.4 43,03
60.6 1.44328 1.2974 725.7 559.3 43,12
60.7 1.44351 1.2980 727.2 560.2 43,21
60.8 1.44374 1.2986 728.6 561.1 43,29
60.9 1.44396 1.2992 730.1 562.0 43,38
61.0 1.44419 1.2998 731.6 562.8 43,47
61.1 1.44442 1.3004 733.1 563.8 43,56
61.2 1.44465 1.3011 734.7 564.7 43,65
61.3 1.44488 1.3017 736.2 565.6 43,74
61.4 1.44511 1.3023 737.8 566.5 43,84
61.5 1.44533 1.3030 739.4 567.4 43,93
61.6 1.44556 1.3036 740.9 568.4 44,02
61.7 1.44579 1.3042 742.5 569.3 44,12
61.8 1.44602 1.3048 744.0 570.2 44,21
61.9 1.44625 1.3055 745.6 571.1 44,30
62.0 1.44648 1.3061 747.1 572.0 44,39
62.1 1.44671 1.3067 748.6 572.9 44,48
62.2 1.44694 1.3073 750.2 573.8 44,57
62.3 1.44717 1.3080 751.7 574.7 44,66
62.4 1.44740 1.3086 753.3 575.6 44,76
62.5 1.44764 1.3092 754.8 576.5 44,85
62.6 1.44787 1.3098 756.3 577.4 44,94
62.7 1.44810 1.3104 757.9 578.3 45,03
62.8 1.44833 1.3111 759.4 579.2 45,12
62.9 1.44856 1.3117 761.0 580.1 45,21
63.0 1.44879 1.3123 762.5 581.0 45,31
63.1 1.44902 1.3130 764.1 582.0 45,40
63.2 1.44926 1.3136 765.7 582.9 45,49
63.3 1.44949 1.3143 767.3 583.8 45,59
63.4 1.44972 1.3149 768.9 584.8 45,69
63.5 1.44996 1.3156 770.6 585.7 45,79
63.6 1.45019 1.3162 772.2 586.6 45,88
63.7 1.45042 1.3169 773.8 587.6 45,98
63.8 1.45065 1.3175 775.4 588.5 46,07
63.9 1.45089 1.3182 777.0 589.4 46,17
64.0 1.45112 1.3188 778.6 590.4 46,26
64.1 1.45135 1.3195 780.1 591.3 46,35
64.2 1.45159 1.3201 781.7 592.1 46,45
64.3 1.45183 1.3207 783.2 593.0 46,53
64.4 1.45206 1.3213 784.8 593.9 46,63
64.5 1.45230 1.3219 786.3 594.8 46,72
64.6 1.45253 1.3226 787.8 595.7 46,81
64.7 1.45276 1.3232 789.4 596.6 46,90
64.8 1.45300 1.3238 790.9 597.5 46,99
64.9 1.45324 1.3244 792.5 598.3 47,091990R2676 — EN — 18.03.2003 — 007.001 — 29
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
65.0 1.45347 1.3251 794.0 599.2 47,18
65.1 1.45371 1.3257 795.6 600.1 47,27
65.2 1.45394 1.3264 797.2 601.1 47,37
65.3 1.45418 1.3270 798.8 602.0 47,46
65.4 1.45441 1.3277 800.4 602.9 47,56
65.5 1.45465 1.3283 802.1 603.8 47,66
65.6 1.45489 1.3290 803.7 604.7 47,75
65.7 1.45512 1.3296 805.3 605.6 47,85
65.8 1.45536 1.3303 806.9 606.6 47,94
65.9 1.45559 1.3309 808.5 607.5 48,04
66.0 1.45583 1.3316 810.1 608.4 48,13
66.1 1.45607 1.3322 811.6 609.3 48,22
66.2 1.45630 1.3328 813.2 610.1 48,32
66.3 1.45654 1.3335 814.8 611.0 48,41
66.4 1.45678 1.3341 816.3 611.9 48,50
66.5 1.45702 1.3347 817.9 612.8 48,60
66.6 1.45725 1.3353 819.4 613.6 48,69
66.7 1.45749 1.3360 820.9 614.5 48,77
66.8 1.45773 1.3366 822.5 615.4 48,87
66.9 1.45796 1.3372 824.1 616.2 48,97
67.0 1.45820 1.3378 825.6 617.1 49,05
67.1 1.45844 1.3385 827.2 618.0 49,15
67.2 1.45868 1.3391 828.8 618.9 49,24
67.3 1.45892 1.3398 830.4 619.8 49,34
67.4 1.45916 1.3404 832.0 620.7 49,43
67.5 1.45940 1.3411 833.7 621.6 49,53
67.6 1.45964 1.3418 835.3 622.5 49,63
67.7 1.45988 1.3424 836.9 623.4 49,73
67.8 1.46012 1.3431 838.5 624.3 49,82
67.9 1.46036 1.3437 840.1 625.2 49,92
68.0 1.46060 1.3444 841.7 626.1 50,01
68.1 1.46084 1.3450 843.4 627.0 50,11
68.2 1.46108 1.3457 845.1 628.0 50,21
68.3 1.46132 1.3464 846.7 628.9 50,31
68.4 1.46156 1.3471 848.4 629.8 50,41
68.5 1.46181 1.3478 850.1 630.8 50,51
68.6 1.46205 1.3484 851.8 631.7 50,61
68.7 1.46229 1.3491 853.5 632.6 50,71
68.8 1.46253 1.3498 855.1 633.5 50,81
68.9 1.46277 1.3505 856.8 634.5 50,91
69.0 1.46301 1.3512 858.5 635.4 51,01
69.1 1.46325 1.3518 860.1 636.3 51,10
69.2 1.46350 1.3525 861.7 637.2 51,20
69.3 1.46374 1.3531 863.3 638.0 51,29
69.4 1.46398 1.3538 864.9 638.9 51,39
69.5 1.46423 1.3544 866.6 639.8 51,49
69.6 1.46447 1.3551 868.2 640.7 51,58
69.7 1.46471 1.3557 869.8 641.6 51,68
69.8 1.46495 1.3564 871.4 642.4 51,78
69.9 1.46520 1.3570 873.0 643.3 51,871990R2676 — EN — 18.03.2003 — 007.001 — 30
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
70.0 1.46544 1.3577 874.6 644.2 51,97
70.1 1.46568 1.3583 876.2 645.1 52,06
70.2 1.46593 1.3590 877.8 645.9 52,15
70.3 1.46618 1.3596 879.4 646.8 52,25
70.4 1.46642 1.3603 881.0 647.7 52,35
70.5 1.46667 1.3609 882.7 648.6 52,45
70.6 1.46691 1.3616 884.3 649.4 52,54
70.7 1.46715 1.2622 885.9 650.3 52,64
70.8 1.46740 1.3629 887.5 651.2 52,73
70.9 1.46765 1.3635 889.1 652.1 52,83
71.0 1.46789 1.3642 890.7 652.9 52,92
71.1 1.46814 1.3649 892.4 653.8 53,02
71.2 1.46838 1.3655 894.1 654.7 53,12
71.3 1.46863 1.3662 895.7 655.6 53,22
71.4 1.46888 1.3669 897.4 656.5 53,32
71.5 1.46913 1.3676 899.1 657.4 53,42
71.6 1.46937 1.3683 900.8 658.3 53,52
71.7 1.46962 1.3689 902.5 659.2 53,62
71.8 1.46987 1.3696 904.1 660.1 53,72
71.9 1.47011 1.3703 905.8 661.0 53,82
72.0 1.47036 1.3710 907.5 661.9 53,92
72.1 1.47061 1.3717 909.2 662.8 54,02
72.2 1.47086 1.3723 910.8 663.7 54,12
72.3 1.47110 1.3730 912.5 664.6 54,22
72.4 1.47135 1.3737 914.2 665.5 54,32
72.5 1.47160 1.3744 915.9 666.4 54,42
72.6 1.47185 1.3750 917.5 667.3 54,51
72.7 1.47210 1.3757 919.2 668.2 54,62
72.8 1.47234 1.3764 920.9 669.0 54,72
72.9 1.47259 1.3771 922.5 669.9 54,81
73.0 1.47284 1.3777 924.2 670.8 54,91
73.1 1.47309 1.3784 925.9 671.7 55,01
73.2 1.47334 1.3791 927.6 672.6 55,11
73.3 1.47359 1.3798 929.2 673.5 55,21
73.4 1.47384 1.3804 930.9 674.4 55,31
73.5 1.47409 1.3811 932.6 675.2 55,41
73.6 1.47434 1.3818 934.3 676.1 55,51
73.7 1.47459 1.3825 936.0 677.0 55,61
73.8 1.47484 1.3832 937.6 677.9 55,71
73.9 1.47509 1.3838 939.3 678.8 55,81
74.0 1.47534 1.3845 941.0 679.7 55,91
74.1 1.47559 1.3852 942.7 680.5 56,01
74.2 1.47584 1.3859 944.4 681.4 56,11
74.3 1.47609 1.3866 946.0 682.3 56,21
74.4 1.47634 1.3872 947.7 683.2 56,31
74.5 1.47660 1.3879 949.4 684.0 56,41
74.6 1.47685 1.3886 951.1 684.9 56,51
74.7 1.47710 1.3893 952.8 685.8 56,61
74.8 1.47735 1.3900 954.4 686.7 56,71
74.9 1.47760 1.3906 956.1 687.5 56,811990R2676 — EN — 18.03.2003 — 007.001 — 31
▼B
TABLE III
Table giving the sugar (1)concentration in rectified concentrated must in grams per litre and
grams per kilogram, determined by means of a refractometer graduated either in percentage
bymassofsucroseat20ºCorinrefractive indexat20ºC.Thedensityat20ºCisalsogiven
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
50.0 1.42008 1.2342 627.6 508.5 37,28
50.1 1.42029 1.2348 629.3 509.6 37,38
50.2 1.42050 1.2355 630.9 510.6 37,48
50.3 1.42071 1.2362 632.4 511.6 37,56
50.4 1.42092 1.2367 634.1 512.7 37,66
50.5 1.42113 1.2374 635.7 513.7 37,76
50.6 1.42135 1.2381 637.3 514.7 37,85
50.7 1.42156 1.2386 638.7 515.7 37,94
50.8 1.42177 1.2391 640.4 516.8 38,04
50.9 1.42198 1.2396 641.9 517.8 38,13
51.0 1.42219 1.2401 643.4 518.8 38,22
51.1 1.42240 1.2406 645.0 519.9 38,31
51.2 1.42261 1.2411 646.5 520.9 38,40
51.3 1.42282 1.2416 648.1 522.0 38,50
51.4 1.42304 1.2421 649.6 523.0 38,59
51.5 1.42325 1.2427 651.2 524.0 38,68
51.6 1.42347 1.2434 652.9 525.1 38,78
51.7 1.42368 1.2441 654.5 526.1 38,88
51.8 1.42389 1.2447 656.1 527.1 38,97
51.9 1.42410 1.2454 657.8 528.2 39,07
52.0 1.42432 1.2461 659.4 529.2 39,17
52.1 1.42453 1.2466 661.0 530.2 39,26
52.2 1.42475 1.2470 662.5 531.3 39,35
52.3 1.42496 1.2475 664.1 532.3 39,45
52.4 1.42517 1.2480 665.6 533.3 39,54
52.5 1.42538 1.2486 667.2 534.4 39,63
52.6 1.42560 1.2493 668.9 535.4 39,73
52.7 1.42581 1.2500 670.5 536.4 39,83
52.8 1.42603 1.2506 672.2 537.5 39,93
52.9 1.42624 1.2513 673.8 538.5 40,02
53.0 1.42645 1.2520 675.5 539.5 40,12
53.1 1.42667 1.2525 677.1 540.6 40,22
53.2 1.42689 1.2530 678.5 541.5 40,30
53.3 1.42711 1.2535 680.2 542.6 40,40
53.4 1.42733 1.2540 681.8 543.7 40,50
53.5 1.42754 1.2546 683.4 544.7 40,59
53.6 1.42776 1.2553 685.1 545.8 40,69
53.7 1.42797 1.2560 686.7 546.7 40,79
53.8 1.42819 1.2566 688.4 547.8 40,89
53.9 1.42840 1.2573 690.1 548.9 40,99
54.0 1.42861 1.2580 691.7 549.8 41,09
54.1 1.42884 1.2585 693.3 550.9 41,18
54.2 1.42906 1.2590 694.9 551.9 41,28
54.3 1.42927 1.2595 696.5 553.0 41,37
54.4 1.42949 1.2600 698.1 554.0 41,47
54.5 1.42971 1.2606 699.7 555.1 41,56
54.6 1.42993 1.2613 701.4 556.1 41,66
54.7 1.43014 1.2620 703.1 557.1 41,76
54.8 1.43036 1.2625 704.7 558.2 41,86
54.9 1.43058 1.2630 706.2 559.1 41,951990R2676 — EN — 18.03.2003 — 007.001 — 32
(1) The sugar concentration is expressed in terms of invert sugar.
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
55.0 1.43079 1.2635 707.8 560.2 42,04
55.1 1.43102 1.2639 709.4 561.3 42,14
55.2 1.43124 1.2645 711.0 562.3 42,23
55.3 1.43146 1.2652 712.7 563.3 42,33
55.4 1.43168 1.2659 714.4 564.3 42,44
55.5 1.43189 1.2665 716.1 565.4 42,54
55.6 1.43211 1.2672 717.8 566.4 42,64
55.7 1.43233 1.2679 719.5 567.5 42,74
55.8 1.43255 1.2685 721.1 568.5 42,83
55.9 1.43277 1.2692 722.8 569.5 42,93
56.0 1.43298 1.2699 724.5 570.5 43,04
56.1 1.43321 1.2703 726.1 571.6 43,13
56.2 1.43343 1.2708 727.7 572.6 43,23
56.3 1.43365 1.2713 729.3 573.7 43,32
56.4 1.43387 1.2718 730.9 574.7 43,42
56.5 1.43409 1.2724 732.6 575.8 43,52
56.6 1.43431 1.2731 734.3 576.8 43,62
56.7 1.43454 1.2738 736.0 577.8 43,72
56.8 1.43476 1.2744 737.6 578.8 43,81
56.9 1.43498 1.2751 739.4 579.9 43,92
57.0 1.43519 1.2758 741.1 580.9 44,02
57.1 1.43542 1.2763 742.8 582.0 44,12
57.2 1.43564 1.2768 744.4 583.0 44,22
57.3 1.43586 1.2773 745.9 584.0 44,31
57.4 1.43609 1.2778 747.6 585.1 44,41
57.5 1.43631 1.2784 749.3 586.1 44,51
57.6 1.43653 1.2791 751.0 587.1 44,61
57.7 1.43675 1.2798 752.7 588.1 44,71
57.8 1.43698 1.2804 754.4 589.2 44,81
57.9 1.43720 1.2810 756.1 590.2 44,91
58.0 1.43741 1.2818 757.8 591.2 45,01
58.1 1.43764 1.2822 759.5 592.3 45,11
58.2 1.43784 1.2827 761.1 593.4 45,21
58.3 1.43809 1.2832 762.6 594.3 45,30
58.4 1.43832 1.2837 764.3 595.4 45,40
58.5 1.43854 1.2843 766.0 596.4 45,50
58.6 1.43877 1.2850 767.8 597.5 45,61
58.7 1.43899 1.2857 769.5 598.5 45,71
58.8 1.43922 1.2863 771.1 599.5 45,80
58.9 1.43944 1.2869 772.9 600.6 45,91
59.0 1.43966 1.2876 774.6 601.6 46,01
59.1 1.43988 1.2882 776.3 602.6 46,11
59.2 1.44011 1.2889 778.1 603.7 46,22
59.3 1.44034 1.2896 779.8 604.7 46,32
59.4 1.44057 1.2902 781.6 605.8 46,43
59.5 1.44079 1.2909 783.3 606.8 46,53
59.6 1.44102 1.2916 785.2 607.9 46,64
59.7 1.44124 1.2921 786.8 608.9 46,74
59.8 1.44147 1.2926 788.4 609.9 46,83
59.9 1.44169 1.2931 790.0 610.9 46,931990R2676 — EN — 18.03.2003 — 007.001 — 33
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
60.0 1.44192 1.2936 791.7 612.0 47,03
60.1 1.44215 1.2942 793.3 613.0 47,12
60.2 1.44238 1.2949 795.2 614.1 47,23
60.3 1.44260 1.2956 796.9 615.1 47,34
60.4 1.44283 1.2962 798.6 616.1 47,44
60.5 1.44305 1.2969 800.5 617.2 47,55
60.6 1.44328 1.2976 802.2 618.2 47,65
60.7 1.44351 1.2981 803.9 619.3 47,75
60.8 1.44374 1.2986 805.5 620.3 47,85
60.9 1.44397 1.2991 807.1 621.3 47,94
61.0 1.44419 1.2996 808.7 622.3 48,04
61.1 1.44442 1.3002 810.5 623.4 48,14
61.2 1.44465 1.3009 812.3 624.4 48,25
61.3 1.44488 1.3016 814.2 625.5 48,36
61.4 1.44511 1.3022 815.8 626.5 48,46
61.5 1.44534 1.3029 817.7 627.6 48,57
61.6 1.44557 1.3036 819.4 628.6 48,67
61.7 1.44580 1.3042 821.3 629.7 48,79
61.8 1.44603 1.3049 823.0 630.7 48,89
61.9 1.44626 1.3056 824.8 631.7 48,99
62.0 1.44648 1.3062 826.6 632.8 49,10
62.1 1.44672 1.3068 828.3 633.8 49,20
62.2 1.44695 1.3075 830.0 634.8 49,30
62.3 1.44718 1.3080 831.8 635.9 49,40
62.4 1.44741 1.3085 833.4 636.9 49,50
62.5 1.44764 1.3090 835.1 638.0 49,60
62.6 1.44787 1.3095 836.8 639.0 49,71
62.7 1.44810 1.3101 838.5 640.0 49,81
62.8 1.44833 1.3108 840.2 641.0 49,91
62.9 1.44856 1.3115 842.1 642.1 50,02
63.0 1.44879 1.3121 843.8 643.1 50,12
63.1 1.44902 1.3128 845.7 644.2 50,23
63.2 1.44926 1.3135 847.5 645.2 50,34
63.3 1.44949 1.3141 849.3 646.3 50,45
63.4 1.44972 1.3148 851.1 647.3 50,56
63.5 1.44995 1.3155 853.0 648.4 50,67
63.6 1.45019 1.3161 854.7 649.4 50,77
63.7 1.45042 1.3168 856.5 650.4 50,88
63.8 1.45065 1.3175 858.4 651.5 50,99
63.9 1.45088 1.3180 860.0 652.5 51,08
64.0 1.45112 1.3185 861.6 653.5 51,18
64.1 1.45135 1.3190 863.4 654.6 51,29
64.2 1.45158 1.3195 865.1 655.6 51,39
64.3 1.45181 1.3201 866.9 656.7 51,49
64.4 1.45205 1.3208 868.7 657.7 51,60
64.5 1.45228 1.3215 870.6 658.8 51,71
64.6 1.45252 1.3221 872.3 659.8 51,81
64.7 1.45275 1.3228 874.1 660.8 51,92
64.8 1.45299 1.3235 876.0 661.9 52,03
64.9 1.45322 1.3241 877.8 662.9 52,141990R2676 — EN — 18.03.2003 — 007.001 — 34
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
65.0 1.45347 1.3248 879.7 664.0 52,25
65.1 1.45369 1.3255 881.5 665.0 52,36
65.2 1.45393 1.3261 883.2 666.0 52,46
65.3 1.45416 1.3268 885.0 667.0 52,57
65.4 1.45440 1.3275 886.9 668.1 52,68
65.5 1.45463 1.3281 888.8 669.2 52,79
65.6 1.45487 1.3288 890.6 670.2 52,90
65.7 1.45510 1.3295 892.4 671.2 53,01
65.8 1.45534 1.3301 894.2 672.3 53,12
65.9 1.45557 1.3308 896.0 673.3 53,22
66.0 1.45583 1.3315 898.0 674.4 53,34
66.1 1.45605 1.3320 899.6 675.4 53,44
66.2 1.45629 1.3325 901.3 676.4 53,54
66.3 1.45652 1.3330 903.1 677.5 53,64
66.4 1.45676 1.3335 904.8 678.5 53,75
66.5 1.45700 1.3341 906.7 679.6 53,86
66.6 1.45724 1.3348 908.5 680.6 53,96
66.7 1.45747 1.3355 910.4 681.7 54,08
66.8 1.45771 1.3361 912.2 682.7 54,18
66.9 1.45795 1.3367 913.9 683.7 54,29
67.0 1.45820 1.3374 915.9 684.8 54,40
67.1 1.45843 1.3380 917.6 685.8 54,51
67.2 1.45867 1.3387 919.6 686.9 54,62
67.3 1.45890 1.3395 921.4 687.9 54,73
67.4 1.45914 1.3400 923.1 688.9 54,83
67.5 1.45938 1.3407 925.1 690.0 54,95
67.6 1.45962 1.3415 927.0 691.0 55,06
67.7 1.45986 1.3420 928.8 692.1 55,17
67.8 1.46010 1.3427 930.6 693.1 55,28
67.9 1.46034 1.3434 932.6 694.2 55,40
68.0 1.46060 1.3440 934.4 695.2 55,50
68.1 1.46082 1.3447 936.2 696.2 55,61
68.2 1.46106 1.3454 938.0 697.2 55,72
68.3 1.46130 1.3460 939.9 698.3 55,83
68.4 1.46154 1.3466 941.8 699.4 55,94
68.5 1.46178 1.3473 943.7 700.4 56,06
68.6 1.46202 1.3479 945.4 701.4 56,16
68.7 1.46226 1.3486 947.4 702.5 56,28
68.8 1.46251 1.3493 949.2 703.5 56,38
68.9 1.46275 1.3499 951.1 704.6 56,50
69.0 1.46301 1.3506 953.0 705.6 56,61
69.1 1.46323 1.3513 954.8 706.6 56,72
69.2 1.46347 1.3519 956.7 707.7 56,83
69.3 1.46371 1.3526 958.6 708.7 56,94
69.4 1.46396 1.3533 960.6 709.8 57,06
69.5 1.46420 1.3539 962.4 710.8 57,17
69.6 1.46444 1.3546 964.3 711.9 57,28
69.7 1.46468 1.3553 966.2 712.9 57,39
69.8 1.46493 1.3560 968.2 714.0 57,51
69.9 1.46517 1.3566 970.0 715.0 57,621990R2676 — EN — 18.03.2003 — 007.001 — 35
▼B
Sucrose
% (m/m)Refractive index
at 20 ºCDensity at 20 ºCSugar
ing/lSugar
ing/kgAlcoholic
strength
% vol at 20 ºC
70.0 1.46544 1.3573 971.8 716.0 57,72
70.1 1.46565 1.3579 973.8 717.1 57,84
70.2 1.46590 1.3586 975.6 718.1 57,95
70.3 1.46614 1.3593 977.6 719.2 58,07
70.4 1.46639 1.3599 979.4 720.2 58,18
70.5 1.46663 1.3606 981.3 721.2 58,29
70.6 1.46688 1.3613 983.3 722.3 58,41
70.7 1.46712 1.3619 985.2 723.4 58,52
70.8 1.46737 1.3626 987.1 724.4 58,63
70.9 1.46761 1.3633 988.9 725.4 58,74
71.0 1.46789 1.3639 990.9 726.5 58,86
71.1 1.46810 1.3646 992.8 727.5 58,97
71.2 1.46835 1.3653 994.8 728.6 59,09
71.3 1.46859 1.3659 996.6 729.6 59,20
71.4 1.46884 1.3665 998.5 730.7 59,31
71.5 1.46908 1.3672 1 000.4 731.7 59,42
71.6 1.46933 1.3678 1 002.2 732.7 59,53
71.7 1.46957 1.3685 1 004.2 733.8 59,65
71.8 1.46982 1.3692 1 006.1 734.8 59,76
71.9 1.47007 1.3698 1 008.0 735.9 59,88
72.0 1.47036 1.3705 1 009.9 736.9 59,99
72.1 1.47056 1.3712 1 012.0 738.0 60,11
72.2 1.47081 1.3718 1 013.8 739.0 60,22
72.3 1.47106 1.3725 1 015.7 740.0 60,33
72.4 1.47131 1.3732 1 017.7 741.1 60,45
72.5 1.47155 1.3738 1 019.5 742.1 60,56
72.6 1.47180 1.3745 1 021.5 743.2 60,68
72.7 1.47205 1.3752 1 023.4 744.2 60,79
72.8 1.47230 1.3758 1 025.4 745.3 60,91
72.9 1.47254 1.3765 1 027.3 746.3 61,02
73.0 1.47284 1.3772 1 029.3 747.4 61,14
73.1 1.47304 1.3778 1 031.2 748.4 61,25
73.2 1.47329 1.3785 1 033.2 749.5 61,37
73.3 1.47354 1.3792 1 035.1 750.5 61,48
73.4 1.47379 1.3798 1 037.1 751.6 61,60
73.5 1.47404 1.3805 1 039.0 752.6 61,72
73.6 1.47429 1.3812 1 040.9 753.6 61,83
73.7 1.47454 1.3818 1 042.8 754.7 61,94
73.8 1.47479 1.3825 1 044.8 755.7 62,06
73.9 1.47504 1.3832 1 046.8 756.8 62,18
74.0 1.47534 1.3838 1 048.6 757.8 62,28
74.1 1.47554 1.3845 1 050.7 758.9 62,41
74.2 1.47579 1.3852 1 052.6 759.9 62,52
74.3 1.47604 1.3858 1 054.6 761.0 62,64
74.4 1.47629 1.3865 1 056.5 762.0 62,76
74.5 1.47654 1.3871 1 058.5 763.1 62,87
74.6 1.47679 1.3878 1 060.4 764.1 62,99
74.7 1.47704 1.3885 1 062.3 765.1 63,10
74.8 1.47730 1.3892 1 064.4 766.2 63,23
74.9 1.47755 1.3898 1 066.3 767.2 63,33
75.0 1.47785 1.3905 1 068.3 768.3 63,461990R2676 — EN — 18.03.2003 — 007.001 — 36
▼B
3.ALCOHOLICSTRENGTHBYVOLUME
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 ºC. It is expressed by the symbol ‘% vol’.
Note:Homologues of ethanol, together with the ethanol and ethanol homo-
logues in ethyl esters, are included in the alcoholic strength since
they occur inthe distillate.
2. PRINCIPLE OF METHODS
2.1. Distillation of wine made alkaline by a suspension of calcium hydro-
xide. Measurement of the alcoholic strength of the distillate.
2.2.Referencemethod: measurement of the density of the distillate with
a pycnometer.
2.3.Usualmethods:
2.3.1. Measurement of the alcoholic strength of the distillate with a hydro-
meter.
2.3.2. Measurement of the alcoholic strength of the distillate by densimetry
using a hydrostatic balance.
2.3.3. Measurement of the alcoholic strength of the distillate by refracto-
metry.
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 beencalculated from the International Tables of Alco-
holic 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 DISTILLATE3.1.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 anethan ol-water mixture with analcoholic stren gth of 10 %
vol five times insuccession . The distillate should have analcoholic
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.1990R2676 — EN — 18.03.2003 — 007.001 — 37
▼B
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 ºC on to 120 g of quicklime
(CaO).
3.3.Preparationofsample
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-distillationapparatus. Rin se 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 inthe 200-ml graduated flask used to measure
the wine.
Collect a volume of about three-quarters of the initial volume if
distillationis used an d a volume of 198 to 199 ml of distillate if
steam distillationis used. Make up to 200 ml with distilled water,
keeping the distillate at a temperature within 2 ºC 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 condi-
tions described above, but replacing the suspension of calcium
hydroxide with 1 M sulphuric acid diluted to 10 parts in100 (vol/
vol).
4. REFERENCE METHOD
Measurement of the alcoholic strength of the distillate using a pycn-
ometer.
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 tºC 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.Expressionofresults
4.3.1. Method of calculation
Find the alcoholic strength at 20 ºC using Table I. In the horizontal
line of this table corresponding to the temperature T(expressed as a
whole number) immediately below tºC,find the smallest density
greater than ρt. Use the tabular difference just below this density to
calculate the density ρat this temperature T.
Onthe lin e of the temperature T, find the density ρimmediately
above ρ′and calculate the difference between the densities ρand
ρ′. Divide this difference by the tabular difference just to the right
of the density ρ′. The quotient gives the decimal part of the alco-
holic strength, while the whole number part of this strength is
shownat the head of the columninwhich the den sity ρ′is located.
Anexample of the calculationof analcoholic stren gth is givenin
Appendix I to this chapter of the Annex.1990R2676 — EN — 18.03.2003 — 007.001 — 38
▼B
Note:
This temperature correctionhas beenin corporated ina 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.
5. USUAL METHODS
5.1.Hydrometry
5.1.1. Apparatus
5.1.1.1. Alcoholmeter.
The alcoholmeter must conform to the specifications for class I or
class II equipment defined in International Recommendation No 44,
Alcoholmeters and Alcohol Hydrometers , of the ILMO (International
Legal Metrology Organization).
5.1.1.2. Thermometer graduated indegrees an d in0,1 ºC from 0 to 40 ºC
certified to within1/20th degree.
5.1.1.3. Measuring cylinder of diameter 36 mm and height 320 mm held
vertically by supporting levelling screws.
5.1.2. Procedure
Pour the distillate (3.4) into the measuring cylinder. Ensure that the
cylinder is kept vertical. Insert the thermometer and alcoholmeter.
Read the temperature on the thermometer one minute after stirring
to equilibrate the temperature of the measuring cylinder, the thermo-
meter, the alcoholmeter and the distillate. Remove the thermometer
and read the apparent alcoholic strength after one minute. Take at
least three readings using a magnifying glass. Correct the apparent
strength measured at tºC for the effect of temperature using Table
II.
The temperature of the liquid must differ very little from ambient
temperature (at most, by 5 ºC).
5.2.Densimetryusingahydrostaticbalance
5.2.1. Apparatus
5.2.1.1. The hydrostatic balance is used as described in the chapter ‘Density
and specific gravity’.
5.2.2. Procedure
The apparent density of the distillate at tºC is measured as described
in the chapter ‘Density and specific gravity’, section 5.2.2.
5.2.3. Expression of results
Find the alcoholic strength at 20 ºC by following the method
described in 4.3.1, using Table I if the float is of Pyrex glass and
Table III if it is of ordinary glass.
5.3.Refractometry
5.3.1. Apparatus
5.3.1.1. Refractometer enabling refractive indices to be measured in the
range 1,330 to 1,346. Depending on the type of equipment, measure-
ments are made:
— either at 20 ºC with a suitable instrument,
— or at ambient temperature tºC by an instrument fitted with a
thermometer enabling the temperature to be determined to withinat least 0,05 ºC. A table giving temperature corrections will be
provided with the instrument.
5.3.2. Procedure
The refractive index of the wine distillate obtained as in 3.3 above is
measured by following the procedure prescribed for the type of
instrument used.1990R2676 — EN — 18.03.2003 — 007.001 — 39
▼B
5.3.3. Expression of results
Table IV is used to find the alcoholic strength corresponding to the
refractive index at 20 ºC.
Note:Table IV gives the alcoholic strengths corresponding to refractive
indices for both pure alcohol-water mixtures and for wine distillates.
In the case of wine distillates, it takes into account the presence of
impurities in the distillate (mainly higher alcohols). The presence of
methanol lowers the refractive index and thus the alcoholic strength.
6. EXAMPLE OF THE CALCULATION OF THE ALCOHOLIC
STRENGTH OF A WINE
6.1.Measurementbypycnometeronatwin-panbalance
6.1.1. The constants of the pycnometer have been determined and calcu-
lated as described in chapter 1, ‘Density and specific gravity’,
section6.1.1.
6.1.2. Weighing of pycnometer filled with distillate
Numeric example
Tare = pycnometer + distillate at tºC +p″/C144/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C146tºC = 18,90 ºC
tºC corrected = 18,70 ºC
p″ = 2,8074 g
p+m−p″= mass of distillate at tºC 105,0698 − 2,8074 = 102,2624 g
Apparent density at tºC
ρ
t¼pþm/C0p00
volume of pycnometer at 20 ºC/C144/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C146ρ
18;70 ºC¼102 ;2624
104 ;0229¼0;983076
6.1.3. Calculation of alcoholic strength
Refer to the table of apparent densities of
water-alcohol mixtures at different
temperatures, as indicated above/C144/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C146On the line 18 ºC of the table of apparent
densities, the smallest density greater
thanthe observed den sity of 0,983076 is
0,98398 incolumn11 % vol
The density at 18 ºC is:
(98307,6 + 0,7 × 22) 10
−5= 0,98323
0,98398 − 0,98323 = 0,00075
The decimal portionof the % vol of alco-
holic strength is 75/114 = 0,65
The alcoholic strength is 11,65 % vol
6.2.Measurementbypycnometeronasinglepanbalance
6.2.1. The constants of the pycnometer have been determined and calcu-
lated as described in chapter 1, ‘Density and specific gravity’,
section6.2.1.
6.2.2. Weighing of the pycnometer filled with distillate
Weight of tare bottle at the time of
measurement in grams: T1= 171,9178
Pycnometer filled with distillate at
20,50 ºC ingrams: P2= 167,8438
Variationinthe buoyan cy of air: d T= 171,9178 − 171,9160
= + 0,0018
Mass of the distillate at 20,50 ºC: Lt= 167,8438 − (67,6695 + 0,0018)
= 100,17251990R2676 — EN — 18.03.2003 — 007.001 — 40
▼B
Apparent density of the distillate: ρ20;50 ºC¼100 ;1725
100 ;8194¼0;983825
6.2.3. Calculation of alcoholic strength
Refer to the table of apparent densities of
water-alcohol mixtures at different
temperatures, as indicated above/C144/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C146On the line 20 ºC of the table of apparent
densities, the smallest density greater
thanobserved den sity of 0,983825 is
0,98471 incolumn10 % vol
The density at 20 ºC is:
(98382,5 + 0,5 × 24) 10
−5= 0,983945
0,98471 − 0,983945 = 0,000765
The decimal portionof the % vol of alco-
holic strength is 76,5/119 = 0,64
The alcoholic strength is 10,64 % vol1990R2676 — EN — 18.03.2003 — 007.001 — 41
▼B
FORMULA FROM WHICH TABLES OF ALCOHOLIC STRENGHTS OF
ETHANOL-WATER MIXTURES ARE CALCULATED
The density ρinkilograms per metre cubed (kg/m3) of anethan ol-water mixture
at temperature tindegrees Celsius is givenby the formula below as a fun ction
of:
— the alcoholic strength by weight pexpressed as a decimal (1),
— the temperature tinºC (EIPT 68),
— the numerical coefficients below.
The formula is valid for temperatures between− 20 an d +40 ºC.
Numerical coefficients in the formula
kAk
kg/m3Bk
1 9,982 012 300 · 102− 2,061 851 3 · 10−1kg/(m3· ºC)
2 − 1,929 769 495 · 102− 5,268 254 2 · 10−3kg/(m3·º C2)
3 3,891 238 958 · 1023,613 001 3 · 10−5kg/(m3·º C3)
4 − 1,668 103 923 · 103− 3,895 770 2 · 10−7kg/(m3·º C4)
5 1,352 215 441 · 1047,169 354 0 · 10−9kg/(m3·º C5)
6 − 8,829 278 388 · 104− 9,973 923 1 · 10−11kg/(m3·º C6)
7 3,062 874 042 · 105
8 − 6,138 381 234 · 105
9 7,470 172 998 · 105
10 − 5,478 461 354 · 105
11 2,234 460 334 · 105
12 − 3,903 285 426 · 104
kC1,k
kg/(m3· ºC)C2,k
kg/(m3·º C2)
1 1,693 443 461 530 087 · 10−1− 1,193 013 005 057 010 · 10−2
2 − 1,046 914 743 455 169 · 1012,517 399 633 803 461 · 10−1
3 7,196 353 469 546 523 · 101− 2,170 575 700 536 993
4 − 7,047 478 054 272 792 · 1021,353 034 988 843 029 · 101
5 3,924 090 430 035 045 · 103− 5,029 988 758 547 014 · 101
6 − 1,210 164 659 068 747 · 1041,096 355 666 577 570 · 102
7 2,248 646 550 400 788 · 104− 1,422 753 946 421 155 · 102
8 − 2,605 562 982 188 164 · 1041,080 435 942 856 230 · 102
9 1,852 373 922 069 467 · 104− 4,414 153 236 817 392 · 101
10 − 7,420 201 433 430 137 · 1037,442 971 530 188 783
11 1,285 617 841 998 974 · 103
kC3,k
kg/(m3·º C3)C4,k
kg/(m3·º C4)C5,k
kg/(m3·º C5)
1 − 6,802 995 733 503 803 · 10−44,075 376 675 622 027 · 10−6− 2,788 074 354 782 409 · 10−8
2 1,876 837 790 289 664 · 10−2− 8,763 058 573 471 110 · 10−61,345 612 883 493 354 · 10−8
3 − 2,002 561 813 734 156 · 10−16,515 031 360 099 368 · 10−6
4 1,022 992 966 719 220 − 1,515 784 836 987 210 · 10−6
5 − 2,895 696 483 903 638
6 4,810 060 584 300 675
7 − 4,672 147 440 794 683
8 2,458 043 105 903 461
9 − 5,411 227 621 436 812 · 10−11990R2676 — EN — 18.03.2003 — 007.001 — 42
(1) For example, for analcoholic stren gth of 12 % by weight, p= 0,12.
▼B
TABLE I
INTERNATIONAL ALCOHOLIC STRENGTH AT 20 ºC
Tableofapparentdensitiesofethanol-watermixtures—Pyrexpycnometer
Densities at tºC corrected for air buoyancy
tºAlcoholic strength by % vol
01234567891 0 1 1
0º 999,64 1,50 998,14 1,44 996,70 1,40 995,30 1,35 993,95 1,30 992,65 1,24 991,41 1,19 990,22 1,14 989,08 1,10 987,98 1,05 986,93 1,00 985,93 0,95
−0,07 −0,06 −0,06 −0,06 −0,06 −0,06 −0,06 −0,05 −0,04 −0,03 −0,02 −0,01
1º 999,71 1,51 998,20 1,44 996,76 1,40 995,36 1,35 994,01 1,30 992,71 1,24 991,47 1,20 990,27 1,15 989,12 1,11 988,01 1,06 986,95 1,01 985,94 0,97
−0,05 −0,05 −0,04 −0,04 −0,04 −0,04 −0,03 −0,03 −0,02 −0,02 −0,01 0,00
2º 999,76 1,51 998,25 1,45 996,80 1,40 995,40 1,35 994,05 1,30 992,75 1,25 991,50 1,20 990,30 1,16 989,14 1,11 988,03 1,07 986,96 1,02 985,94 0,98
−0,03 −0,03 −0,03 −0,02 −0,02 −0,02 −0,02 −0,01 −0,01 0,00 0,01 0,02
3º 999,79 1,51 998,28 1,45 996,83 1,41 995,42 1,35 994,07 1,30 992,77 1,25 991,52 1,21 990,31 1,16 989,15 1,12 988,03 1,08 986,95 1,03 985,92 1,00
−0,02 −0,02 −0,01 −0,02 −0,01 −0,01 0,00 0,00 0,01 0,02 0,03 0,04
4º 999,81 1,51 998,30 1,46 996,84 1,40 995,44 1,36 994,08 1,30 992,78 1,26 991,52 1,21 990,31 1,17 989,14 1,13 988,01 1,09 986,92 1,04 985,88 1,00
0,00 0,00 0,00 0,00 0,01 0,02 0,02 0,02 0,02 0,03 0,04 0,05
5º 999,81 1,51 998,30 1,46 996,84 1,40 995,44 1,37 994,07 1,31 992,76 1,26 991,50 1,21 990,29 1,17 989,12 1,14 987,98 1,10 986,88 1,05 985,83 1,01
0,01 0,01 0,01 0,02 0,01 0,02 0,03 0,04 0,05 0,05 0,05 0,06
6º 999,80 1,51 998,29 1,46 996,83 1,41 995,42 1,36 994,06 1,32 992,74 1,27 991,47 1,22 990,25 1,18 989,07 1,14 987,93 1,10 986,83 1,06 985,77 1,03
0,03 0,03 0,03 0,03 0,04 0,04 0,04 0,05 0,06 0,07 0,08 0,09
7º 999,77 1,51 998,26 1,46 996,80 1,41 995,39 1,37 994,02 1,32 992,70 1,27 991,43 1,23 990,20 1,19 989,01 1,15 987,86 1,11 986,75 1,07 985,68 1,03
0,05 0,04 0,04 0,05 0,05 0,05 0,05 0,06 0,06 0,07 0,08 0,09
8º 999,72 1,50 998,22 1,46 996,76 1,42 995,34 1,37 993,97 1,32 992,65 1,27 991,38 1,24 990,14 1,19 988,95 1,16 987,79 1,12 986,67 1,08 985,59 1,05
0,05 0,06 0,06 0,06 0,06 0,06 0,07 0,07 0,08 0,09 0,10 0,11
9º 999,67 1,51 998,16 1,46 996,70 1,42 995,28 1,37 993,91 1,32 992,59 1,28 991,31 1,24 990,07 1,20 988,87 1,17 987,70 1,13 986,57 1,09 985,48 1,06
0,07 0,07 0,07 0,07 0,07 0,08 0,08 0,09 0,09 0,10 0,11 0,12
10º 999,60 1,51 998,09 1,46 996,63 1,42 995,21 1,37 993,84 1,33 992,51 1,28 991,23 1,25 989,98 1,20 988,78 1,17 987,60 1,14 986,46 1,10 985,36 1,06
0,09 0,09 0,09 0,08 0,09 0,09 0,10 0,10 0,11 0,11 0,12 0,13
11º 999,51 1,51 998,00 1,46 996,54 1,41 995,13 1,38 993,75 1,33 992,42 1,29 991,13 1,25 989,88 1,21 988,67 1,18 987,49 1,15 986,34 1,11 985,23 1,07
0,10 0,09 0,09 0,10 0,10 0,11 0,11 0,11 0,12 0,13 0,13 0,14
12º 999,41 1,50 997,91 1,46 996,45 1,42 995,03 1,38 993,65 1,34 992,31 1,29 991,02 1,25 989,77 1,22 988,55 1,19 987,36 1,15 986,21 1,12 985,09 1,09
0,11 0,11 0,11 0,11 0,11 0,11 0,12 0,12 0,13 0,14 0,15 0,16
13º 999,30 1,50 997,80 1,46 996,34 1,42 994,92 1,38 993,54 1,34 992,20 1,30 990,90 1,25 989,65 1,23 988,42 1,20 987,22 1,16 986,06 1,13 984,93 1,09
0,12 0,12 0,12 0,13 0,13 0,13 0,13 0,14 0,14 0,15 0,16 0,16
14º 999,18 1,50 997,68 1,46 996,22 1,43 994,79 1,38 993,41 1,34 992,07 1,30 990,77 1,26 989,51 1,23 988,28 1,21 987,07 1,17 985,90 1,13 984,77 1,11
0,13 0,14 0,14 0,13 0,13 0,14 0,14 0,15 0,16 0,16 0,17 0,18
15º 999,05 1,51 997,54 1,46 996,08 1,42 994,66 1,38 993,28 1,35 991,93 1,30 990,63 1,27 989,36 1,24 988,12 1,21 986,91 1,18 985,73 1,14 984,59 1,12
0,15 0,14 0,14 0,15 0,15 0,15 0,16 0,16 0,17 0,17 0,18 0,19
16º 998,90 1,50 997,40 1,46 995,94 1,43 994,51 1,38 993,13 1,35 991,78 1,31 990,47 1,27 989,20 1,25 987,95 1,21 986,74 1,19 985,55 1,15 984,40 1,13
0,16 0,16 0,16 0,16 0,16 0,17 0,17 0,18 0,18 0,19 0,19 0,20
17º 998,74 1,50 997,24 1,46 995,78 1,43 994,35 1,38 992,97 1,36 991,61 1,31 990,30 1,28 989,02 1,25 987,77 1,22 986,55 1,19 985,36 1,16 984,20 1,14
0,17 0,17 0,17 0,16 0,17 0,17 0,18 0,18 0,19 0,20 0,21 0,22
18º 998,57 1,50 997,07 1,46 995,61 1,42 994,19 1,39 992,80 1,36 991,44 1,32 990,12 1,28 988,84 1,26 987,58 1,23 986,35 1,20 985,15 1,17 983,98 1,14
0,18 0,18 0,18 0,19 0,19 0,19 0,19 0,20 0,20 0,20 0,21 0,22
19º 998,39 1,50 996,89 1,46 995,43 1,43 994,00 1,39 992,61 1,36 991,25 1,32 989,93 1,29 988,64 1,26 987,38 1,23 986,15 1,21 984,94 1,10 983,76 1,16
0,19 0,19 0,19 0,19 0,19 0,19 0,20 0,20 0,21 0,22 0,23 0,24
20º 998,20 1,50 996,70 1,46 995,24 1,43 993,81 1,39 992,42 1,36 991,06 1,33 989,73 1,29 988,44 1,27 987,17 1,24 985,93 1,22 984,71 1,19 983,52 1,16
tºAlcoholic strength by % vol
01234567891 0 1 1
20º 998,20 1,50 996,70 1,46 995,24 1,43 993,81 1,39 992,42 1,36 991,06 1,33 989,73 1,29 988,44 1,27 987,17 1,24 985,93 1,22 984,71 1,19 983,52 1,16
0,20 0,20 0,20 0,20 0,21 0,21 0,21 0,22 0,22 0,23 0,24 0,24
21º 998,00 1,50 996,50 1,46 995,04 1,43 993,61 1,40 992,21 1,36 990,85 1,33 989,52 1,30 988,22 1,27 986,95 1,25 985,70 1,23 984,47 1,19 983,28 1,18
0,21 0,21 0,21 0,21 0,21 0,22 0,22 0,23 0,24 0,24 0,24 0,26
22º 997,79 1,50 996,29 1,46 994,83 1,43 993,40 1,40 992,00 1,37 990,63 1,33 989,30 1,31 987,99 1,28 986,71 1,25 985,46 1,23 984,23 1,21 983,02 1,18
0,22 0,22 0,23 0,23 0,23 0,23 0,24 0,24 0,24 0,25 0,26 0,25
23º 997,57 1,50 996,07 1,47 994,60 1,43 993,17 1,40 991,77 1,37 990,40 1,34 989,06 1,31 987,75 1,28 986,47 1,26 985,21 1,24 983,97 1,20 982,77 1,20
0,24 0,23 0,23 0,23 0,24 0,24 0,24 0,25 0,26 0,26 0,27 0,29
24º 997,33 1,49 995,84 1,47 994,37 1,43 992,94 1,41 991,53 1,37 990,16 1,34 988,82 1,32 987,50 1,29 986,21 1,26 984,95 1,25 983,70 1,22 982,48 1,20
0,24 0,25 0,24 0,25 0,24 0,25 0,26 0,26 0,26 0,27 0,28 0,28
25º 997,09 1,50 995,59 1,46 994,13 1,44 992,69 1,40 991,29 1,38 989,91 1,35 988,56 1,32 987,24 1,29 985,95 1,27 984,68 1,26 983,42 1,22 982,20 1,21
0,25 0,25 0,26 0,25 0,26 0,26 0,26 0,26 0,28 0,28 0,28 0,30
26º 996,84 1,50 995,34 1,47 993,87 1,43 992,44 1,41 991,03 1,38 989,65 1,35 988,30 1,32 986,98 1,31 985,67 1,27 984,40 1,26 983,14 1,24 981,90 1,22
0,26 0,26 0,26 0,27 0,27 0,27 0,27 0,28 0,28 0,29 0,30 0,30
27º 996,58 1,50 995,08 1,47 993,61 1,44 992,17 1,41 990,76 1,38 989,38 1,35 988,03 1,33 986,70 1,31 985,39 1,28 984,11 1,27 982,84 1,24 981,60 1,23
0,27 0,27 0,27 0,27 0,28 0,28 0,29 0,29 0,29 0,30 0,31 0,32
28º 996,31 1,50 994,81 1,47 993,34 1,44 991,90 1,42 990,48 1,38 989,10 1,36 987,74 1,33 986,41 1,31 985,10 1,29 983,81 1,28 982,53 1,25 981,28 1,23
0,28 0,28 0,28 0,29 0,28 0,29 0,29 0,30 0,31 0,31 0,31 0,32
29º 996,03 1,50 994,53 1,47 993,06 1,45 991,61 1,41 990,20 1,39 988,81 1,36 987,45 1,34 986,11 1,32 984,79 1,29 983,50 1,28 982,22 1,26 980,96 1,24
0,28 0,29 0,29 0,29 0,30 0,30 0,31 0,31 0,31 0,32 0,32 0,33
30º 995,75 1,51 994,24 1,47 992,77 1,45 991,32 1,42 989,90 1,39 988,51 1,37 987,14 1,34 985,80 1,32 984,48 1,30 983,18 1,28 981,90 1,27 980,63 1,25
0,30 0,30 0,30 0,30 0,31 0,31 0,31 0,31 0,32 0,33 0,34 0,34
31º 995,45 1,51 993,94 1,47 992,47 1,45 991,02 1,43 989,59 1,39 988,20 1,37 986,83 1,34 985,49 1,33 984,16 1,31 982,85 1,29 981,56 1,27 980,29 1,26
0,31 0,31 0,31 0,32 0,31 0,32 0,32 0,33 0,33 0,34 0,35 0,36
32º 995,14 1,51 993,63 1,47 992,16 1,46 990,70 1,42 989,28 1,40 987,88 1,37 986,51 1,35 985,16 1,33 983,83 1,32 982,51 1,30 981,21 1,28 979,93 1,26
0,31 0,31 0,32 0,32 0,32 0,33 0,33 0,34 0,35 0,35 0,35 0,35
33º 994,83 1,51 993,32 1,48 991,84 1,46 990,38 1,42 988,96 1,41 987,55 1,37 986,18 1,36 984,82 1,34 983,48 1,32 982,16 1,30 980,86 1,28 979,58 1,28
0,32 0,33 0,33 0,33 0,35 0,34 0,35 0,35 0,34 0,35 0,36 0,37
34º 994,51 1,52 992,99 1,48 991,51 1,46 990,05 1,44 988,61 1,40 987,21 1,38 985,83 1,36 984,47 1,33 983,14 1,33 981,81 1,31 980,50 1,29 979,21 1,28
0,33 0,33 0,34 0,35 0,34 0,35 0,35 0,35 0,36 0,36 0,36 0,37
35º 994,18 1,52 992,66 1,49 991,17 1,47 989,70 1,43 988,27 1,41 986,86 1,38 985,48 1,36 984,12 1,34 982,78 1,33 981,45 1,31 980,14 1,30 978,84 1,29
0,34 0,35 0,35 0,35 0,35 0,35 0,35 0,36 0,36 0,37 0,37 0,38
36º 993,84 1,53 992,31 1,49 990,82 1,47 989,35 1,43 987,92 1,41 986,51 1,38 985,13 1,37 983,76 1,34 982,42 1,34 981,08 1,31 979,77 1,31 978,46 1,29
0,35 0,35 0,36 0,35 0,36 0,36 0,37 0,37 0,38 0,37 0,39 0,39
37º 993,49 1,53 991,96 1,50 990,46 1,46 989,00 1,44 987,56 1,41 986,15 1,39 984,76 1,37 983,39 1,35 982,04 1,33 980,71 1,33 979,38 1,31 978,07 1,30
0,36 0,36 0,36 0,37 0,37 0,37 0,37 0,37 0,38 0,39 0,38 0,39
38º 993,13 1,53 991,60 1,50 990,10 1,47 988,63 1,44 987,19 1,41 985,78 1,39 984,39 1,37 983,02 1,36 981,66 1,34 980,32 1,32 979,00 1,32 977,68 1,31
0,36 0,37 0,37 0,37 0,38 0,38 0,38 0,39 0,38 0,39 0,40 0,40
39º 992,77 1,54 991,23 1,50 989,73 1,47 988,26 1,45 986,81 1,41 985,40 1,39 984,01 1,38 982,63 1,35 981,28 1,35 979,93 1,33 978,60 1,32 977,28 1,32
0,37 0,37 0,38 0,39 0,38 0,39 0,39 0,39 0,40 0,39 0,40 0,41
40º 992,40 1,54 990,86 1,51 989,35 1,48 987,87 1,44 986,43 1,42 985,01 1,39 983,62 1,38 982,24 1,36 980,88 1,34 979,54 1,34 978,20 1,33 976,87 1,321990R2676 — EN — 18.03.2003 — 007.001 — 43
▼B
tºAlcoholic strength by % vol
10 11 12 13 14 15 16 17 18 19 20 21
0 986,93 1,00 985,93 0,95 984,98 0,92 984,06 0,88 983,18 0,84 982,34 0,80 981,54 0,78 980,76 0,75 980,01 0,73 979,28 0,72 978,56 0,70 977,86 0,70
−0,02 −0,01 0,01 0,01 0,03 0,04 0,07 0,08 0,10 0,12 0,14 0,17
1 986,95 1,01 985,94 0,97 984,97 0,92 984,05 0,90 983,15 0,85 982,30 0,83 981,47 0,79 980,68 0,77 979,91 0,75 979,16 0,74 978,42 0,73 977,69 0,72
−0,01 0,00 0,01 0,03 0,04 0,07 0,08 0,10 0,12 0,14 0,16 0,18
2 986,96 1,02 985,94 0,98 984,96 0,94 984,02 0,91 983,11 0,88 982,23 0,84 981,39 0,81 980,58 0,79 979,79 0,77 979,02 0,76 978,26 0,75 977,51 0,74
0,01 0,02 0,04 0,05 0,06 0,07 0,09 0,11 0,13 0,15 0,17 0,19
3 986,95 1,03 985,92 1,00 984,92 0,95 983,97 0,92 983,05 0,89 982,16 0,86 981,30 0,83 980,47 0,81 979,66 0,79 978,87 0,78 978,09 0,77 977,32 0,77
0,03 0,04 0,04 0,06 0,07 0,09 0,10 0,12 0,14 0,16 0,18 0,20
4 986,92 1,04 985,88 1,00 984,88 0,97 983,91 0,93 982,98 0,91 982,07 0,87 981,20 0,85 980,35 0,83 979,52 0,81 978,71 0,80 977,91 0,79 977,12 0,79
0,04 0,05 0,06 0,07 0,09 0,10 0,12 0,14 0,15 0,17 0,19 0,22
5 986,88 1,05 985,83 1,01 984,82 0,98 983,84 0,95 982,89 0,92 981,97 0,89 981,08 0,87 980,21 0,84 979,37 0,83 978,54 0,82 977,72 0,82 976,90 0,80
0,05 0,06 0,08 0,09 0,10 0,12 0,13 0,14 0,17 0,19 0,21 0,22
6 986,83 1,06 985,77 1,03 984,74 0,99 983,75 0,96 982,79 0,94 981,85 0,90 980,95 0,88 980,07 0,87 979,20 0,85 978,35 0,84 977,51 0,83 976,68 0,83
0,08 0,09 0,09 0,10 0,12 0,13 0,15 0,16 0,18 0,19 0,21 0,23
7 986,75 1,07 985,68 1,03 984,65 1,00 983,65 0,98 982,67 0,95 981,72 0,92 980,80 0,89 979,91 0,89 979,02 0,86 978,16 0,86 977,30 0,85 976,45 0,85
0,08 0,09 0,11 0,13 0,13 0,14 0,15 0,18 0,19 0,21 0,23 0,25
8 986,67 1,08 985,59 1,05 984,54 1,02 983,52 0,98 982,54 0,96 981,58 0,93 980,65 0,92 979,73 0,90 978,83 0,88 977,95 0,88 977,07 0,87 976,20 0,87
0,10 0,11 0,12 0,12 0,14 0,16 0,18 0,19 0,21 0,22 0,24 0,26
9 986,57 1,09 985,48 1,06 984,42 1,02 983,40 1,00 982,40 0,98 981,42 0,95 980,47 0,93 979,54 0,92 978,62 0,89 977,73 0,90 976,83 0,89 975,94 0,89
0,11 0,12 0,12 0,14 0,16 0,17 0,18 0,20 0,20 0,23 0,24 0,26
10 986,46 1,10 985,36 1,06 984,30 1,04 983,26 1,02 982,24 0,99 981,25 0,96 980,29 0,95 979,34 0,92 978,42 0,92 977,50 0,91 976,59 0,91 975,68 0,91
0,12 0,13 0,14 0,16 0,16 0,17 0,19 0,20 0,23 0,25 0,27 0,29
11 986,34 1,11 985,23 1,07 984,16 1,06 983,10 1,02 982,08 1,00 981,08 0,98 980,10 0,96 979,14 0,95 978,19 0,94 977,25 0,93 976,32 0,93 975,39 0,92
0,13 0,14 0,16 0,16 0,18 0,19 0,21 0,22 0,24 0,25 0,27 0,28
12 986,21 1,12 985,09 1,09 984,00 1,06 982,94 1,04 981,90 1,01 980,89 1,00 979,89 0,97 978,92 0,97 977,95 0,95 977,00 0,95 976,05 0,94 975,11 0,95
0,15 0,16 0,16 0,18 0,19 0,20 0,21 0,23 0,24 0,26 0,28 0,30
13 986,06 1,13 984,93 1,09 983,84 1,08 982,76 1,05 981,71 1,02 980,69 1,01 979,68 0,99 978,69 0,98 977,71 0,97 976,74 0,97 975,77 0,96 974,81 0,96
0,16 0,16 0,18 0,18 0,20 0,22 0,23 0,24 0,26 0,27 0,28 0,30
14 985,90 1,13 984,77 1,11 983,66 1,08 982,58 1,07 981,51 1,04 980,47 1,02 979,45 1,00 978,45 1,00 977,45 0,98 976,47 0,98 975,49 0,98 975,51 0,98
0,17 0,18 0,19 0,20 0,21 0,22 0,24 0,25 0,26 0,28 0,30 0,32
15 985,73 1,14 984,59 1,12 983,47 1,09 982,38 1,08 981,30 1,05 960,25 1,04 979,21 1,01 978,20 1,01 977,19 1,00 976,19 1,00 975,19 1,00 974,19 1,00
0,18 0,19 0,20 0,22 0,22 0,24 0,24 0,27 0,28 0,30 0,31 0,32
16 985,55 1,15 984,40 1,13 983,27 1,11 982,16 1,08 981,08 1,07 980,01 1,04 978,97 1,04 977,93 1,02 976,91 1,02 975,89 1,01 974,88 1,01 973,87 1,02
0,19 0,20 0,21 0,22 0,23 0,24 0,26 0,27 0,29 0,30 0,32 0,33
17 985,36 1,16 984,20 1,14 983,06 1,12 981,94 1,09 980,85 1,08 979,77 1,06 978,71 1,05 977,66 1,04 976,62 1,03 975,59 1,03 974,56 1,02 973,54 1,04
0,21 0,22 0,22 0,23 0,25 0,26 0,27 0,28 0,29 0,31 0,32 0,35
18 985,15 1,17 983,98 1,14 982,84 1,13 981,71 1,11 980,60 1,09 979,51 1,07 978,44 1,06 977,38 1,05 976,33 1,05 975,28 1,04 974,24 1,05 973,19 1,05
0,21 0,22 0,24 0,24 0,25 0,26 0,28 0,29 0,31 0,32 0,34 0,35
19 984,94 1,18 983,76 1,16 982,60 1,13 981,47 1,12 980,35 1,10 979,25 1,09 978,16 1,07 977,09 1,07 976,02 1,06 974,96 1,06 973,90 1,06 972,84 1,06
0,23 0,24 0,24 0,26 0,27 0,28 0,29 0,30 0,31 0,33 0,34 0,36
20 984,71 1,19 983,52 1,16 982,36 1,15 981,21 1,13 980,08 1,11 978,97 1,10 977,87 1,08 976,79 1,08 975,71 1,08 974,63 1,07 973,56 1,08 972,48 1,08
tºAlcoholic strength by % vol
10 11 12 13 14 15 16 17 18 19 20 21
20º 984,71 1,19 983,52 1,16 982,36 1,15 981,21 1,13 980,08 1,11 978,97 1,10 977,87 1,08 976,79 1,08 975,71 1,08 974,63 1,07 973,56 1,08 972,48 1,08
0,24 0,24 0,26 0,26 0,27 0,28 0,29 0,31 0,33 0,34 0,36 0,37
21º 984,47 1,19 983,28 1,18 982,10 1,15 980,95 1,14 978,81 1,12 978,69 1,11 977,58 1,10 976,48 1,10 975,38 1,09 974,29 1,09 973,20 1,09 972,11 1,09
0,24 0,26 0,28 0,29 0,30 0,31 0,33 0,33 0,35 0,35 0,36 0,37
22º 984,23 1,21 983,02 1,18 981,84 1,17 980,67 1,15 979,52 1,13 978,39 1,12 977,27 1,12 976,15 1,10 975,05 1,11 973,94 1,10 972,84 1,10 971,74 1,12
0,26 0,26 0,27 0,28 0,29 0,31 0,32 0,33 0,35 0,35 0,37 0,39
23º 983,97 1,20 982,77 1,20 981,57 1,18 980,39 1,16 979,23 1,15 978,08 1,13 976,95 1,13 975,82 1,12 974,70 1,11 973,59 1,12 972,47 1,12 972,47 1,12
0,27 0,29 0,29 0,29 0,30 0,31 0,33 0,33 0,35 0,37 0,38 0,40
24º 983,70 1,22 982,48 1,20 981,28 1,18 980,10 1,17 978,93 1,16 977,77 1,15 976,62 1,13 975,49 1,14 974,35 1,13 973,22 1,13 972,09 1,14 970,95 1,14
0,28 0,28 0,29 0,31 0,32 0,33 0,33 0,35 0,36 0,37 0,39 0,40
25º 983,42 1,22 982,20 1,21 980,99 1,20 979,79 1,18 978,61 1,17 977,44 1,15 976,29 1,15 975,14 1,15 973,99 1,14 972,85 1,15 971,70 1,15 970,55 1,16
0,28 0,30 0,31 0,31 0,32 0,33 0,35 0,36 0,37 0,39 0,40 0,41
26º 983,14 1,24 981,90 1,22 980,68 1,20 979,48 1,19 978,29 1,18 977,11 1,17 975,94 1,16 974,78 1,16 973,62 1,16 972,46 1,16 971,30 1,16 970,14 1,17
0,30 0,30 0,31 0,32 0,33 0,34 0,35 0,36 0,38 0,39 0,40 0,42
27º 982,84 1,24 981,60 1,23 980,37 1,21 979,16 1,20 977,96 1,19 976,77 1,18 975,59 1,17 974,42 1,18 973,24 1,17 972,07 1,17 970,90 1,18 969,72 1,18
0,31 0,32 0,32 0,33 0,34 0,35 0,36 0,38 0,38 0,40 0,41 0,43
28º 982,53 1,25 981,28 1,23 980,05 1,22 978,83 1,21 977,62 1,20 976,42 1,19 975,23 1,19 974,04 1,18 972,86 1,19 971,67 1,18 970,49 1,20 969,29 1,20
0,31 0,32 0,33 0,34 0,35 0,36 0,37 0,38 0,40 0,40 0,42 0,43
29º 982,22 1,26 980,96 1,24 979,72 1,23 978,49 1,22 977,27 1,21 976,06 1,20 974,86 1,20 973,66 1,20 972,46 1,19 971,27 1,20 970,07 1,21 968,86 1,22
0,32 0,33 0,34 0,35 0,36 0,37 0,38 0,40 0,41 0,43 0,44 0,45
30º 981,90 1,27 980,63 1,25 979,38 1,24 978,14 1,23 976,91 1,22 975,69 1,21 974,48 1,22 973,26 1,21 972,05 1,21 970,84 1,21 969,63 1,22 968,41 1,23
0,34 0,34 0,35 0,36 0,37 0,38 0,40 0,40 0,41 0,42 0,44 0,45
31º 981,56 1,27 980,29 1,26 979,03 1,25 977,78 1,24 976,54 1,23 975,31 1,23 974,08 1,22 972,86 1,22 971,64 1,22 970,42 1,23 969,19 1,23 967,96 1,24
0,35 0,36 0,36 0,37 0,38 0,39 0,39 0,40 0,42 0,43 0,44 0,46
32º 981,21 1,28 979,93 1,26 978,67 1,26 977,41 1,25 976,16 1,24 974,92 1,23 973,69 1,23 972,46 1,24 971,22 1,23 969,99 1,24 968,75 1,25 967,50 1,25
0,35 0,35 0,37 0,37 0,38 0,39 0,40 0,42 0,42 0,44 0,45 0,46
33º 980,86 1,28 979,58 1,28 978,30 1,26 977,04 1,26 975,78 1,25 974,53 1,24 973,29 1,25 972,04 1,24 970,80 1,25 969,55 1,25 968,30 1,26 967,04 1,27
0,36 0,37 0,37 0,38 0,39 0,40 0,41 0,42 0,43 0,44 0,46 0,47
34º 980,50 1,29 979,21 1,28 977,93 1,27 976,66 1,27 975,39 1,26 974,13 1,25 972,88 1,26 971,62 1,25 970,37 1,26 969,11 1,27 967,84 1,27 966,57 1,29
0,36 0,37 0,38 0,39 0,39 0,40 0,42 0,42 0,44 0,46 0,46 0,48
35º 980,14 1,30 978,84 1,29 977,55 1,28 976,27 1,27 975,00 1,27 973,73 1,27 972,46 1,26 971,20 1,27 969,93 1,28 968,65 1,27 967,38 1,29 966,09 1,30
0,37 0,38 0,38 0,39 0,40 0,41 0,42 0,44 0,45 0,45 0,47 0,48
36º 979,77 1,31 978,46 1,29 977,17 1,29 975,88 1,28 974,60 1,28 973,32 1,28 972,04 1,28 970,76 1,28 969,48 1,28 968,20 1,29 966,91 1,30 965,61 1,32
0,39 0,39 0,40 0,40 0,41 0,42 0,43 0,44 0,45 0,47 0,48 0,49
37º 978,38 1,31 978,07 1,30 976,77 1,29 975,48 1,29 974,19 1,29 972,90 1,29 971,61 1,29 970,32 1,29 969,03 1,30 967,73 1,30 966,43 1,31 965,12 1,33
0,38 0,39 0,40 0,41 0,42 0,43 0,44 0,45 0,46 0,47 0,49 0,50
38º 979,00 1,32 977,68 1,31 976,37 1,30 975,07 1,30 973,77 1,30 972,47 1,30 971,17 1,30 969,87 1,30 968,57 1,31 967,26 1,32 965,94 1,32 964,62 1,34
0,40 0,40 0,41 0,42 0,42 0,43 0,44 0,45 0,47 0,48 0,49 0,50
39º 978,60 1,32 977,28 1,32 975,96 1,31 974,65 1,30 973,35 1,31 972,04 1,31 970,73 1,31 969,42 1,32 968,10 1,32 966,78 1,33 965,45 1,33 964,12 1,36
0,40 0,41 0,41 0,42 0,43 0,44 0,45 0,46 0,47 0,48 0,49 0,51
40º 978,20 1,33 976,87 1,32 975,55 1,32 974,23 1,31 972,92 1,32 971,60 1,32 970,28 1,32 968,96 1,33 967,63 1,33 966,30 1,34 964,96 1,35 963,61 1,371990R2676 — EN — 18.03.2003 — 007.001 — 44
▼B
tºAlcoholic strength by % vol
20 21 22 23 24 25 26 27 28 29 30 31
0 978,56 0,70 977,86 0,70 977,16 0,69 976,47 0,71 975,76 0,71 975,05 0,72 974,33 0,75 973,58 0,77 972,81 0,80 972,01 0,83 971,18 0,87 970,31 0,90
0,14 0,17 0,19 0,22 0,24 0,26 0,29 0,31 0,34 0,36 0,39 0,41
1 978,42 0,73 977,69 0,72 976,97 0,72 976,25 0,73 975,52 0,73 974,79 0,75 974,04 0,77 973,27 0,80 972,47 0,82 971,65 0,86 970,79 0,89 969,90 0,92
0,16 0,18 0,20 0,23 0,25 0,28 0,30 0,32 0,34 0,37 0,39 0,41
2 978,26 0,75 977,51 0,74 976,77 0,75 976,02 0,75 975,27 0,76 974,51 0,77 973,74 0,79 972,95 0,82 972,13 0,85 971,28 0,88 970,40 0,91 969,49 0,95
0,17 0,19 0,22 0,23 0,26 0,28 0,31 0,33 0,36 0,38 0,40 0,42
3 978,09 0,77 977,32 0,77 976,55 0,76 975,79 0,78 975,01 0,78 974,23 0,80 973,43 0,81 972,62 0,85 971,77 0,87 970,90 0,90 970,00 0,93 969,07 0,98
0,18 0,20 0,22 0,25 0,27 0,29 0,31 0,34 0,36 0,38 0,40 0,43
4 977,91 0,79 977,12 0,79 976,33 0,79 975,54 0,80 974,94 0,80 973,94 0,82 973,12 0,84 972,28 0,87 971,41 0,89 970,52 0,92 969,60 0,96 968,64 1,00
0,19 0,22 0,23 0,26 0,27 0,30 0,33 0,35 0,37 0,39 0,42 0,44
5 977,72 0,82 976,90 0,80 976,10 0,82 975,28 0,81 974,47 0,83 973,64 0,85 972,79 0,86 971,93 0,89 971,04 0,91 970,13 0,95 969,18 0,98 968,20 1,01
0,21 0,22 0,25 0,26 0,29 0,31 0,33 0,35 0,37 0,40 0,42 0,44
6 977,51 0,83 976,68 0,83 975,85 0,83 975,02 0,84 974,18 0,85 973,33 0,87 972,46 0,86 971,58 0,91 970,67 0,94 969,73 0,97 968,76 1,00 967,76 1,03
0,21 0,23 0,25 0,28 0,30 0,32 0,34 0,36 0,38 0,40 0,42 0,44
7 977,30 0,85 976,45 0,85 975,60 0,86 974,74 0,86 973,88 0,87 973,01 0,89 972,12 0,90 971,22 0,93 970,29 0,96 969,33 0,99 968,34 1,02 967,32 1,06
0,23 0,25 0,27 0,28 0,31 0,33 0,35 0,37 0,40 0,42 0,43 0,46
8 977,07 0,87 976,20 0,87 975,33 0,87 974,46 0,89 973,57 0,89 972,68 0,91 971,77 0,92 970,85 0,96 969,89 0,98 968,91 1,00 967,91 1,05 966,86 1,07
0,24 0,26 0,28 0,30 0,31 0,34 0,35 0,38 0,39 0,41 0,44 0,46
9 976,83 0,89 975,94 0,89 975,05 0,89 974,16 0,90 973,26 0,92 972,34 0,92 971,42 0,95 970,47 0,97 969,50 1,00 968,50 1,03 967,47 1,07 966,40 1,09
0,24 0,26 0,28 0,30 0,33 0,34 0,37 0,39 0,41 0,43 0,45 0,46
10 976,59 0,91 975,68 0,91 974,77 0,91 973,86 0,93 972,93 0,93 972,00 0,95 971,05 0,97 970,08 0,99 969,09 1,02 968,07 1,05 967,02 1,08 965,94 1,12
0,27 0,29 0,30 0,33 0,34 0,36 0,38 0,40 0,42 0,44 0,46 0,47
11 976,32 0,93 975,39 0,92 974,47 0,94 973,53 0,94 972,59 0,95 971,64 0,97 970,67 0,99 969,68 1,01 968,67 1,04 967,63 1,07 966,56 1,09 965,47 1,13
0,27 0,28 0,31 0,32 0,34 0,36 0,38 0,40 0,42 0,44 0,45 0,48
12 976,05 0,94 975,11 0,95 974,16 0,95 973,21 0,96 972,25 0,97 971,28 0,99 970,29 1,01 969,28 1,03 968,25 1,06 967,19 1,08 966,11 1,12 964,99 1,15
0,28 0,30 0,31 0,33 0,35 0,37 0,39 0,41 0,43 0,45 0,47 0,49
13 975,77 0,96 974,81 0,96 973,85 0,97 972,88 0,98 971,90 0,99 970,91 1,01 969,90 1,03 968,87 1,05 967,82 1,08 966,74 1,10 965,64 1,14 964,50 1,17
0,28 0,30 0,32 0,34 0,36 0,38 0,40 0,41 0,43 0,45 0,47 0,49
14 975,49 0,98 974,51 0,98 973,53 0,99 972,54 1,00 971,54 1,01 970,53 1,03 969,50 1,04 968,46 1,07 967,39 1,10 966,29 1,12 965,17 1,16 964,01 1,19
0,30 0,32 0,34 0,35 0,37 0,39 0,40 0,42 0,44 0,46 0,48 0,49
15 975,19 1,00 974,19 1,00 973,19 1,00 972,19 1,02 971,17 1,03 970,14 1,04 969,10 1,06 968,04 1,09 966,95 1,12 965,83 1,14 964,69 1,17 963,52 1,21
0,31 0,32 0,34 0,36 0,37 0,39 0,41 0,43 0,45 0,46 0,48 0,51
16 974,88 1,01 973,87 1,02 972,85 1,02 971,83 1,03 970,80 1,05 969,75 1,06 968,69 1,08 967,61 1,11 966,50 1,13 965,37 1,16 964,21 1,20 963,01 1,22
0,32 0,33 0,35 0,37 0,39 0,40 0,42 0,44 0,45 0,48 0,50 0,50
17 974,56 1,02 973,54 1,04 972,50 1,04 971,46 1,05 970,41 1,06 969,35 1,08 968,27 1,10 967,17 1,12 966,05 1,16 964,89 1,18 963,71 1,20 962,51 1,24
0,32 0,35 0,36 0,37 0,39 0,41 0,43 0,45 0,47 0,48 0,49 0,52
18 974,24 1,05 973,19 1,05 972,14 1,05 971,09 1,07 970,02 1,08 968,94 1,10 967,84 1,12 966,72 1,14 965,58 1,17 964,41 1,19 963,22 1,23 961,99 1,25
0,34 0,35 0,36 0,39 0,40 0,42 0,43 0,45 0,47 0,48 0,50 0,52
19 973,90 1,06 972,84 1,06 971,78 1,08 970,70 1,08 969,62 1,10 968,52 1,11 967,41 1,14 966,27 1,16 965,11 1,18 963,93 1,21 962,72 1,25 961,47 1,27
0,34 0,36 0,38 0,39 0,41 0,42 0,45 0,46 0,47 0,49 0,51 0,52
20 973,56 1,08 972,48 1,08 971,40 1,09 970,31 1,10 969,21 1,11 968,10 1,14 966,96 1,15 965,81 1,17 964,64 1,20 963,44 1,23 962,21 1,26 960,95 1,29
tºAlcoholic strength by % vol
20 21 22 23 24 25 26 27 28 29 30 31
20 973,56 1,08 972,48 1,08 971,40 1,09 970,31 1,10 969,21 1,11 968,10 1,14 966,96 1,15 965,81 1,17 964,64 1,20 963,44 1,23 962,21 1,26 960,95 1,29
0,36 0,37 0,38 0,40 0,42 0,44 0,45 0,46 0,49 0,50 0,52 0,53
21 973,20 1,09 972,11 1,09 971,02 1,11 969,91 1,12 968,79 1,13 967,66 1,15 966,51 1,16 965,35 1,20 964,15 1,21 962,94 1,25 961,69 1,27 960,42 1,31
0,36 0,37 0,40 0,41 0,42 0,44 0,45 0,48 0,49 0,51 0,52 0,54
22 972,84 1,10 971,74 1,12 970,62 1,12 969,50 1,13 968,37 1,15 967,22 1,16 966,06 1,19 964,87 1,21 963,66 1,23 962,43 1,26 961,17 1,29 959,88 1,32
0,37 0,39 0,40 0,42 0,43 0,45 0,47 0,48 0,49 0,51 0,53 0,55
23 972,47 1,12 971,35 1,13 970,22 1,14 969,08 1,14 967,94 1,17 966,77 1,18 965,59 1,20 964,39 1,22 963,17 1,25 961,92 1,28 960,64 1,31 959,33 1,33
0,38 0,40 0,41 0,42 0,44 0,45 0,47 0,49 0,51 0,52 0,54 0,55
24 972,09 1,14 970,95 1,14 969,81 1,15 968,66 1,16 967,50 1,18 966,32 1,20 965,12 1,22 963,90 1,24 962,66 1,26 961,40 1,30 960,10 1,32 958,78 1,35
0,39 0,40 0,42 0,43 0,45 0,47 0,48 0,49 0,51 0,53 0,54 0,55
25 971,70 1,15 970,55 1,16 969,39 1,16 968,23 1,18 967,05 1,20 965,85 1,21 964,64 1,23 963,41 1,26 962,15 1,28 960,87 1,31 959,56 1,33 958,23 1,37
0,40 0,41 0,42 0,44 0,46 0,47 0,49 0,50 0,51 0,53 0,54 0,57
26 971,30 1,16 970,14 1,17 968,97 1,18 967,79 1,20 966,59 1,21 965,38 1,23 964,15 1,24 962,91 1,27 961,64 1,30 960,34 1,32 959,02 1,36 957,66 1,38
0,40 0,42 0,43 0,45 0,46 0,48 0,49 0,51 0,53 0,54 0,56 0,56
27 970,90 1,18 969,72 1,18 968,54 1,20 967,34 1,21 966,13 1,23 964,90 1,24 963,66 1,26 962,40 1,29 961,11 1,31 959,80 1,34 958,46 1,36 957,10 1,40
0,41 0,43 0,45 0,46 0,47 0,48 0,50 0,52 0,54 0,56 0,57 0,59
28 970,49 1,20 969,29 1,20 968,09 1,21 966,88 1,22 965,66 1,24 964,42 1,26 963,16 1,28 961,88 1,31 960,57 1,33 959,24 1,35 957,89 1,38 956,51 1,41
0,42 0,43 0,45 0,47 0,49 0,50 0,52 0,53 0,53 0,55 0,56 0,58
29 970,07 1,21 968,86 1,22 967,64 1,23 966,41 1,24 965,17 1,25 963,92 1,28 962,64 1,29 961,35 1,31 960,04 1,35 958,69 1,36 957,33 1,40 955,93 1,42
0,44 0,45 0,46 0,47 0,49 0,50 0,51 0,53 0,55 0,55 0,58 0,58
30 969,63 1,22 968,41 1,23 967,18 1,24 965,94 1,26 964,68 1,26 963,42 1,29 962,13 1,31 960,82 1,33 959,49 1,35 958,14 1,39 956,75 1,40 955,35 1,44
0,44 0,45 0,46 0,48 0,49 0,51 0,52 0,53 0,55 0,57 0,58 0,60
31 969,19 1,23 967,96 1,24 966,72 1,26 965,46 1,27 964,19 1,28 962,91 1,30 961,61 1,32 960,29 1,35 958,94 1,37 957,57 1,40 956,17 1,42 954,75 1,44
0,44 0,46 0,47 0,48 0,50 0,51 0,53 0,54 0,55 0,57 0,58 0,59
32 968,75 1,25 967,50 1,25 966,25 1,27 964,98 1,29 963,69 1,29 962,40 1,32 961,08 1,33 959,75 1,36 958,39 1,39 957,00 1,41 955,59 1,43 954,16 1,46
0,45 0,46 0,48 0,49 0,50 0,52 0,53 0,55 0,57 0,57 0,59 0,61
33 968,30 1,26 967,04 1,27 965,77 1,28 964,49 1,30 963,19 1,31 961,88 1,33 960,55 1,35 959,20 1,38 957,82 1,39 956,43 1,43 955,00 1,45 953,55 1,47
0,46 0,47 0,49 0,50 0,51 0,53 0,54 0,56 0,56 0,59 0,59 0,60
34 967,84 1,27 966,57 1,29 965,28 1,29 963,99 1,31 962,68 1,33 961,35 1,34 960,01 1,37 958,64 1,38 957,26 1,42 955,84 1,43 954,41 1,46 952,95 1,49
0,46 0,48 0,49 0,51 0,52 0,53 0,55 0,56 0,58 0,58 0,60 0,62
35 967,38 1,29 966,09 1,30 964,79 1,31 963,48 1,32 962,16 1,34 960,82 1,36 959,46 1,38 958,08 1,40 956,68 1,42 955,26 1,45 953,81 1,48 952,33 1,50
0,47 0,48 0,50 0,51 0,53 0,54 0,55 0,57 0,58 0,60 0,61 0,62
36 966,91 1,30 965,61 1,32 964,29 1,32 962,97 1,34 961,63 1,35 960,28 1,37 958,91 1,40 957,51 1,41 956,10 1,44 954,66 1,46 953,20 1,49 951,71 1,51
0,48 0,49 0,50 0,52 0,53 0,55 0,56 0,57 0,59 0,60 0,61 0,62
37 966,43 1,31 965,12 1,33 963,79 1,34 962,45 1,35 961,10 1,37 959,73 1,38 958,35 1,41 956,94 1,43 955,51 1,45 954,06 1,47 952,59 1,50 951,09 1,53
0,49 0,50 0,51 0,52 0,54 0,55 0,57 0,58 0,59 0,60 0,62 0,63
38 965,94 1,32 964,62 1,34 963,28 1,35 961,93 1,37 960,56 1,38 959,18 1,40 957,78 1,42 956,36 1,44 954,92 1,46 953,46 1,49 951,97 1,51 950,46 1,54
0,49 0,50 0,52 0,53 0,54 0,56 0,57 0,58 0,60 0,61 0,62 0,64
39 965,45 1,33 964,12 1,36 962,76 1,36 961,40 1,38 960,02 1,40 958,62 1,41 957,21 1,43 955,78 1,46 954,32 1,47 952,85 1,50 951,35 1,53 949,82 1,55
0,49 0,51 0,52 0,54 0,55 0,56 0,58 0,59 0,60 0,62 0,63 0,64
40 964,96 1,35 963,61 1,37 962,24 1,38 960,86 1,39 959,47 1,41 958,06 1,43 956,63 1,44 955,19 1,47 953,72 1,49 952,23 1,51 950,72 1,54 949,18 1,571990R2676 — EN — 18.03.2003 — 007.001 — 45
▼B
TABLE II
INTERNATIONAL ALCOHOLIC STRENGTH AT 20 ºC
Tableofcorrections tobeappliedtotheapparentalcoholicstrengthtocorrect fortheeffect oftemperature
Add or subtract from the apparent alcoholic strength at tºC (ordinary glass alcoholmeter) the correction indicated
below
Apparent alcoholic strength at tºC
01234567891 0 1 1 1 2 1 3 1 4 1 5 1 6Temperature (ºC)0º
Add0,76 0,77 0,82 0,87 0,95 1,04 1,16 1,31 1,49 1,70 1,95 2,26 2,62 3,03 3,49 4,02 4,56
1º 0,81 0,83 0,87 0,92 1,00 1,09 1,20 1,35 1,52 1,73 1,97 2,26 2,59 2,97 3,40 3,87 4,36
2º 0,85 0,87 0,92 0,97 1,04 1,13 1,24 1,38 1,54 1,74 1,97 2,24 2,54 2,89 3,29 3,72 4,173º 0,88 0,91 0,95 1,00 1,07 1,15 1,26 1,39 1,55 1,73 1,95 2,20 2,48 2,80 3,16 3,55 3,954º 0,90 0,92 0,97 1,02 1,09 1,17 1,27 1,40 1,55 1,72 1,92 2,15 2,41 2,71 3,03 3,38 3,75
5º 0,91 0,93 0,98 1,03 1,10 1,17 1,27 1,39 1,53 1,69 1,87 2,08 2,33 2,60 2,89 3,21 3,54
6º 0,92 0,94 0,98 1,02 1,09 1,16 1,25 1,37 1,50 1,65 1,82 2,01 2,23 2,47 2,74 3,02 3,32
7º 0,91 0,93 0,97 1,01 1,07 1,14 1,23 1,33 1,45 1,59 1,75 1,92 2,12 2,34 2,58 2,83 3,10
8º 0,89 0,91 0,94 0,98 1,04 1,11 1,19 1,28 1,39 1,52 1,66 1,82 2,00 2,20 2,42 2,65 2,889º 0,86 0,88 0,91 0,95 1,01 1,07 1,14 1,23 1,33 1,44 1,57 1,71 1,87 2,05 2,24 2,44 2,65
10º 0,82 0,84 0,87 0,91 0,96 1,01 1,08 1,16 1,25 1,35 1,47 1,60 1,74 1,89 2,06 2,24 2,43
11º 0,78 0,79 0,82 0,86 0,90 0,95 1,01 1,08 1,16 1,25 1,36 1,47 1,60 1,73 1,88 2,03 2,20
12º 0,72 0,74 0,76 0,79 0,83 0,88 0,93 0,99 1,07 1,15 1,24 1,34 1,44 1,56 1,69 1,82 1,96
13º 0,66 0,67 0,69 0,72 0,76 0,80 0,84 0,90 0,96 1,03 1,11 1,19 1,28 1,38 1,49 1,61 1,73
14º 0,59 0,60 0,62 0,64 0,67 0,71 0,74 0,79 0,85 0,91 0,97 1,04 1,12 1,20 1,29 1,39 1,49
15º 0,51 0,52 0,53 0,55 0,58 0,61 0,64 0,68 0,73 0,77 0,83 0,89 0,95 1,02 1,09 1,16 1,24
16º 0,42 0,43 0,44 0,46 0,48 0,50 0,53 0,56 0,60 0,63 0,67 0,72 0,77 0,82 0,88 0,94 1,00
17º 0,33 0,33 0,34 0,35 0,37 0,39 0,41 0,43 0,46 0,48 0,51 0,55 0,59 0,62 0,67 0,71 0,7518º 0,23 0,23 0,23 0,24 0,25 0,26 0,27 0,29 0,31 0,33 0,35 0,37 0,40 0,42 0,45 0,48 0,5119º 0,12 0,12 0,12 0,12 0,13 0,13 0,14 0,15 0,16 0,17 0,18 0,19 0,20 0,21 0,23 0,24 0,25
Apparent alcoholic strength at tºC
01234567891 0 1 1 1 2 1 3 1 4 1 5 1 6Temperature (º)21º
Subtract0,13 0,13 0,13 0,14 0,14 0,15 0,16 0,17 0,18 0,19 0,19 0,20 0,22 0,23 0,25 0,26
22º 0,26 0,27 0,28 0,29 0,30 0,31 0,32 0,34 0,36 0,37 0,39 0,41 0,44 0,47 0,49 0,5223º 0,40 0,41 0,42 0,44 0,45 0,47 0,49 0,51 0,54 0,57 0,60 0,63 0,66 0,70 0,74 0,7824º 0,55 0,56 0,58 0,60 0,62 0,64 0,67 0,70 0,73 0,77 0,81 0,85 0,89 0,94 0,99 1,04
25º 0,69 0,71 0,73 0,76 0,79 0,82 0,85 0,89 0,93 0,97 1,02 1,07 1,13 1,19 1,25 1,3126º 0,85 0,87 0,90 0,93 0,96 1,00 1,04 1,08 1,13 1,18 1,24 1,30 1,36 1,43 1,50 1,57
27º 1,03 1,07 1,11 1,15 1,19 1,23 1,28 1,34 1,40 1,46 1,53 1,60 1,68 1,76 1,8428º 1,21 1,25 1,29 1,33 1,38 1,43 1,49 1,55 1,62 1,69 1,77 1,85 1,93 2,02 2,1129º 1,39 1,43 1,47 1,52 1,58 1,63 1,70 1,76 1,84 1,92 2,01 2,10 2,19 2,29 2,39
30º 1,57 1,61 1,66 1,72 1,78 1,84 1,91 1,98 2,07 2,15 2,25 2,35 2,45 2,56 2,67
31º 1,75 1,80 1,86 1,92 1,98 2,05 2,13 2,21 2,30 2,39 2,49 2,60 2,71 2,83 2,94
32º 1,94 2,00 2,06 2,13 2,20 2,27 2,35 2,44 2,53 2,63 2,74 2,86 2,97 3,09 3,22
33º 2,20 2,27 2,34 2,42 2,50 2,58 2,67 2,77 2,88 2,99 3,12 3,24 3,37 3,51
34º 2,41 2,48 2,56 2,64 2,72 2,81 2,91 3,02 3,13 3,25 3,38 3,51 3,65 3,79
35º 2,62 2,70 2,78 2,86 2,95 3,05 3,16 3,27 3,39 3,51 3,64 3,78 3,93 4,08
36º 2,83 2,91 3,00 3,09 3,19 3,29 3,41 3,53 3,65 3,78 3,91 4,05 4,21 4,37
37º 3,13 3,23 3,33 3,43 3,54 3,65 3,78 3,91 4,04 4,18 4,33 4,49 4,6538º 3,36 3,47 3,57 3,68 3,79 3,91 4,03 4,17 4,31 4,46 4,61 4,77 4,94
39º 3,59 3,70 3,81 3,93 4,05 4,17 4,30 4,44 4,58 4,74 4,90 5,06 5,23
40º 3,82 3,94 4,06 4,18 4,31 4,44 4,57 4,71 4,86 5,02 5,19 5,36 5,531990R2676 — EN — 18.03.2003 — 007.001 — 46
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Apparent alcoholic strength at tºC
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Temperature (ºC)0º
Add3,49 4,02 4,56 5,11 5,65 6,16 6,63 7,05 7,39 7,67 7,91 8,07 8,20 8,30 8,36 8,39 8,40
1º 3,40 3,87 4,36 4,86 5,35 5,82 6,26 6,64 6,96 7,23 7,45 7,62 7,75 7,85 7,91 7,95 7,96
2º 3,29 3,72 4,17 4,61 5,05 5,49 5,89 6,25 6,55 6,81 7,02 7,18 7,31 7,40 7,47 7,51 7,533º 3,16 3,55 3,95 4,36 4,77 5,17 5,53 5,85 6,14 6,39 6,59 6,74 6,86 6,97 7,03 7,07 7,094º 3,03 3,38 3,75 4,11 4,48 4,84 5,17 5,48 5,74 5,97 6,16 6,31 6,43 6,53 6,59 6,63 6,66
5º 2,89 3,21 3,54 3,86 4,20 4,52 4,83 5,11 5,35 5,56 5,74 5,89 6,00 6,10 6,16 6,20 6,236º 2,74 3,02 3,32 3,61 3,91 4,21 4,49 4,74 4,96 5,16 5,33 5,47 5,58 5,67 5,73 5,77 5,80
7º 2,58 2,83 3,10 3,36 3,63 3,90 4,15 4,38 4,58 4,77 4,92 5,05 5,15 5,24 5,30 5,34 5,378º 2,42 2,65 2,88 3,11 3,35 3,59 3,81 4,02 4,21 4,38 4,52 4,64 4,74 4,81 4,87 4,92 4,959º 2,24 2,44 2,65 2,86 3,07 3,28 3,48 3,67 3,84 3,99 4,12 4,23 4,32 4,39 4,45 4,50 4,53
10º 2,06 2,24 2,43 2,61 2,80 2,98 3,16 3,33 3,48 3,61 3,73 3,83 3,91 3,98 4,03 4,08 4,11
11º 1,88 2,03 2,20 2,36 2,52 2,68 2,83 2,98 3,12 3,24 3,34 3,43 3,50 3,57 3,62 3,66 3,69
12º 1,69 1,82 1,96 2,10 2,24 2,38 2,51 2,64 2,76 2,87 2,96 3,04 3,10 3,16 3,21 3,25 3,27
13º 1,49 1,61 1,73 1,84 1,96 2,08 2,20 2,31 2,41 2,50 2,58 2,65 2,71 2,76 2,80 2,83 2,8514º 1,29 1,39 1,49 1,58 1,68 1,78 1,88 1,97 2,06 2,13 2,20 2,26 2,31 2,36 2,39 2,42 2,44
15º 1,09 1,16 1,24 1,32 1,40 1,48 1,56 1,64 1,71 1,77 1,83 1,88 1,92 1,96 1,98 2,01 2,03
16º 0,88 0,94 1,00 1,06 1,12 1,19 1,25 1,31 1,36 1,41 1,46 1,50 1,53 1,56 1,58 1,60 1,62
17º 0,67 0,71 0,75 0,80 0,84 0,89 0,94 0,98 1,02 1,05 1,09 1,12 1,14 1,17 1,18 1,20 1,2118º 0,45 0,48 0,51 0,53 0,56 0,59 0,62 0,65 0,68 0,70 0,72 0,74 0,76 0,78 0,79 0,80 0,81
19º 0,23 0,24 0,25 0,27 0,28 0,30 0,31 0,33 0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,40 0,41
Apparent alcoholic strength at tºC
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Temperature (ºC)21º
Subtract0,23 0,25 0,26 0,28 0,29 0,30 0,31 0,33 0,34 0,35 0,35 0,37 0,38 0,38 0,39 0,39 0,40
22º 0,47 0,49 0,52 0,55 0,57 0,60 0,62 0,65 0,67 0,70 0,72 0,74 0,75 0,76 0,78 0,79 0,8023º 0,70 0,74 0,78 0,82 0,86 0,90 0,93 0,97 1,01 1,04 1,07 1,10 1,12 1,15 1,17 1,18 1,1924º 0,94 0,99 1,04 1,10 1,15 1,20 1,25 1,29 1,34 1,39 1,43 1,46 1,50 1,53 1,55 1,57 1,59
25º 1,19 1,25 1,31 1,37 1,43 1,49 1,56 1,62 1,68 1,73 1,78 1,83 1,87 1,90 1,94 1,97 1,99
26º 1,43 1,50 1,57 1,65 1,73 1,80 1,87 1,94 2,01 2,07 2,13 2,19 2,24 2,28 2,32 2,35 2,38
27º 1,68 1,76 1,84 1,93 2,01 2,10 2,18 2,26 2,34 2,41 2,48 2,55 2,61 2,66 2,70 2,74 2,77
28º 1,93 2,02 2,11 2,21 2,31 2,40 2,49 2,58 2,67 2,76 2,83 2,90 2,98 3,03 3,08 3,13 3,1729º 2,19 2,29 2,39 2,50 2,60 2,70 2,81 2,91 3,00 3,09 3,18 3,26 3,34 3,40 3,46 3,51 3,55
30º 2,45 2,56 2,67 2,78 2,90 3,01 3,12 3,23 3,34 3,44 3,53 3,62 3,70 3,77 3,84 3,90 3,95
31º 2,71 2,83 2,94 3,07 3,19 3,31 3,43 3,55 3,67 3,78 3,88 3,98 4,07 4,15 4,22 4,28 4,33
32º 2,97 3,09 3,22 3,36 3,49 3,62 3,74 3,87 4,00 4,11 4,22 4,33 4,43 4,51 4,59 4,66 4,72
33º 3,24 3,37 3,51 3,65 3,79 3,92 4,06 4,20 4,33 4,45 4,57 4,68 4,79 4,88 4,97 5,04 5,10
34º 3,51 3,65 3,79 3,94 4,09 4,23 4,37 4,52 4,66 4,79 4,91 5,03 5,15 5,25 5,34 5,42 5,49
35º 3,78 3,93 4,08 4,23 4,38 4,53 4,69 4,84 4,98 5,12 5,26 5,38 5,50 5,61 5,71 5,80 5,87
36º 4,05 4,21 4,37 4,52 4,68 4,84 5,00 5,16 5,31 5,46 5,60 5,73 5,86 5,97 6,08 6,17 6,25
37º 4,33 4,49 4,65 4,82 4,98 5,15 5,31 5,48 5,64 5,80 5,95 6,09 6,22 6,33 6,44 6,54 6,6338º 4,61 4,77 4,94 5,12 5,29 5,46 5,63 5,80 5,97 6,13 6,29 6,43 6,57 6,69 6,81 6,92 7,01
39º 4,90 5,06 5,23 5,41 5,59 5,77 5,94 6,12 6,30 6,47 6,63 6,78 6,93 7,06 7,18 7,29 7,39
40º 5,19 5,36 5,53 5,71 5,90 6,08 6,26 6,44 6,62 6,80 6,97 7,13 7,28 7,41 7,54 7,66 7,761990R2676 — EN — 18.03.2003 — 007.001 — 47
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TABLE III
INTERNATIONAL ALCOHOLIC STRENGTH AT 20 ºC
Tableofapparentdensitiesofethanol-watermixtures—Ordinaryglassapparatus
Densities at tºC corrected for air buoyancy
tºAlcoholic strength by % vol
01234567891 0 1 1
0 999,34 1,52 997,82 1,45 996,37 1,39 994,98 1,35 993,63 1,29 992,34 1,24 991,10 1,18 989,92 1,15 988,77 1,09 987,68 1,05 986,63 1,00 985,63 0,96
−0,09 −0,09 −0,09 −0,08 −0,08 −0,08 −0,07 −0,05 −0,05 −0,04 −0,03 −0,02
1 999,43 1,52 997,91 1,45 996,46 1,40 995,06 1,35 993,71 1,29 992,42 1,25 991,17 1,20 989,97 1,15 988,82 1,10 987,72 1,06 986,66 1,01 985,65 0,97
−0,06 −0,06 −0,06 −0,06 −0,06 −0,05 −0,05 −0,04 −0,03 −0,02 −0,02 −0,01
2 999,49 1,52 997,97 1,40 996,52 1,40 995,12 1,35 993,77 1,30 992,47 1,25 991,22 1,21 990,01 1,16 988,85 1,11 987,74 1,06 986,68 1,02 985,66 0,98
−0,05 −0,05 −0,04 −0,04 −0,04 −0,04 −0,03 −0,03 −0,03 −0,02 0,00 0,01
3 999,54 1,52 998,02 1,46 996,56 1,40 995,16 1,35 993,81 1,30 992,51 1,26 991,25 1,21 990,04 1,16 988,88 1,12 987,76 1,08 986,68 1,03 985,65 0,99
−0,03 −0,03 −0,03 −0,03 −0,02 −0,02 −0,02 −0,01 0,00 0,01 0,01 0,02
4 999,57 1,52 998,05 1,46 996,59 1,40 995,19 1,36 993,83 1,30 992,53 1,26 991,27 1,22 990,05 1,17 988,88 1,13 987,75 1,08 986,67 1,04 985,63 1,00
−0,02 −0,02 −0,02 −0,02 −0,02 −0,01 0,00 0,00 0,00 0,01 0,02 0,03
5 999,59 1,52 998,07 1,46 996,61 1,40 995,21 1,36 993,85 1,31 992,54 1,27 991,27 1,22 990,05 1,17 988,88 1,14 987,74 1,09 986,65 1,05 985,60 1,02
0,00 0,00 0,00 0,01 0,01 0,01 0,01 0,02 0,03 0,03 0,04 0,06
6 999,59 1,52 998,07 1,46 996,61 1,41 995,20 1,36 993,84 1,31 992,53 1,27 991,26 1,23 990,03 1,18 988,85 1,14 987,71 1,10 986,61 1,07 985,54 1,02
0,01 0,01 0,01 0,01 0,01 0,02 0,02 0,02 0,03 0,04 0,05 0,06
7 999,58 1,52 998,06 1,46 996,60 1,41 995,19 1,36 993,83 1,32 992,51 1,27 991,24 1,23 990,01 1,19 988,82 1,15 987,67 1,11 986,56 1,08 985,48 1,04
0,03 0,03 0,03 0,03 0,04 0,04 0,05 0,05 0,06 0,07 0,07 0,08
8 999,55 1,52 998,03 1,46 996,57 1,41 995,16 1,37 993,79 1,32 992,47 1,28 991,19 1,23 989,96 1,20 988,76 1,16 987,60 1,11 986,49 1,09 985,40 1,05
0,04 0,04 0,04 0,04 0,04 0,04 0,05 0,06 0,06 0,06 0,08 0,08
9 999,51 1,52 997,99 1,46 996,53 1,41 995,12 1,37 993,75 1,32 992,43 1,29 991,14 1,24 989,90 1,20 988,70 1,16 987,54 1,13 986,41 1,09 985,32 1,06
0,06 0,06 0,06 0,06 0,06 0,07 0,07 0,07 0,08 0,09 0,10 0,11
10 999,45 1,52 997,93 1,46 996,47 1,41 995,06 1,37 993,69 1,33 992,36 1,29 991,07 1,24 989,83 1,21 988,62 1,17 987,45 1,14 986,31 1,10 985,21 1,07
0,07 0,06 0,06 0,07 0,07 0,07 0,07 0,08 0,09 0,10 0,10 0,11
11 999,38 1,51 997,87 1,46 996,41 1,42 994,99 1,37 993,62 1,33 992,29 1,29 991,00 1,25 989,75 1,22 988,53 1,18 987,35 1,14 986,21 1,11 985,10 1,08
0,09 0,09 0,09 0,09 0,09 0,09 0,10 0,11 0,11 0,11 0,12 0,13
12 999,29 1,51 997,78 1,46 996,32 1,42 994,90 1,37 993,53 1,33 992,20 1,30 990,90 1,26 989,64 1,22 988,42 1,18 987,24 1,15 986,09 1,12 984,97 1,09
0,09 0,09 0,09 0,09 0,10 0,10 0,10 0,10 0,11 0,12 0,13 0,14
13 999,20 1,51 997,69 1,46 996,23 1,42 994,81 1,38 993,43 1,33 992,10 1,30 990,80 1,26 989,54 1,23 988,31 1,19 987,12 1,16 985,96 1,13 984,83 1,10
0,11 0,11 0,11 0,11 0,11 0,12 0,12 0,13 0,13 0,14 0,15 0,16
14 999,09 1,51 997,58 1,46 996,12 1,42 994,70 1,38 993,32 1,34 991,98 1,30 990,68 1,27 989,41 1,23 988,18 1,20 986,98 1,17 985,81 1,14 984,67 1,11
0,12 0,12 0,12 0,12 0,12 0,12 0,13 0,13 0,14 0,14 0,15 0,16
15 998,97 1,51 997,46 1,46 996,00 1,42 994,58 1,38 993,20 1,34 991,86 1,31 990,55 1,27 989,28 1,24 988,04 1,20 986,84 1,18 985,66 1,15 984,51 1,12
0,13 0,13 0,13 0,13 0,14 0,14 0,14 0,15 0,15 0,17 0,17 0,18
16 998,84 1,51 997,33 1,46 995,87 1,42 994,45 1,39 993,06 1,34 991,72 1,31 990,41 1,28 989,13 1,24 987,89 1,22 986,67 1,18 985,49 1,16 984,33 1,13
0,14 0,14 0,14 0,14 0,14 0,15 0,15 0,15 0,16 0,17 0,17 0,18
17 998,70 1,51 997,19 1,46 995,73 1,42 994,31 1,39 992,92 1,35 991,57 1,31 990,26 1,28 988,98 1,25 987,73 1,22 986,50 1,18 985,32 1,17 984,15 1,14
0,15 0,15 0,16 0,16 0,16 0,16 0,17 0,17 0,18 0,18 0,19 0,19
18 998,55 1,51 997,04 1,47 995,57 1,42 994,15 1,39 992,76 1,35 991,41 1,32 990,09 1,28 988,81 1,26 987,55 1,23 986,32 1,19 985,13 1,17 983,96 1,15
0,17 0,16 0,16 0,16 0,16 0,16 0,17 0,18 0,18 0,19 0,20 0,21
19 998,38 1,50 996,88 1,47 995,41 1,42 993,99 1,39 992,60 1,35 991,25 1,33 989,92 1,29 988,63 1,26 987,37 1,24 986,13 1,20 984,93 1,18 983,75 1,16
0,18 0,18 0,18 0,18 0,19 0,19 0,19 0,20 0,21 0,22 0,22 0,23
20 998,20 1,50 996,70 1,47 995,23 1,42 993,81 1,40 992,41 1,35 991,06 1,33 989,73 1,30 988,43 1,27 987,16 1,24 985,92 1,21 984,71 1,19 983,52 1,17
tºAlcoholic strength by % vol
01234567891 0 1 1
20 998,20 1,50 996,70 1,47 995,23 1,42 993,81 1,40 992,41 1,35 991,06 1,33 989,73 1,30 988,43 1,27 987,16 1,24 985,92 1,21 984,71 1,19 983,52 1,17
0,19 0,19 0,19 0,19 0,19 0,20 0,20 0,21 0,21 0,22 0,23 0,23
21 998,01 1,50 996,51 1,47 995,04 1,42 993,62 1,40 992,22 1,36 990,86 1,33 989,53 1,31 988,22 1,27 986,95 1,25 985,70 1,22 984,48 1,19 983,29 1,17
0,20 0,20 0,19 0,20 0,20 0,20 0,21 0,21 0,22 0,22 0,23 0,24
22 987,81 1,50 996,31 1,46 994,85 1,43 993,42 1,40 992,02 1,36 990,66 1,34 989,32 1,31 988,01 1,28 986,73 1,25 985,48 1,23 984,25 1,20 983,05 1,18
0,21 0,21 0,21 0,21 0,21 0,22 0,22 0,22 0,23 0,24 0,24 0,25
23 997,60 1,50 996,10 1,46 994,64 1,43 993,21 1,40 991,81 1,37 990,44 1,34 989,10 1,31 987,79 1,29 986,50 1,26 985,24 1,23 984,01 1,21 982,80 1,19
0,21 0,21 0,22 0,22 0,22 0,22 0,23 0,23 0,23 0,24 0,25 0,26
24 997,39 1,50 995,89 1,47 994,42 1,43 992,99 1,40 991,59 1,37 990,22 1,35 988,87 1,31 987,56 1,29 986,27 1,27 985,00 1,24 983,76 1,22 982,54 1,20
0,23 0,23 0,23 0,23 0,24 0,24 0,24 0,25 0,25 0,25 0,26 0,27
25 997,16 1,50 995,66 1,47 994,19 1,43 992,76 1,41 991,35 1,37 989,98 1,35 988,63 1,32 987,31 1,29 986,02 1,27 984,75 1,25 983,50 1,23 982,27 1,21
0,23 0,23 0,23 0,24 0,24 0,24 0,24 0,25 0,26 0,27 0,27 0,28
26 996,93 1,50 995,43 1,47 993,96 1,44 992,52 1,41 991,11 1,37 989,74 1,35 988,39 1,33 987,06 1,30 985,76 1,28 984,48 1,25 983,23 1,24 981,99 1,22
0,25 0,25 0,25 0,25 0,25 0,26 0,26 0,26 0,27 0,28 0,29 0,29
27 996,68 1,50 995,18 1,47 993,71 1,44 992,27 1,41 990,86 1,38 989,48 1,35 988,13 1,33 986,80 1,31 985,49 1,29 984,20 1,26 982,94 1,24 981,70 1,23
0,25 0,25 0,26 0,26 0,26 0,26 0,27 0,28 0,28 0,28 0,29 0,30
28 996,43 1,50 994,93 1,48 993,45 1,44 992,01 1,41 990,60 1,38 989,22 1,36 987,86 1,34 986,52 1,31 985,21 1,29 983,92 1,27 982,65 1,25 981,40 1,23
0,26 0,27 0,27 0,27 0,27 0,28 0,28 0,28 0,29 0,29 0,30 0,31
29 996,17 1,51 994,66 1,48 993,18 1,44 991,74 1,41 990,33 1,39 988,94 1,36 987,58 1,34 986,24 1,32 984,92 1,29 983,63 1,28 982,35 1,26 981,09 1,24
0,27 0,27 0,27 0,28 0,28 0,28 0,28 0,29 0,29 0,30 0,31 0,32
30 995,90 1,51 994,39 1,48 992,91 1,45 991,46 1,41 990,05 1,39 988,66 1,37 987,29 1,34 985,95 1,32 984,63 1,30 983,33 1,29 982,04 1,27 980,77 1,25
0,29 0,29 0,29 0,29 0,30 0,30 0,30 0,31 0,31 0,32 0,32 0,32
31 995,61 1,51 994,10 1,48 992,62 1,45 991,17 1,42 989,75 1,39 988,36 1,37 986,99 1,35 985,64 1,33 984,31 1,30 983,01 1,29 981,72 1,27 980,45 1,26
0,29 0,29 0,29 0,29 0,30 0,31 0,31 0,31 0,31 0,32 0,33 0,34
32 995,32 1,51 993,81 1,48 992,33 1,45 990,88 1,42 989,45 1,40 988,05 1,37 986,68 1,35 985,33 1,33 984,00 1,31 982,69 1,30 981,39 1,28 980,11 1,26
0,30 0,31 0,31 0,31 0,31 0,31 0,31 0,32 0,33 0,33 0,34 0,34
33 995,02 1,52 993,50 1,48 992,02 1,45 990,57 1,43 989,14 1,40 987,74 1,37 986,37 1,36 985,01 1,34 983,67 1,31 982,36 1,31 981,05 1,28 979,77 1,27
0,30 0,31 0,31 0,31 0,31 0,32 0,33 0,33 0,33 0,34 0,34 0,35
34 994,72 1,53 993,19 1,48 991,71 1,45 990,26 1,43 988,83 1,41 987,42 1,38 986,04 1,36 984,68 1,34 983,34 1,32 982,02 1,31 980,71 1,29 979,42 1,28
0,32 0,32 0,32 0,33 0,33 0,33 0,33 0,33 0,33 0,34 0,34 0,35
35 994,40 1,53 992,87 1,48 991,39 1,46 989,93 1,43 988,50 1,41 987,09 1,38 985,71 1,36 984,35 1,34 983,01 1,33 981,68 1,31 980,37 1,30 979,07 1,29
0,32 0,32 0,33 0,33 0,33 0,33 0,34 0,34 0,35 0,35 0,36 0,37
36 994,08 1,53 992,55 1,49 991,06 1,46 989,60 1,43 988,17 1,41 986,76 1,39 985,37 1,36 984,01 1,35 982,66 1,33 981,33 1,32 980,01 1,31 978,70 1,29
0,33 0,34 0,34 0,34 0,35 0,35 0,35 0,35 0,36 0,36 0,36 0,37
37 993,75 1,54 992,21 1,49 990,72 1,46 989,26 1,44 987,82 1,41 986,41 1,39 985,02 1,37 983,65 1,35 982,30 1,33 980,97 1,32 979,65 1,32 978,33 1,30
0,34 0,34 0,35 0,36 0,36 0,36 0,36 0,36 0,37 0,38 0,38 0,38
38 993,41 1,54 991,87 1,50 990,37 1,47 988,90 1,44 987,46 1,41 986,05 1,39 984,66 1,37 983,29 1,36 981,93 1,34 980,59 1,32 979,27 1,32 977,95 1,31
0,35 0,35 0,36 0,36 0,36 0,37 0,37 0,37 0,37 0,38 0,38 0,39
39 993,06 1,54 991,52 1,51 990,01 1,47 988,54 1,44 987,10 1,41 985,68 1,39 984,29 1,37 982,92 1,36 981,56 1,34 980,22 1,33 978,89 1,33 977,56 1,31
0,35 0,36 0,36 0,37 0,38 0,38 0,38 0,38 0,38 0,39 0,39 0,39
40 992,71 1,55 991,16 1,51 989,65 1,48 988,17 1,45 986,72 1,42 985,30 1,39 983,91 1,37 982,54 1,36 981,18 1,35 979,83 1,33 978,50 1,33 977,17 1,321990R2676 — EN — 18.03.2003 — 007.001 — 48
▼B
tºAlcoholic strength by % vol
10 11 12 13 14 15 16 17 18 19 20 21
0 986,63 1,00 985,63 0,96 984,67 0,92 983,75 0,87 982,88 0,84 982,04 0,81 981,23 0,77 980,46 0,75 979,71 0,73 978,98 0,72 978,26 0,70 977,56 0,70
−0,03 −0,02 −0,01 0,00 0,02 0,04 0,05 0,07 0,09 0,11 0,13 0,15
1 986,66 1,01 985,65 0,97 984,68 0,93 983,75 0,89 982,86 0,86 982,00 0,82 981,18 0,79 980,39 0,77 979,62 0,75 978,87 0,74 978,13 0,72 977,41 0,72
−0,02 −0,01 0,00 0,01 0,03 0,04 0,06 0,08 0,10 0,12 0,14 0,17
2 986,68 1,02 985,66 0,98 984,68 0,94 983,74 0,91 982,83 0,87 981,96 0,84 981,12 0,81 980,31 0,79 979,52 0,77 978,75 0,76 977,99 0,75 977,24 0,74
0,00 0,01 0,02 0,04 0,05 0,06 0,08 0,10 0,12 0,14 0,16 0,18
3 986,68 1,03 985,65 0,99 984,66 0,96 983,70 0,92 982,78 0,88 981,90 0,86 981,04 0,83 980,21 0,81 979,40 0,79 978,61 0,78 977,83 0,77 977,06 0,76
0,01 0,02 0,03 0,04 0,05 0,07 0,08 0,10 0,12 0,14 0,16 0,18
4 986,67 1,04 985,63 1,00 984,63 0,97 983,66 0,93 982,73 0,90 981,83 0,87 980,96 0,85 980,11 0,83 979,28 0,81 978,47 0,80 977,67 0,79 976,88 0,79
0,02 0,03 0,05 0,06 0,08 0,09 0,11 0,13 0,14 0,16 0,18 0,20
5 986,65 1,05 985,60 1,02 984,58 0,98 983,60 0,95 982,65 0,91 981,74 0,89 980,85 0,87 979,98 0,84 979,14 0,83 978,31 0,82 977,49 0,81 976,68 0,81
0,04 0,06 0,06 0,07 0,08 0,10 0,11 0,13 0,15 0,17 0,19 0,21
6 986,61 1,07 985,54 1,02 984,52 0,99 983,53 0,96 982,57 0,93 981,64 0,90 980,74 0,89 979,85 0,86 978,99 0,85 978,14 0,84 977,30 0,83 976,47 0,83
0,05 0,06 0,08 0,09 0,10 0,12 0,14 0,15 0,17 0,19 0,20 0,22
7 986,56 1,08 985,48 1,04 984,44 1,00 983,44 0,97 982,47 0,95 981,52 0,92 980,60 0,90 979,70 0,88 978,82 0,87 977,95 0,85 977,10 0,85 976,25 0,85
0,07 0,08 0,09 0,10 0,11 0,12 0,14 0,16 0,18 0,19 0,21 0,23
8 986,49 1,09 985,40 1,05 984,35 1,01 983,34 0,98 982,36 0,96 981,40 0,94 980,46 0,92 979,54 0,90 978,64 0,88 977,76 0,87 976,89 0,87 976,02 0,87
0,08 0,08 0,09 0,11 0,13 0,14 0,15 0,16 0,18 0,20 0,22 0,24
9 986,41 1,09 985,32 1,06 984,26 1,03 983,23 1,00 982,23 0,97 981,26 0,95 980,31 0,93 979,38 0,92 978,48 0,90 977,56 0,89 976,67 0,89 975,78 0,89
0,10 0,11 0,12 0,13 0,14 0,16 0,17 0,18 0,19 0,21 0,23 0,25
10 986,31 1,10 985,21 1,07 984,14 1,04 983,10 1,01 982,09 0,99 981,10 0,96 980,14 0,94 979,20 0,93 978,27 0,92 977,35 0,91 976,44 0,91 975,53 0,91
0,10 0,11 0,12 0,13 0,15 0,16 0,17 0,19 0,21 0,23 0,25 0,27
11 986,21 1,11 985,10 1,08 984,02 1,05 982,97 1,03 981,94 1,00 980,94 0,97 979,97 0,96 979,01 0,95 978,06 0,94 977,12 0,93 976,19 0,93 975,26 0,92
0,12 0,13 0,14 0,15 0,16 0,17 0,19 0,21 0,22 0,24 0,26 0,27
12 986,09 1,12 984,97 1,09 983,88 1,06 982,82 1,04 981,78 1,01 980,77 0,99 979,78 0,98 978,80 0,96 977,84 0,96 976,88 0,95 975,93 0,94 974,99 0,94
0,13 0,14 0,15 0,16 0,17 0,19 0,20 0,21 0,23 0,24 0,26 0,28
13 985,96 1,13 984,83 1,10 983,73 1,07 982,66 1,05 981,61 1,03 980,58 1,00 979,58 0,99 978,59 0,98 977,61 0,97 976,64 0,97 975,67 0,96 974,71 0,96
0,15 0,16 0,17 0,18 0,19 0,20 0,22 0,23 0,24 0,26 0,27 0,29
14 985,81 1,14 984,67 1,11 983,56 1,08 982,48 1,06 981,42 1,04 980,38 1,02 979,36 1,00 978,36 0,99 977,37 0,99 976,38 0,98 975,40 0,98 974,42 0,98
0,15 0,16 0,17 0,18 0,19 0,20 0,22 0,24 0,26 0,27 0,28 0,30
15 985,66 1,15 984,51 1,12 983,39 1,09 982,30 1,07 981,23 1,05 980,18 1,04 979,14 1,02 978,12 1,01 977,11 1,00 976,11 0,99 975,12 1,00 974,12 1,00
0,17 0,18 0,19 0,20 0,21 0,22 0,23 0,25 0,26 0,28 0,30 0,31
16 985,49 1,16 984,33 1,13 983,20 1,10 982,10 1,08 981,02 1,06 979,96 1,05 978,91 1,04 977,87 1,02 976,85 1,02 975,83 1,01 974,82 1,01 973,81 1,02
0,17 0,18 0,19 0,20 0,21 0,23 0,24 0,25 0,27 0,29 0,30 0,31
17 985,32 1,17 984,15 1,14 983,01 1,11 981,90 1,09 980,81 1,08 979,73 1,06 978,67 1,05 977,62 1,04 976,58 1,04 975,54 1,02 974,52 1,02 973,50 1,04
0,19 0,19 0,20 0,22 0,24 0,25 0,26 0,27 0,28 0,29 0,31 0,33
18 985,13 1,17 983,96 1,15 982,81 1,13 981,68 1,11 980,57 1,09 979,48 1,07 978,41 1,06 977,35 1,05 976,30 1,05 975,25 1,04 974,21 1,04 973,17 1,05
0,20 0,21 0,22 0,23 0,24 0,25 0,26 0,27 0,29 0,30 0,32 0,34
19 984,93 1,18 983,75 1,16 982,59 1,14 981,45 1,12 980,33 1,10 979,23 1,08 978,15 1,07 977,08 1,07 976,01 1,06 974,94 1,05 973,89 1,06 972,83 1,06
0,22 0,23 0,24 0,24 0,25 0,26 0,28 0,29 0,30 0,31 0,33 0,35
20 984,71 1,19 983,52 1,17 982,35 1,14 981,21 1,13 980,08 1,11 978,97 1,10 977,87 1,08 976,79 1,08 975,71 1,08 974,63 1,07 973,56 1,08 972,48 1,08
tºAlcoholic strength by % vol
10 11 12 13 14 15 16 17 18 19 20 21
20º 984,71 1,19 983,52 1,17 982,35 1,14 981,21 1,13 980,08 1,11 978,97 1,10 977,87 1,08 976,79 1,08 975,71 1,08 974,63 1,07 973,56 1,08 972,48 1,08
0,23 0,23 0,23 0,25 0,26 0,28 0,29 0,31 0,32 0,33 0,35 0,36
21º 984,48 1,19 983,29 1,17 982,12 1,16 980,96 1,14 979,82 1,13 978,69 1,11 977,58 1,10 976,48 1,09 975,39 1,09 974,30 1,09 973,21 1,09 972,12 1,09
0,23 0,24 0,25 0,26 0,27 0,28 0,29 0,31 0,32 0,33 0,35 0,36
22 984,25 1,20 983,05 1,18 981,97 1,17 980,70 1,15 979,55 1,14 978,41 1,12 977,29 1,12 976,17 1,10 975,07 1,10 973,97 1,10 972,86 1,10 971,76 1,11
0,24 0,25 0,26 0,27 0,28 0,29 0,30 0,31 0,33 0,34 0,35 0,37
23 984,01 1,21 982,80 1,19 981,61 1,18 980,43 1,16 979,27 1,15 978,12 1,13 976,99 1,13 975,86 1,12 974,74 1,11 973,63 1,12 972,51 1,12 971,39 1,13
0,25 0,26 0,27 0,28 0,29 0,30 0,31 0,32 0,33 0,35 0,36 0,38
24 983,76 1,22 982,54 1,20 981,34 1,19 980,15 1,17 978,98 1,16 977,82 1,14 976,68 1,14 975,54 1,13 974,41 1,13 973,28 1,13 972,15 1,14 971,01 1,14
0,26 0,27 0,28 0,29 0,30 0,31 0,32 0,33 0,35 0,36 0,38 0,39
25 983,50 1,23 982,27 1,21 981,06 1,20 979,86 1,18 978,68 1,17 977,51 1,16 976,36 1,15 975,21 1,15 974,06 1,14 972,92 1,15 971,77 1,15 970,62 1,15
0,27 0,28 0,29 0,29 0,30 0,31 0,33 0,34 0,35 0,37 0,38 0,39
26 983,23 1,24 981,99 1,22 980,77 1,20 979,57 1,19 978,38 1,18 977,20 1,17 976,03 1,16 974,87 1,16 973,71 1,16 972,55 1,16 971,39 1,16 970,23 1,17
0,29 0,29 0,30 0,31 0,32 0,33 0,34 0,36 0,37 0,38 0,39 0,41
27 982,94 1,24 981,70 1,23 980,47 1,21 979,26 1,20 978,06 1,19 976,87 1,18 975,69 1,18 974,51 1,17 973,34 1,17 972,17 1,17 921,00 1,18 969,82 1,18
0,29 0,30 0,30 0,31 0,32 0,33 0,35 0,36 0,38 0,39 0,40 0,41
28 982,65 1,25 981,40 1,23 980,17 1,22 978,95 1,21 977,74 1,20 976,54 1,20 975,34 1,19 974,15 1,19 972,96 1,18 971,78 1,18 970,60 1,19 969,41 1,20
0,30 0,31 0,32 0,33 0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,42
29 982,35 1,26 981,09 1,24 979,85 1,23 978,62 1,22 977,40 1,21 976,19 1,21 974,98 1,20 973,78 1,20 972,58 1,19 971,39 1,19 970,20 1,21 968,99 1,21
0,31 0,32 0,33 0,34 0,35 0,36 0,37 0,38 0,38 0,40 0,42 0,43
30 982,04 1,27 980,77 1,25 979,52 1,24 978,28 1,23 977,05 1,22 975,83 1,21 974,62 1,21 973,41 1,21 972,20 1,21 970,99 1,21 969,78 1,22 968,56 1,23
0,32 0,32 0,33 0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,42 0,43
31 981,72 1,27 980,45 1,26 979,19 1,25 977,94 1,24 976,70 1,23 975,47 1,22 974,25 1,22 973,03 1,22 971,81 1,22 970,59 1,23 969,36 1,23 968,13 1,24
0,33 0,34 0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,42 0,43 0,45
32 981,39 1,28 980,11 1,26 978,85 1,26 977,59 1,25 976,34 1,24 975,10 1,23 973,87 1,23 972,64 1,23 971,41 1,24 970,17 1,24 968,93 1,25 967,68 1,26
0,34 0,34 0,35 0,35 0,36 0,37 0,39 0,40 0,41 0,42 0,43 0,45
33 981,05 1,28 979,77 1,27 978,50 1,26 977,24 1,26 975,78 1,25 974,73 1,25 973,48 1,24 972,24 1,24 971,00 1,25 969,75 1,25 968,50 1,27 967,23 1,27
0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,41 0,42 0,43 0,45 0,45
34 980,71 1,29 979,42 1,28 978,14 1,27 976,87 1,27 975,60 1,26 974,34 1,26 973,08 1,25 971,83 1,25 970,58 1,26 969,32 1,27 968,05 1,27 966,78 1,29
0,34 0,35 0,36 0,37 0,38 0,39 0,40 0,41 0,43 0,44 0,45 0,47
35 980,37 1,30 979,07 1,29 977,78 1,28 976,50 1,28 975,22 1,27 973,95 1,27 972,68 1,26 971,42 1,27 970,15 1,27 968,88 1,28 967,60 1,29 966,31 1,30
0,36 0,37 0,37 0,38 0,38 0,39 0,40 0,42 0,43 0,44 0,45 0,47
36 980,01 1,31 978,70 1,29 977,41 1,29 976,12 1,28 974,84 1,28 973,56 1,28 972,28 1,28 971,00 1,28 969,72 1,28 968,44 1,29 967,15 1,31 965,84 1,31
0,36 0,37 0,38 0,39 0,40 0,41 0,42 0,43 0,44 0,45 0,46 0,47
37 979,65 1,32 978,33 1,30 977,03 1,30 975,73 1,29 974,44 1,29 973,15 1,29 971,86 1,29 970,57 1,29 969,28 1,29 967,99 1,30 966,69 1,32 965,37 1,32
0,38 0,38 0,39 0,39 0,40 0,41 0,42 0,43 0,44 0,46 0,47 0,48
38 979,27 1,32 977,95 1,31 976,64 1,30 975,34 1,30 974,04 1,30 972,74 1,30 971,44 1,30 970,14 1,30 968,84 1,31 967,53 1,31 966,22 1,33 964,89 1,34
0,38 0,39 0,39 0,40 0,41 0,42 0,43 0,44 0,45 0,46 0,48 0,49
39 978,89 1,33 977,56 1,31 976,25 1,31 974,94 1,31 973,63 1,31 972,32 1,31 971,01 1,31 969,70 1,31 968,39 1,32 967,07 1,33 965,74 1,34 964,40 1,36
0,39 0,39 0,40 0,41 0,42 0,42 0,43 0,45 0,47 0,48 0,49 0,50
40 978,50 1,33 977,17 1,32 975,85 1,32 974,53 1,32 973,21 1,31 971,90 1,32 970,58 1,33 969,25 1,33 967,92 1,33 966,59 1,34 965,25 1,35 963,90 1,371990R2676 — EN — 18.03.2003 — 007.001 — 49
▼B
tºAlcoholic strength by % vol
20 21 22 23 24 25 26 27 28 29 30 31
0 978,26 0,70 977,56 0,70 976,86 0,69 976,17 0,70 975,47 0,72 974,75 0,72 974,03 0,74 973,29 0,77 972,52 0,80 971,72 0,83 970,89 0,87 970,02 0,90
0,13 0,15 0,17 0,20 0,22 0,24 0,27 0,30 0,32 0,35 0,37 0,39
1 978,13 0,72 977,41 0,72 976,69 0,72 975,97 0,72 975,25 0,74 974,51 0,75 973,76 0,77 972,99 0,79 972,20 0,83 971,37 0,85 970,52 0,89 969,63 0,93
0,14 0,17 0,19 0,21 0,24 0,26 0,29 0,31 0,34 0,36 0,38 0,41
2 977,99 0,75 977,24 0,74 976,50 0,74 975,76 0,75 975,01 0,76 974,25 0,78 973,47 0,79 972,68 0,82 971,86 0,85 971,01 0,87 970,14 0,92 960,22 0,96
0,16 0,18 0,20 0,23 0,25 0,27 0,29 0,32 0,34 0,36 0,38 0,40
3 977,83 0,77 977,06 0,76 976,30 0,77 975,53 0,77 974,76 0,78 973,98 0,80 973,18 0,82 972,36 0,84 971,52 0,87 970,65 0,89 969,76 0,94 968,82 0,98
0,16 0,18 0,21 0,23 0,25 0,28 0,30 0,32 0,34 0,36 0,39 0,42
4 977,67 0,79 976,88 0,79 976,09 0,79 975,30 0,79 974,51 0,81 973,70 0,82 972,88 0,84 972,04 0,86 971,18 0,89 970,29 0,92 969,37 0,96 968,40 1,00
0,18 0,20 0,22 0,24 0,26 0,28 0,30 0,33 0,35 0,38 0,40 0,41
5 977,49 0,81 976,68 0,81 975,87 0,81 975,06 0,81 974,25 0,83 973,42 0,84 972,58 0,86 971,71 0,88 970,83 0,92 969,91 0,94 968,97 0,98 967,99 1,02
0,19 0,21 0,23 0,25 0,27 0,30 0,33 0,34 0,37 0,39 0,41 0,43
6 977,30 0,83 976,47 0,83 975,64 0,83 974,81 0,84 973,97 0,85 973,12 0,87 972,25 0,88 971,37 0,91 970,46 0,94 969,52 0,96 968,56 1,00 967,56 1,04
0,20 0,22 0,24 0,26 0,28 0,30 0,32 0,35 0,37 0,39 0,41 0,43
7 976,10 0,85 976,25 0,85 975,40 0,85 974,55 0,86 973,69 0,87 972,82 0,89 971,93 0,91 971,02 0,93 970,09 0,96 969,13 0,98 968,15 1,02 967,13 1,06
0,21 0,23 0,25 0,27 0,29 0,31 0,33 0,35 0,37 0,39 0,42 0,44
8 976,89 0,87 976,02 0,87 975,15 0,87 974,28 0,88 973,40 0,89 972,51 0,91 971,60 0,93 970,67 0,95 969,72 0,98 968,74 1,01 967,73 1,04 966,69 1,08
0,22 0,24 0,26 0,28 0,30 0,32 0,34 0,36 0,39 0,41 0,43 0,45
9 976,67 0,89 975,78 0,89 974,89 0,89 974,00 0,90 973,10 0,91 972,19 0,93 971,26 0,95 970,31 0,98 969,33 1,00 968,33 1,03 967,30 1,06 966,24 1,09
0,23 0,25 0,27 0,29 0,31 0,33 0,35 0,37 0,39 0,41 0,43 0,45
10 976,44 0,91 975,53 0,91 974,62 0,91 973,71 0,92 972,79 0,93 971,86 0,95 970,91 0,97 969,94 1,00 968,94 1,02 967,92 1,05 966,87 1,08 965,79 1,11
0,25 0,27 0,28 0,30 0,32 0,34 0,36 0,38 0,40 0,42 0,44 0,45
11 976,11 0,93 975,26 0,92 974,34 0,93 973,41 0,94 972,47 0,95 971,52 0,97 970,55 0,99 969,56 1,02 968,54 1,04 967,50 1,07 966,43 1,09 965,34 1,13
0,26 0,27 0,29 0,31 0,33 0,35 0,37 0,39 0,40 0,42 0,44 0,46
12 975,93 0,94 974,99 0,94 974,05 0,95 973,10 0,96 972,14 0,97 971,17 0,99 970,18 1,01 969,17 1,03 968,14 1,06 967,08 1,09 965,99 1,11 964,88 1,15
0,26 0,28 0,30 0,32 0,34 0,36 0,38 0,39 0,41 0,43 0,45 0,47
13 975,67 0,96 974,71 0,96 973,75 0,97 972,78 0,98 971,80 0,99 970,81 1,01 969,80 1,02 968,78 1,05 967,73 1,08 966,65 1,11 965,54 1,13 964,41 1,17
0,27 0,29 0,31 0,33 0,35 0,37 0,38 0,40 0,42 0,44 0,45 0,47
14 975,40 0,98 974,42 0,98 973,44 0,99 972,45 1,00 971,45 1,01 970,44 1,02 969,42 1,04 968,38 1,07 967,31 1,10 966,21 1,12 965,09 1,15 963,94 1,19
0,28 0,30 0,32 0,33 0,35 0,37 0,39 0,41 0,43 0,45 0,47 0,49
15 975,12 1,00 974,12 1,00 973,12 1,00 972,12 1,02 971,10 1,03 970,07 1,04 969,03 1,06 967,97 1,09 966,88 1,12 965,76 1,14 964,62 1,17 963,45 1,20
0,30 0,31 0,33 0,35 0,36 0,38 0,40 0,42 0,44 0,45 0,47 0,49
16 974,82 1,01 973,81 1,02 972,79 1,02 971,77 1,03 970,74 1,05 969,69 1,06 968,63 1,08 967,55 1,11 966,44 1,13 965,31 1,16 964,15 1,19 962,96 1,22
0,30 0,31 0,33 0,35 0,37 0,38 0,40 0,42 0,43 0,45 0,47 0,49
17 974,52 1,02 973,50 1,04 972,46 1,04 971,42 1,05 970,37 1,06 969,31 1,08 968,23 1,10 967,13 1,12 966,01 1,15 964,86 1,18 963,68 1,21 962,47 1,24
0,31 0,33 0,34 0,36 0,38 0,40 0,42 0,43 0,45 0,47 0,48 0,50
18 974,21 1,04 973,17 1,05 972,12 1,06 971,06 1,07 969,99 1,08 968,91 1,10 967,81 1,11 966,70 1,14 965,56 1,17 964,39 1,19 963,20 1,23 961,97 1,26
0,32 0,34 0,35 0,36 0,38 0,40 0,42 0,44 0,46 0,47 0,49 0,50
19 973,89 1,06 972,83 1,06 971,77 1,07 970,70 1,09 969,61 1,10 968,51 1,11 967,39 1,13 966,26 1,16 965,10 1,18 963,92 1,21 962,71 1,24 961,47 1,28
0,33 0,35 0,37 0,39 0,40 0,41 0,42 0,45 0,46 0,48 0,50 0,52
20 973,56 1,08 972,48 1,08 971,40 1,09 970,31 1,10 969,21 1,11 968,10 1,13 966,97 1,14 965,81 1,17 964,64 1,20 963,44 1,23 962,21 1,26 960,95 1,29
tºAlcoholic strength by % vol
20 21 22 23 24 25 26 27 28 29 30 31
20º 973,56 1,08 972,48 1,08 971,40 1,09 970,31 1,10 969,21 1,11 968,10 1,13 966,97 1,16 965,81 1,17 964,64 1,20 963,44 1,23 962,21 1,26 960,95 1,29
0,35 0,36 0,37 0,39 0,40 0,42 0,44 0,45 0,47 0,49 0,50 0,52
21º 973,21 1,09 972,12 1,09 971,03 1,11 969,92 1,11 968,81 1,13 967,68 1,15 966,53 1,17 965,36 1,19 964,17 1,22 962,95 1,24 961,71 1,28 960,43 1,31
0,35 0,36 0,38 0,39 0,41 0,43 0,44 0,46 0,48 0,49 0,51 0,52
22 972,86 1,10 971,76 1,11 970,65 1,12 969,53 1,13 968,40 1,15 967,25 1,16 966,09 1,19 964,90 1,21 963,69 1,23 962,46 1,26 961,20 1,29 959,91 1,32
0,35 0,37 0,39 0,40 0,42 0,43 0,45 0,46 0,48 0,50 0,52 0,53
23 972,51 1,12 971,39 1,13 970,26 1,13 969,13 1,15 967,98 1,16 966,82 1,18 965,64 1,20 964,44 1,23 963,21 1,25 961,96 1,28 960,68 1,30 959,38 1,33
0,36 0,38 0,39 0,41 0,42 0,44 0,46 0,48 0,49 0,51 0,53 0,54
24 972,15 1,14 971,01 1,14 969,87 1,15 968,72 1,16 967,56 1,18 966,38 1,20 965,18 1,22 963,96 1,24 962,72 1,27 961,45 1,29 960,16 1,32 958,84 1,34
0,38 0,39 0,40 0,42 0,44 0,45 0,46 0,48 0,50 0,51 0,53 0,54
25 971,77 1,15 970,62 1,15 969,47 1,17 968,30 1,18 967,12 1,19 965,93 1,21 964,72 1,24 963,48 1,26 962,22 1,28 960,94 1,31 959,63 1,33 958,30 1,36
0,38 0,39 0,41 0,42 0,44 0,46 0,48 0,49 0,50 0,52 0,53 0,55
26 971,39 1,16 970,23 1,17 969,06 1,18 967,88 1,20 966,68 1,21 965,47 1,23 964,24 1,25 962,99 1,27 961,72 1,30 960,42 1,32 959,10 1,35 957,75 1,38
0,39 0,41 0,42 0,44 0,45 0,46 0,48 0,50 0,51 0,52 0,53 0,55
27 971,00 1,18 969,82 1,18 968,64 1,20 967,44 1,21 966,23 1,22 965,01 1,25 963,76 1,27 962,49 1,28 961,21 1,31 959,90 1,33 958,57 1,37 957,20 1,40
0,40 0,41 0,43 0,44 0,46 0,48 0,49 0,50 0,52 0,53 0,55 0,56
28 970,60 1,19 969,41 1,20 968,21 1,21 967,00 1,23 965,77 1,24 964,53 1,26 963,27 1,28 961,99 1,30 960,69 1,32 959,37 1,35 958,02 1,38 956,64 1,41
0,40 0,42 0,43 0,45 0,46 0,48 0,49 0,50 0,52 0,54 0,55 0,56
29 970,20 1,21 968,99 1,21 967,78 1,23 966,55 1,24 965,31 1,26 964,05 1,27 962,78 1,29 961,49 1,32 960,17 1,34 958,83 1,36 957,47 1,39 956,08 1,43
0,42 0,43 0,45 0,46 0,47 0,48 0,50 0,52 0,53 0,54 0,56 0,58
30 969,78 1,22 968,56 1,23 967,33 1,24 966,09 1,25 964,84 1,27 963,57 1,29 962,28 1,31 960,97 1,33 959,64 1,35 958,29 1,38 956,91 1,41 955,50 1,44
0,42 0,43 0,44 0,45 0,47 0,49 0,51 0,52 0,53 0,55 0,56 0,58
31 969,36 1,23 968,13 1,24 966,89 1,25 965,64 1,27 964,37 1,29 963,08 1,31 961,77 1,32 960,45 1,34 959,11 1,37 957,74 1,39 956,35 1,43 954,92 1,45
0,43 0,45 0,46 0,48 0,49 0,50 0,51 0,52 0,54 0,56 0,57 0,58
32 968,93 1,25 967,68 1,25 966,43 1,27 965,16 1,28 963,88 1,30 962,58 1,32 961,26 1,33 959,93 1,36 958,57 1,39 957,18 1,40 955,78 1,44 954,34 1,47
0,43 0,45 0,47 0,48 0,50 0,51 0,52 0,54 0,55 0,56 0,58 0,59
33 968,50 1,27 967,23 1,27 965,96 1,28 964,68 1,30 963,38 1,31 962,07 1,33 960,74 1,35 959,39 1,37 958,02 1,40 956,62 1,42 955,20 1,45 953,75 1,48
0,45 0,45 0,47 0,49 0,50 0,51 0,52 0,54 0,55 0,56 0,58 0,60
34 968,05 1,27 966,78 1,29 965,49 1,30 964,19 1,31 962,88 1,32 961,56 1,34 960,22 1,37 958,85 1,38 957,47 1,41 956,06 1,44 954,62 1,47 953,15 1,49
0,45 0,47 0,48 0,49 0,50 0,52 0,54 0,55 0,57 0,58 0,59 0,60
35 967,60 1,29 996,31 1,30 965,01 1,31 963,70 1,32 962,38 1,34 961,04 1,36 959,68 1,38 958,30 1,40 956,90 1,42 955,48 1,45 954,03 1,48 952,55 1,50
0,45 0,47 0,48 0,49 0,51 0,53 0,54 0,55 0,57 0,59 0,60 0,61
36 967,15 1,31 965,84 1,31 964,53 1,32 963,21 1,34 961,87 1,36 960,51 1,37 959,14 1,39 957,75 1,42 956,33 1,44 954,89 1,46 953,43 1,49 951,94 1,51
0,46 0,47 0,48 0,50 0,52 0,53 0,55 0,56 0,57 0,58 0,60 0,61
37 966,69 1,32 965,37 1,32 964,05 1,34 962,71 1,36 961,35 1,37 959,98 1,39 958,59 1,40 957,19 1,43 955,76 1,45 954,31 1,48 952,83 1,50 951,33 1,52
0,47 0,48 0,50 0,51 0,52 0,54 0,55 0,57 0,58 0,59 0,60 0,61
38 966,22 1,33 964,89 1,34 963,55 1,35 962,20 1,37 960,83 1,39 959,44 1,40 958,04 1,42 956,62 1,44 955,18 1,46 953,72 1,49 952,23 1,51 950,72 1,54
0,48 0,49 0,51 0,52 0,53 0,54 0,56 0,57 0,58 0,60 0,61 0,62
39 965,74 1,34 964,40 1,36 963,04 1,36 961,68 1,38 960,30 1,40 958,90 1,42 957,48 1,43 956,05 1,45 954,60 1,48 953,12 1,50 951,62 1,52 950,10 1,55
0,49 0,50 0,51 0,53 0,54 0,55 0,56 0,58 0,60 0,61 0,62 0,64
40 965,25 1,35 963,90 1,37 962,53 1,38 961,15 1,39 959,76 1,41 958,35 1,43 956,92 1,45 955,47 1,47 954,00 1,49 952,51 1,51 951,00 1,54 949,49 1,561990R2676 — EN — 18.03.2003 — 007.001 — 50
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TABLE IV
Tablegivingtherefractiveindicesofpureethanol-watermixturesanddistillatesat20ºCandthecorresponding
alcoholic strengthsat20ºC
1990R2676 — EN — 18.03.2003 — 007.001 — 51
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4.TOTALDRYEXTRACT
Totaldrymatter
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 under-
goes as little alterationas possible while the test is bein g 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
an d the total sugars inexcess of 1 g/l, potassium sulphate inexcess
of 1 g/l, any mannitol present and any other chemical substances
which may have beenadded to the win e.
The residual extract is the sugar-free extract less the fixed acidity
expressed as tartaric acid.
The extract is expressed ingrams per litre an d it should be deter-
mined 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 interms of the quan tity of sucrose
which, when dissolved in water and made up to a volume of one
litre, gives a solutionof 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 drof the ‘alcohol-free wine’ is calculated
using the following formula:
dr=dv−da+ 1,000
where:
dv= specific gravity of the wine at 20 ºC (corrected for volatile
acidity) (1),
da= specific gravity at 20 ºC of a water-alcohol mixture of the
same alcoholic strength as the wine.
drmay also be calculated from the densities at 20 ºC, ρvof the wine
andρaof the water-alcohol mixture of the same alcoholic strength by
the formula:
dr= 1,0018 ( ρv−ρa) + 1,000
where the coefficient 1,0018 approximates to 1 when ρvis below
1,05, which is most oftenthe 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 drof the alcohol-free wine or from
the specific gravity d20º C20º Cof the must.
The total dry extract is expressed ing/l to on e decimal place.1990R2676 — EN — 18.03.2003 — 007.001 — 52
(1) 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 acidityusing the formula:d
v=d20º C20º C− 0,0000086 aorρv=ρ20− 0,0000086 a
whereais the volatile acidity expressed inmilliequivalen ts per litre.
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TABLE I
forthecalculationofthecontenttotaldryextract(g/l)
Specific gravity
to two decimal
placesThird decimal place of the specific gravity
0123456789
Grams of extract per litre
1,00 0 2,6 5,1 7,7 10,3 12,9 15,4 18,0 20,6 23,2
1,01 25,8 28,4 31,0 33,6 36,2 38,8 41,3 43,9 46,5 49,1
1,02 51,7 54,3 56,9 59,5 62,1 64,7 67,3 69,9 72,5 75,1
1,03 77,7 80,3 82,9 85,5 88,1 90,7 93,3 95,9 98,5 101,1
1,04 103,7 106,3 109,0 111,6 114,2 116,8 119,4 122,0 124,6 127,2
1,05 129,8 132,4 135,0 137,6 140,3 142,9 145,5 148,1 150,7 153,3
1,06 155,9 158,6 161,2 163,8 166,4 169,0 171,6 174,3 176,9 179,5
1,07 182,1 184,8 187,4 190,0 192,6 195,2 197,8 200,5 203,1 205,81,08 208,4 211,0 213,6 216,2 218,9 221,5 224,1 226,8 229,4 232,0
1,09 234,7 237,3 239,9 242,5 245,2 247,8 250,4 253,1 255,7 258,4
1,10 261,0 263,6 266,3 268,9 271,5 274,2 276,8 279,5 282,1 284,8
1,11 287,4 290,0 292,7 295,3 298,0 300,6 303,3 305,9 308,6 311,2
1,12 313,9 316,5 319,2 321,8 324,5 327,1 329,8 332,4 335,1 337,8
1,13 340,4 343,0 345,7 348,3 351,0 353,7 356,3 359,0 361,6 364,3
1,14 366,9 369,6 372,3 375,0 377,6 380,3 382,9 385,6 388,3 390,9
1,15 393,6 396,2 398,9 401,6 404,3 406,9 409,6 412,3 415,0 417,6
1,16 420,3 423,0 425,7 428,3 431,0 433,7 436,4 439,0 441,7 444,4
1,17 447,1 449,8 452,4 455,2 457,8 460,5 463,2 465,9 468,6 471,3
1,18 473,9 476,6 479,3 482,0 484,7 487,4 490,1 492,8 495,5 498,2
1,19 500,9 503,5 506,2 508,9 511,6 514,3 517,0 519,7 522,4 525,1
1,20 527,8 —————————
Interpolation table
Fourth decimal
place of the
specific gravityGrams of extract
per litreFourth decimal
place of the
specific gravityGrams of extract
per litreFourth decimal
place of the
specific gravityGrams of extract
per litre
1 0,3 4 1,0 7 1,8
2 0,5 5 1,3 8 2,1
3 0,8 6 1,6 9 2,31990R2676 — EN — 18.03.2003 — 007.001 — 53
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5.REDUCINGSUGARS
1. DEFINITION
Reducing sugars comprise all the sugars exhibiting ketonic and alde-
hydic functions and are determined by their reducing action on an
alkaline solution of a copper salt.
2. PRINCIPLE OF THE METHODS2.1.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 between0,5 an d 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 withinthe limits prescribed inthe followin g table:
Description Sugar content (g/l) Density Dilution (%)
Musts and mistelles > 125 > 1,038 1
Sweet wines,
whether fortified or
not25 to 125 1,005 to 1,038 4
Semi-sweet wines 5 to 25 0,997 to 1,005 20
Dry wines < 5 < 0,997 No dilution
3.1.Reference method
3.1.1. Reagents
3.1.1.1. 1 M solutionhydrochloric acid (HCl);
3.1.1.2. 1 M solutionsodium hydroxide (NaOH);3.1.1.3. 4 M solutionacetic acid (CH
3COOH);
3.1.1.4. 2 M solutionsodium hydroxide (NaOH);
3.1.1.5. Anion exchange resin (Dowex 3 (20-50 mesh) or equivalent resin).
Preparation of the anion exchange resin columnPlace a small plug of glass wool and 15 ml of the anion exchange
resin(3.1.1.5) inthe bottom of the burette.
Before the resinis used, subject it to two complete cycles of regen -
eration by passing alternately the 1 M solutions of hydrochloric acid
(3.1.1.1) and sodium hydroxide (3.1.1.2) through it. After rinsingwith 50 ml of distilled water, transfer the resin to a beaker, add
50 ml of the 4 M solutionof acetic acid (3.1.1.3) an d 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 resinstored ina bottle filled with this 4 M acetic
acid solution.) Wash the column with distilled water until the
effluent is neutral.1990R2676 — EN — 18.03.2003 — 007.001 — 54
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Regeneration of the resin
Pour 150 ml of a 2 M sodium hydroxide solutionthrough 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 solu-
tion through it. Wash the column until the effluent is neutral.
3.1.1.6. Neutral lead acetate solution(approximately saturated)
Neutral lead acetate [Pb (CH3COO)2·3H2O], 250 g;
very hot water to 500 ml;stir until dissolved.
3.1.1.7. Calcium carbonate (Ca CO
3)
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 solu-
tion(3.1.1.2) ( nbeing 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) an d
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 % solutionof 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 ºC 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 volu-
metric 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.
3.2.Usualmethods
3.2.1. Clarification by neutral lead acetate
3.2.1.1. Reagents
Solutionof n eutral lead acetate (approximately saturated) (see
3.1.1.6).
Calcium carbonate.
3.2.1.2. Procedure3.2.1.2.1. Dry wines: place 50 ml of the wine in a 100 ml volumetric flask;
add ½ ( n− 0,5) ml of a 1 M solutionof sodium hydroxide
(3.1.1.2) (where nis the volume of a 0,1 M solutionof sodium1990R2676 — EN — 18.03.2003 — 007.001 — 55
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hydroxide used to determine the total acidity in 10 ml of wine). Add,
while stirring, 2,5 ml of saturated lead acetate solution (3.1.1.6) and
0,5 g calcium carbonate (3.1.1.7). Shake several times and allow to
stand for at least 15 minutes. Make up to the mark with water. Filter.
1 ml of the filtrate corresponds to 0,5 ml of the wine.
3.2.1.2.2. Musts, mistelles, sweet and semi-sweet wines: into a 100 ml volu-
metric flask, place the following volumes of wine (or must or
mistelle), the dilutions being given for guidance:
1.Musts and mistelles: prepare a 10 % solutionof 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 between 0,997 and 1,005:
take 20 ml of the undiluted wine.
Add 0,5 g calcium carbonate, about 60 ml water and 0,5, 1 or
2 ml of saturated lead acetate solution. Stir and leave to stand
for at least 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.
3.2.2. Clarification by zinc 2-hexacyanoferrate
This clarification process should be used only for white wines,
lightly coloured sweet wines and musts.
3.2.2.1. Reagents3.2.2.1.1. SolutionI, potassium 2-hexacyan oferrate:
Potassium 2-hexacyanoferrate (K
4Fe (CN)6·3H2O), 150 g;
Water to 1 000 ml.
3.2.2.1.2. SolutionII, zin c sulphate:
Zinc sulphate (Zn SO4·7H2O), 300 g;
Water to 1 000 ml.
3.2.2.2. Procedure
Into a 100 ml volumetric flask, place the following volumes of wine
(or must or mistelle), the dilutions being given for guidance:
1.Musts and mistelles: prepare a 10 % solutionof 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 between 0,997 and 1,005:
take 20 ml of the undiluted wine.
4.Dry wines: take 50 ml of undiluted wine.
Add 5 ml of solutionI, potassium 2-hexacyan oferrate
(3.2.2.1.1), and 5 ml of solution II, zinc sulphate (3.2.2.1.2).
Mix. Make up to the mark with water. Wait 10 minutes. 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.4. 1 ml of filtrate corresponds to 0,50 ml of dry wine.1990R2676 — EN — 18.03.2003 — 007.001 — 56
▼B
4. DETERMINATION OF SUGARS
4.1.Reagents
4.1.1 Alkaline copper salt solution:
copper sulphate, pure, CuSO4·5 H2O2 5 g
citric acid (C6H8O7·H2O) 50 g
crystalline sodium carbonate, Na2CO3· 10H2O 388 g
water to 1 000 ml
Dissolve the copper sulphate in100 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:
potassium iodide (KI) 30 g
water to 100 ml
Store ina coloured glass bottle.
4.1.3. 25 % sulphuric acid:
concentrated sulphuric acid, (H2SO4)ρ20= 1,84 g/ml 25 g
water to 100 ml
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 inwith about 500 ml of water. Brin g 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.
Sodium thiosulphate, 0,1 M solution
Invert sugar solution, 5 g/l, to be used for checking the method of
determination:
Place the following into a 200 ml volumetric flask:
pure dry sucrose (C12H22O11) 4,75 g
water, approximately 100 ml
concentrated hydrochloric acid (HCl) ( ρ20= 1,16 − 1,19 g/ml) 5 ml
Heat the flask in a water-bath maintained at 60 ºC until the tempera-
ture of the solutionreaches 50 ºC; thenkeep the flask an d solutionat
50 ºC for 15 minutes. Allow the flask to cool naturally for 30
minutes and then immerse it in a cold water-bath. Transfer the solu-
tion to a one-litre volumetric flask and make up to one litre. This
solutionkeeps satisfactorily for a mon th. Whenit 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 solutionina 300 ml con ical 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).1990R2676 — EN — 18.03.2003 — 007.001 — 57
▼B
Titrate with 0,1 M sodium thiosulphate solution(4.1.5) Let nbe 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.Expressionofresults
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 dilu-
tion made during clarification and of the volume of the test sample.
4.3.2. Repeatability
r= 0,015xi
xi= concentration of inverted sugar in g/l per sample
4.3.3. Reproducibility
R= 0,058xi
xi= concentration of inverted sugar in g/l per sample
Relationbetweenthe volume of 0,1 M sodium thiosulphate solution ,
(n′−n )ml, and the quantity of reducing sugars in mg
Na2S2O3(ml 0,1 M)Reducing
sugars (mg)Differ-
enceNa2S2O3(ml 0,1 M)Reducing sugars
(mg)Differ-
ence
1 2,4 2,4 13 33,0 2,7
2 4,8 2,4 14 35,7 2,8
3 7,2 2,5 15 38,5 2,8
4 9,7 2,5 16 41,3 2,9
5 12,2 2,5 17 44,2 2,9
6 14,7 2,6 18 47,2 2,9
7 17,2 2,6 19 50,0 3,0
8 19,8 2,6 20 53,0 3,0
9 22,4 2,6 21 56,0 3,1
10 25,0 2,6 22 59,1 3,1
11 27,6 2,7 23 62,2
12 30,3 2,71990R2676 — EN — 18.03.2003 — 007.001 — 58
▼B
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-hydro-
chloric acid at 105 º C.
II. For testing and determination by high-performance liquid chro-
matography: the sucrose is separated ina columnof alkylamin e-
bonded silica and detected by refractometry. The result is quanti-
fied 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 inchapter 42,
point (f).
2. QUALITATIVE TESTING BY THIN-LAYER CHROMATO-
GRAPHY
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. Ovenwith regulationto 105 ± 2 ºC.
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 º 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 ina volumetric flask.
2.3.2. Obtaining the chromatogram
Place ona parallel lin e 2,5 cm from the bottom edge of the plate:
— 10 µl of sample
— 10 µl of standard.1990R2676 — EN — 18.03.2003 — 007.001 — 59
▼B
Place the plate inthe tan k, previously saturated with the vapour from
the mobile phase. Allow the mobile phase to migrate to within1 cm
of the top of the plate. Remove the plate and dry it in a current of
warm air. Repeat the migrationtwo more times, dryin g the plate
each time. Spray the plate uniformly with 15 ml of colouring agent
and place in the oven at 105 ºC for approximately five minutes.
2.4.Results
Saccharose and fructose appear as a deep blue spot on a white back-
ground: 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 columnfilled with the same phase,
5. an arrangement for insulating the guard column and analytical
columns or for maintaining their temperature (30 º 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 (CH
3CN).
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:
— For wines and musts: filter using a 0,45 µm membrane filter.
— For rectified concentrated musts: use the solution obtained by
diluting the rectified concentrated must to 40 % (m/v) as
described inthe chapter ‘Total acidity’, section5.1.2., an d
filtering it using a 0,45 µm membrane filter.
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
solutionan d the sample.
3.4.1. For wines and musts: calculate the concentration in g/l.
3.4.2. For rectified concentrated musts: letCbe the sucrose concentration
in g/l of the 40 % (m/v) solution of rectified concentrated must. The1990R2676 — EN — 18.03.2003 — 007.001 — 60
▼B
sucrose concentration in g/kg of the rectified concentrated must is
then: 2,5C.
3.5.Expressionofresults
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.1990R2676 — EN — 18.03.2003 — 007.001 — 61
▼B
7.GLUCOSEANDFRUCTOSE
1. DEFINITION
Glucose and fructose may be determined individually by an enzy-
matic 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 dinucleo-
tide 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 deter-
mined 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
— A spectrophotometer enabling measurements to be made at
340 nm, the wavelength at which absorption by NADPH is at a
maximum. Absolute measurements are involved (i.e. calibration
plots are not used but standardization is made using the extinc-
tion coefficient of NADPH), so that the wavelength scales of
and absorbence values obtained from the apparatus must be
checked.
If not available, a spectrophotometer using a source with a
discontinuous spectrum which enables measurements to be
made at 334 nm or at 365 nm may be used.
— Glass cells with optical path lengths of 1 cm or single-use cells.— Pipettes for use with enzymatic test solutions, 0,02, 0,05, 0,1,
0,2 ml.
4. REAGENTS
4.1.Solution 1: buffer solution (0,3 M triethanolamine, pH
7,6, 4 × 10
−3Mi nM g2+): dissolve 11,2 g triethanolamine hydro-
chloride ((C2H5)3N · HCl) and 0,2 g MgSO4·7 H2O in150 ml of
doubly distilled water, add about 4 ml of 5 M sodium hydroxide
(NaOH) solutionto obtaina pH value of 7,6 an d make up to 200 ml.
This buffer solutionmay be kept for four weeks at + 4 ºC.
4.2.Solution 2: nicotinamide adenine dinucleotide phosphate solution
(about 11,5 × 10−3M): dissolve 50 mg disodium nicotinamide
adenine dinucleotide phosphate in 5 ml of doubly distilled water.
This solutionmay be kept for four weeks at +4 ºC.1990R2676 — EN — 18.03.2003 — 007.001 — 62
▼B
4.3.Solution 3: adenosine 5 ′-triphosphate solution(about 81 × 10−3M):
dissolve 250 mg disodium adenosine 5 ′-triphosphate and 250 mg
sodium hydrogencarbonate (NaHCO3) in5 ml of doubly distilled
water.
This solutionmay be kept for four weeks at +4 ºC.
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 ºC.
4.5.Solution5: 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 ºC.
Note:All solutions used above are available commercially.
5. PROCEDURE5.1.Preparationofsample
Depending on the estimated amount of glucose + fructose per litre,
dilute the sample as follows:
Measurement at 340 and
334 nmMeasurement at
365 nmDilutionwith water Dilutionfactor F
up to 0,4 g/l 0,8 g/l — —
up to 4,0 g/l 8,0 g/l 1 + 9 10up to 10,0 g/l 20,0 g/l 1 + 24 25up to 20,0 g/l 40,0 g/l 1 + 49 50up to 40,0 g/l 80,0 g/l 1 + 99 100above 40,0 g/l 80,0 g/l 1 + 999 1 000
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 between20 an d 25 ºC.
Into two cells with 1 cm optical paths, place the following:
Reference cell Sample cell
Solution1 (4.1) (takento 20 ºC) 2,50 ml 2,50 ml
Solution2 (4.2) 0,10 ml 0,10 ml
Solution3 (4.3) 0,10 ml 0,10 ml
Sample to be measured 0,20 ml
Doubly distilled water 0,20 ml
Mix, and after about three minutes read off the absorbence of the
solutions ( A
1). Start the reactionby addin g:
Solution4 (4.4) 0,02 ml 0,02 ml
Mix; wait 15 minutes; read off the absorbence and check that the
reactionhas stopped after a further two min utes ( A2).
Add immediately:
Solution5 (4.5) 0,02 ml 0,02 ml
Mix; read off the absorbence after 10 minutes and check that the
reactionhas stopped after a further two min utes ( A3).
Calculate the differences in the absorbences:
A2−A1corresponding to glucose,1990R2676 — EN — 18.03.2003 — 007.001 — 63
▼B
A3−A2corresponding to fructose,
for the reference and sample cells.
Calculate the differences in absorbence for the reference cell ( ∆AR)
and the sample cell ( ∆AS) and then obtain:
for glucose: ∆AG=∆AS−∆AR
for fructose: ∆AF=∆AS−∆AR
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.Expressionofresults
5.3.1. Calculation
The general formula for calculating the concentrations is:
Cg=lðÞ ¼V/C2M
ε/C2d/C2ν/C21000∆A
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 inthe 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 ∆A
G
For fructose: C(g/l) = 0,420 ∆AF
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor F.
Note:
If the measurements are made at 334 or 365 nm, then the following
expressions are obtained:
— measurement at 334 nm: ε= 6,2 (mmole−1×l×c m−1)
For glucose: C(g/l) = 0,425 ∆AG
For fructose: C(g/l) = 0,428 ∆AF
— measurement at 365 nm: ε= 3,4 (mmole × l−1×c m−1)
For glucose: C(g/l) = 0,773 ∆AG
For fructose: C(g/l) = 0,778 ∆AF
5.3.2. Repeatability (r)
r= 0,056xi
5.3.3. Reproducibility (R)
R= 0,12 + 0,076 xi
xi= concentration of glucose or fructose in g/l.1990R2676 — EN — 18.03.2003 — 007.001 — 64
▼B
8.DETECTINGENRICHMENTOFGRAPEMUSTS,CONCENTRATED
GRAPE MUSTS, RECTIFIED CONCENTRATED GRAPE MUSTS
AND WINES BY APPLICATION OF NUCLEARMAGNETIC R ESO-
NANCEOFDEUTERIUM(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:
CH2DC H2OH CH3CHD OH
II I
CH3CH2OD HOD
III IV
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:
Parameters
Wine(D/H)I(D/H)II(D/H)WQR
— Natural
— Enriched:
— beet sugar
— cane sugar
/C148/C147/C147/C147/C149/C147/C147/C147/C150
 — maize sugar
(D/H)I: Isotope ratio associated with molecule I
(D/H)II: Isotope ratio associated with molecule II
(D/H)WQ: Isotope ratio of the water inthe win e.
R= 2(D/H)II/(D/H)Iexpresses the relative distributionof deuterium
inmolecules I an d II; Ris measured directly from the h-inten-
sities of the signals and then R=3 hII/hI.
(D/H)Imainly 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)IIrepresents 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.
(D/H)WQrepresents 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, (D/H)WQand by
determining the ratio13C/12C inthe ethan ol.1990R2676 — EN — 18.03.2003 — 007.001 — 65
▼M5
▼B
3. PREPARATION OF THE SAMPLE FOR ANALYSIS
3.1.Extractionofethanolandwaterinthewine
Note:
Any method for ethanol extraction can be used as long as 98 to
98,5 % of the total alcohol inthe win e is recovered ina distillate
which contains 92 to 93 % mas (95 % vol).
3.1.1. Apparatus and reagents
Apparatus for extracting ethanol (Figure 1) comprising:
— electric heating mantle with voltage regulator,
— one-litre round-bottom flask with ground glass neck joint,
— Cadiot column with rotating band (moving part in Teflon),
— 125 ml conical flasks with ground glass neck joints,— 125 and 60 ml bottles with plastic stoppers.
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 thanthe
nearest 0,05 % vol.
3.1.2.2. Extractionof the ethan ol
Introduce a homogeneous sample of 500 ml of wine of alcoholic
strengthtvinto the flask in the distillation apparatus with a constant
reflux ratio of about 0,9. Place a 125 ml ground conical flask, cali-
brated beforehand, to receive the distillate. Collect the boiling liquid
between78,0 an d 78,2 ºC, i.e. approximately 40 to 60 ml. If the
temperature exceeds 78,5 ºC, discontinue collection for five minutes.
When the temperature returns to 78 ºC, recommence collecting the
distillate until 78,5 ºC and repeat this operation until the tempera-
ture, 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 inthe win e to be recovered ina 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 estab-
lished.
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.
Note:
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.1990R2676 — EN — 18.03.2003 — 007.001 — 66
▼B
Figure 1
Distillationapparatus for the extractionof ethan ol
The strength by mass of the alcohol is given by
tD
m¼p/C0p0
p/C2100
3.2.Fermentationofmusts,concentratedmustsandrectifiedconcen-
tratedmusts
3.2.1. Apparatus and reagents
Tartaric acid
DIFCO Bacto Yeast NitrogenBase without amin o acidsActive dry yeasts (Saccharomyces cerevisæ) .1990R2676 — EN — 18.03.2003 — 007.001 — 67
▼B
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 conver-
sion of fermentable sugars into ethanol should be greater than 98 %.
3.2.2. Procedure
3.2.2.1. Musts
— Fresh musts
Place one litre of must, whose concentration of fermentable
sugars has been previously determined, in the fermentation
vessel. Add 1 g of dry yeast reactivated beforehand. Insert device
to keep it airtight. Allow fermentation to proceed at around 20 ºCuntil the sugar is used up. After determining the alcoholic
strength of the fermentation product and calculating the rate of
conversion of sugars into alcohol, the fermented liquid is centri-
fuged and distilled to extract the ethanol.
— Musts with fermentation prevented by the addition of sulphur
dioxide
De-sulphite a quantity of must slightly in excess of one litre (i.e.
1,2 litre) by bubbling nitrogen through the must in a water bath
at 70 to 80 ºC under reflux until the total sulphur dioxide content
is less than200 mg/l. Take care to see that the must is n ot
concentrated through evaporation of water by using effective
cooling. Place 1 litre of de-sulphited must in the fermentation
vessel and continue as described for fresh must.
Note:
If potassium metabisulphite is used to sulphite the must, 0,25 ml
of sulphuric acid ( ρ
20= 1,84 g/ml) per gram of metabisulphite
used per litre of must should be added to the must before de-
sulphiting.
3.2.2.2. Concentrated musts
PlaceVml 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.
Note:Retain50 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 (D/H)
WQ. The extraction of the water contained in the must may
be very simply carried out by azotropic distillation using toluene.
3.3.PreparationofalcoholsampleforNMRmeasurement
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:
Directorate-General for Science, Research and Development,
Community Bureau of References,200 rue de la Loi, B-1049 Brussels.
3.3.2. Procedure
— 15 mm diameter NMR probe:
ina previously weighed bottle, collect 7 ml alcohol obtain ed as
in 3.1.2 and weigh it to the nearest 0,1 mg (m
A); thentake a 3 ml1990R2676 — EN — 18.03.2003 — 007.001 — 68
▼B
sample of the internal standard (TMU) and weigh to the nearest
0,1 mg (mst). Homogenize by shaking.
— 10 mm diameter NMR probe:
3,2 ml of alcohol and 1,3 ml TMU are sufficient.
Depending on the type of spectrometer and probe used (cf.
section 4), add a sufficient quantity of hexafluorobenzene as a
field-frequency stabilization substance (lock):
Spectrometer10 mm
probe15 mm
probe
7,05 T 150 µl 200 µl
9 , 4T 3 5µ l 5 0µ l
3.4.Preparation of a water sample for the NMRmeasurement, for
thepurposeofapossibledeterminationofitsisotoperatio
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 standar-
dize the ratio Trv (5.2) for each series of wines examined.
4. RECORDING OF2H NMR SPECTRA OF THE ALCOHOL AND
THE WATER
Determination of isotope parameters.
4.1.Apparatus
— NMR spectrometer fitted with a specific ‘deuterium’ probe tuned
to the characteristic frequency Voof the field Bo (e.g. for Bo= 7,05 T, Vo= 46,05 MHz and for Bo= 9,4 T, Vo= 61,4 MHz)
having a proton decoupling channel (B2) and field-frequency
stabilization channel (lock) at the fluorine frequency.
The resolutionmeasured onthe spectrum, tran sformed without
exponential multiplication (i.e. LB = 0) (Figure 2b) and
expressed by the half-width of the methyl and methylene signals
of ethanol and the methyl signal of TMU, must be less than0,5 Hz. The sensitivity, measured with an exponential multi-
plying factor LB equal to 2 (Figure 2a) must be greater than or
equal to 150 for the methyl signal of ethanol of alcoholic
strength 95 % vol (93,5 % mas).
Under these conditions, the confidence interval for the measure-
ment of the signal height, calculated for a 97,5 % probability
(one-sided test) and 10 repetitions of the spectrum, is 0,35 %.
— Automatic sample changer (possibly)
— Data-processing software— 15 mm or 10 mm sample tubes according to spectrometer perfor-
mance.
4.2.Standardizationofspectrometer andchecks
4.2.1. Standardization
Carry out customary standardization for homogeneity and sensitivity
according to the manufacturer's specifications.1990R2676 — EN — 18.03.2003 — 007.001 — 69
▼B
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:
— C: alcohol from cane sugar or maize,
— V: wine spirit,
— B: beet alcohol.
These samples are supplied by the Community Bureau of Refer-
ences.
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)Iand (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 givenfor those parameters for the three stan dard1990R2676 — EN — 18.03.2003 — 007.001 — 70
▼B
alcohols by the Community Bureau of References. If they are not,
carry out the checks again.
4.3.ConditionsforobtainingNMRspectra
Place a sample of alcohol prepared as in3.3 (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:
— a constant probe temperature (e.g. 302 K);
— acquisitiontime of at least 6,8 s for 1 200 Hz spectral width
(16K memory) (i.e. about 20 ppm at 61,4 MHz or 27 ppm at
46,1 MHz);
— 90º pulse;
— adjustment of acquisition time: its value must be of the same
order as the dwell time;
— parabolic detection: fix the offset 01 between the OD and CHD
reference signals for ethanol and between the HOD and TMU
reference signals for water;
— determine the value of the decoupling offset 02 from the proton
spectrum measured by the decoupling coil on the same tube.
Good decoupling is obtained when 02 is located in the middle
of the frequency interval existing between the CH3-a ndC H2-
groups. Use the wide band decoupling mode.
For each spectrum, carry out a number of accumulations NS suffi-
cient 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
onthe types of spectrometer an d probe used (cf. section4). Exam-
ples of the possible choices are:
Spectrometer 10 mm probe 15 mm probe
7,05 T NS = 304 NS = 200
9,4 T NS = 200 NS = 128
5. EXPRESSION OF RESULTS
5.1.Ethanol
For each of the 10 spectra (see NMR spectrum for ethanol, Figure
2a), determine:
—R¼3hII
hI¼3/C2height of signal II CH 3CHD OH ðÞ
height of signal I CH 2DC H2OH ðÞ
—D=HðÞI¼1;5866 /C2TI/C2mst
mA/C2D=HðÞst
tD
m
—D=HðÞH¼2;3799 /C2TII/C2mst
mA/C2D=HðÞst
tD
m
with
—TI¼height of signal I CH 2DC H2OH ðÞ
height of signal of internal standard TMU ðÞ
—TII¼height of signal II CH 3CHD OH ðÞ
height of signal of internal standard TMU ðÞ
—mstand mA, see 3.3.2.
—tD, see 3.1.2.3.
— (D/H)st= isotope ratio of internal standard (TMU) indicated on
the bottle supplied by the Community Bureau of References.
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 reason-
able approximationif applicable (Figure 2b).1990R2676 — EN — 18.03.2003 — 007.001 — 71
▼B
5.2.Water
Whenthe isotope ratio of water is determin ed by NMR from the
water-TMU mixture, the following relationship is used:
—D=HðÞQ
W¼0;9306 /C2TIV/C2m0
st
m0E/C2D=HðÞst
with
—TIV¼Area of HOD ðÞsignal of the water extracted from the wine
Area of the signal from the internal standard TMU ðÞ
—m′standm′E, see 3.4.2.
— (D/H)st= isotope ratio of the internal standard (TMU) indicated
on the bottle supplied by the Community Bureau of References.
5.3. For each of the isotope parameters, calculate the average of 10 deter-
minations 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 devia-
tion, 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 (D/H)iX
measbe the measured value and (D/H)iX
corrbe the corrected
value. This will give:
(D/H)iX
corr= (D/H)iB
st+α[ (D/H)iX
meas− (D/H)iB
meas]
where
α¼D=HðÞVst
i/C0D=HðÞBst
i
D=HðÞVmeas
i/C0D=HðÞBmeas
i
Example:
Standard samples supplied and standardized by the Community
Bureau of References:
(D/H)IV
st= 102,0 ppm (D/H)IB
st= 91,95 ppm
Standard samples measured by the laboratory:
(D/H)IV
meas= 102,8 ppm (D/H)IB
meas= 93,0 ppm
Suspect uncorrected sample: (D/H)IX
meas= 100,2 ppm
α= 1,0255 and (D/H)IX
corr= 99,3 ppm are calculated.
6. INTERPRETATION OF RESULTS
Compare the value RXobtained for the Rratio of the suspect sample
with the ratios obtained for the control wines. If RXdiffers by more
than two standard deviations from the average RTvalue obtained for
the control wine, adulteration may be assumed.
6.1.Additionofbeetsugar,canesugarormaizeglucose
6.1.1. Wines
RXhigher than RT: beet sugar is assumed to have beenadded.
RXless than RT: cane sugar or maize sugar is assumed to have been
added.
Note that (D/H)IXand (D/H)WQXare increased.
Consider (D/H)IX:
— — Beet sugar is assumed to have beenadded: (D/H)IXof the
suspect sample is lower than(D/H)IT, the average value obtained
from the control samples, by more than one standard deviation
— Cane sugar or maize sugar is assumed to have been added:1990R2676 — EN — 18.03.2003 — 007.001 — 72
▼B
(D/H)IXis greater than(D/H)ITby more than one standard devia-
tion
— Calculation of enrichment E expressed in % vol of ethanol:
— Additionof beet sugar:
E % vol ¼tVD=HðÞT
I/C0D=HðÞX
I
D=HðÞT
I/C0D=HðÞB
I
where
(D/H)IB= isotope ratio for the locationI of the beet
alcohol;
(D/H)IB= 92,5 (1)
tV= alcoholic strength of the analysed wine (X).
— Additionof can e sugar or maize sugar:
E % vol ¼tVD=HðÞX
I/C0D=HðÞT
I
D=HðÞC
I/C0D=HðÞT
I
where
(D/H)IC= isotope ratio for the locationI of the can e sugar
or maize sugar;
(D/H)IC= 110,5(1)
tV= alcoholic strength of the analysed wine (X)
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 exam-
ined 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)IandRare changed less than when only one
type of sugar is added.
(D/H)IIis higher, as is (D/H)WQ.
These additions may be confirmed by determining the13C/12C ratio
of the ethanol by mass spectrometry; in that case the ratio is higher.1990R2676 — EN — 18.03.2003 — 007.001 — 73
(1) These values are given pending the creation of a Community data bank of such values.
▼B
9.ASHCONTENT
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 ºC
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
og). Evaporate on the boiling water-bath, and
heat the residue on the hot-plate at 200 ºC 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 ºC. 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 ºC for 10 minutes.
If combustion (oxidation) of the carbonized particles is not complete,
repeat the operations of washing the carbonized particles, evapora-
tion 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 ( P1g).
The weight of the ash inthe sample (20 ml) is then P=(P1−Po)g .
5. EXPRESSION OF RESULTS
5.1. Method of calculation
The weight Pof the ash ingrams per litre will be givento two
decimal places by the expression: P=5 0p1990R2676 — EN — 18.03.2003 — 007.001 — 74
▼B
10.ALKALINITYOFTHEASH
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 standar-
dized 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)
wherenml is the volume of 0,1 M sodium hydroxide used.1990R2676 — EN — 18.03.2003 — 007.001 — 75
▼B
11.CHLORIDES
1. PRINCIPLE
Chlorides are determined directly in the wine by potentiometry using
anAg/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 solutionof n itrate potassium as
electrolyte.
2.4. Microburette graduated in1/100 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, AgNO3are diluted ina 10 % (v/v) alcohol solutionan d
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 frac-
tions 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 forapproximately 30 seconds before reading the corresponding milli-
volts. 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
onthe curve obtain ed.
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 determi-
nation 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 RESULTS5.1.Calculations
Ifnrepresents the number of millilitres of silver nitrate titrating
solution, the chloride content in the tested liquid is:
20 ×nexpressed as milligrams of Cl per litre,
0,5633 × nexpressed as milliequivalents per litre,
32,9 ×nexpressed as milligrams of sodium chloride per litre.1990R2676 — EN — 18.03.2003 — 007.001 — 76
▼B
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 cylind-
rical 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 titrationthe 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:
V¼V
0þ∆Vi∆∆E1
∆∆E1þ∆∆E2
Where:
V= volume of titrating solution at equivalence point;
V′= volume of titrating solution before the largest poten-
tial change;
∆Vi= constant volume of the increments of titrating solu-
tion, i.e. 0,2 ml;
∆∆E1= second difference in potential before the largest
potential change;
∆∆E2= second difference in potential after the largest poten-
tial change.1990R2676 — EN — 18.03.2003 — 007.001 — 77
▼B
Example:
Volume of AgNO3titrating
solutionE potential
inmVDifference
∆ESecond
difference
∆∆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 ( ∆E= 44 mV) occurs inthis
interval. The volume of silver nitrate titrating solution consumed to
measure the chlorides inthe test sample is:
V¼1;6þ0;222
22þ10¼1;74 ml1990R2676 — EN — 18.03.2003 — 007.001 — 78
▼B
12.SULPHATES
1. PRINCIPLE
1.1.Reference method
Precipitation of barium sulphate and weighing. The barium phos-
phate precipitated in the same conditions is eliminated by washing
the precipitate inhydrochloric acid.
Inthe case of musts or win e rich insulphur dioxide, prior de-
sulphiting by boiling in an airtight vessel is recommended.
1.2.Quicktestmethod
Wines are classified into several categories using the so-called limits
method, based onthe precipitationof barium sulphate usin g a
barium iontitran t.
2. REFERENCE METHOD
2.1.Reagents
2.1.1. 2 M solutionof hydrochloric acid.
2.1.2. Barium chloride solutionof 200 g/l of BaCl2·2 H2O.
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 solu-
tionat 200 g/l. Stir with a glass stirrer; rin se 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 ºC
until fully evaporated. The dried precipitate is calcined several times
briefly over a flame until a white residue is obtained. Leave to cool
ina desiccator an d weigh.
Letm= the mass inmilligrams of barium sulphate obtain ed.
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 ( ρ20= 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. Deter-
mine the sulphates in a 40 ml sample as indicated in 2.2.1.
2.3.Expressionofresults
2.3.1. Calculations:
The sulphate content, expressed in milligrams per litre of potassium
sulphate, K2SO4is:
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/l1990R2676 — EN — 18.03.2003 — 007.001 — 79
▼B
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
3. QUICK TEST METHOD
3.1.Reagents
3.1.1. Barium chloride titrating solution
2,804 g of barium chloride, BaCl2·2 H2O and 10 ml of hydrochloric
acid ( ρ20= 1,15 to 1,18 g/ml) are dissolved inen ough water to
obtain one litre of solution, 1 ml of this solution precipitates a quan-
tity of sulphate ions equivalent to 2 mg of potassium sulphate.
3.1.2. Sulphuric acid ( ρ20= 1,84 g/ml), 1/10 (m/v) solution.
3.2.Procedure
Measure 10 ml of musts or wine into three test tubes; add to No 1:
3,5 ml, to No 2: 5 ml and to No 3: 10 ml of the barium chloride
solution. Shake and bring to the boil; leave to stand for one to two
hours. The liquid in each tube is decanted, filtered and divided into
two parts. To one part, add several drops of the dilute sulphuric acid
solution, to the other some drops of the barium chloride solution.
Examine each tube and note whether the liquid is clear or cloudy.
Interpretation of these observations is given in the table below.
Wine Barium chlorideFiltered wine +
diluted sulphuric
acidbarium chloride
solution
First test(ml) (ml) cloudy clear
10 3,5(less than0,7 g K2SO4/l)
clear cloudy
(more than0,7 g K2SO4/l)
Second test 10 5cloudy clear
(less than1 g K2SO4/l)
clear cloudy
(more than1 g K2SO4/l)
Third test 10 10cloudy clear
(less than2 g K2SO4/l)
clear cloudy
(more than2 g K2SO4/l)1990R2676 — EN — 18.03.2003 — 007.001 — 80
▼B
13.TOTALACIDITY
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 solutionpH 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 commer-
cially.
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 indistilled water. The calomel/saturated
potassium chloride electrode must be kept ina saturated potassium
chloride solution. A combined electrode is most frequently used: it
should be kept indistilled water.
4.4. Measuring cylinders 50 ml (wine), 100 ml (rectified concentrated
must).
5. PROCEDURE
5.1.Preparationofsample:
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.1990R2676 — EN — 18.03.2003 — 007.001 — 81
▼B
5.2.Potentiometric titration
5.2.1. Calibration of pH meter
The pH meter is now calibrated for use at 20 ºC, according to the
manufacturer's instructions, with the pH 7,00 buffer solution at 20 ºC.
5.2.2. Method of measurement
Into a measuring cylinder (4.4), introduce a volume of the sample,
prepared as described in5.1, equal to 10 ml inthe case of win e
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 un til the pH is equal to 7 at 20 ºC.
The sodium hydroxide must be added slowly and the solution stirred
continuously. Let nml be the volume of 0,1 M NaOH added.
5.3.Titrationwithindicator (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) an d a volume prepared as
in(5.1) equal to 10 ml inthe case of win e an d 50 ml inthe case of
rectified concentrated must. Add the 0,1 M sodium hydroxide solu-
tion (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) an d a volume of the
sample, prepared as described in5.1, equal to 10 ml inthe 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
inthe prelimin ary test above (5.3.1). Let nml be the volume of
0,1 M sodium hydroxide added.
6. EXPRESSION OF RESULTS6.1.Methodofcalculation
6.1.1. Wines
The total acidity expressed inmilliequivalen ts per litre is givenby:
A=1 0n.
It is recorded to one decimal placeThe total acidity expressed ingrams of tartaric acid per litre is given
by:
A′= 0,075A
It is recorded to one decimal place.
6.1.2. Rectified concentrated musts
— The total acidity expressed inmilliequivalen ts per kilogram of
rectified concentrated must is given by a=5n.
— The total acidity expressed inmilliequivalen ts per kilogram of
total sugars is givenby
A¼
500/C2n
P
P= % concentration (m/m) of total sugars.
It is recorded to one decimal place.
6.2.Repeatability(r) for titrationwith the in dicator:
r= 0,9 meq/litre
r= 0,07 g tartaric acid/litre
for white, rosé and red wines.1990R2676 — EN — 18.03.2003 — 007.001 — 82
▼B
6.3.Reproducibility(R) for titrationwith the in dicator (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/litre1990R2676 — EN — 18.03.2003 — 007.001 — 83
▼B
14.VOLATILEACIDITY
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
Titrationof the volatile acids separated from the win e by steam
distillationan d titrationof the distillate.
Carbondioxide is first removed from the win e.
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 insome coun tries to stabilize the
wines before analysis is present in the distillate. This must be deter-
mined and deducted from the acidity. The method of determination
is giveninsection7 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, stir-
ring continuously and boil for 10 minutes. Add 200 g sodium
chloride. Whencool, make up to on e litre.
3.8. Saturated solutionof sodium borate (Na
2B4O7· 10H2O), i.e. about
55 g/l at 20 ºC.
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 distillationcolumn ;
4. a condenser.
This equipment must pass the following three tests:
(a) Place 20 ml of boiled water inthe 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 solutioninthe flask.
Collect 250 ml of the distillate. Titrate with the 0,1 M sodium
hydroxide solution(3.2): the volume ofthis 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 solutioninthe 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 solutionadded must be less
thanor equal to 1,0 ml (i.e. n ot more than0,5 % of lactic
acid is distilled).
Any apparatus or procedure which passes these tests satisfac-
torily fulfils the requirements of official international
apparatus or procedures.1990R2676 — EN — 18.03.2003 — 007.001 — 84
▼B
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.Steamdistillation
Place 20 ml of win e, freed from carbondioxide as in5.1, inthe
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) usin g two
drops of phenolphthalein (3.3) as indicator. Let nml be the volume
of sodium hydroxide used.
Add four drops of 1/4 dilute hydrochloric acid (3.4), 2 ml starch
solution(3.3) an d 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 colora-
tionreappears. Titrate the combin ed sulphur dioxide with the
0,005 M iodine solution (3.5). Let n″ml be the volume used.
6. EXPRESSION OF RESULTS
6.1.Methodofcalculation
The volatile acidity, expressed in milliequivalents per litre to one
decimal place, is givenby:
A=5(n− 0,1n′− 0,05n″).
The volatile acidity, expressed ingrams of acetic acid per litre to
two decimal places, is givenby:
0,300 (n− 0,1n′− 0,05n″).
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.Winewithsorbicacidpresent
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 onwhich the determin ationof volatile acidity
was carried out. Inthis distillate, the salicylic acid is determin ed by1990R2676 — EN — 18.03.2003 — 007.001 — 85
▼B
a comparative colorimetric method. It is deducted from the acidity of
the volatile acidity distillate.
7.2.Reagents
7.2.1. Hydrochloric acid (HCl) ( ρ20= 1,18 to 1,19 g/l).
7.2.2. Sodium thiosulphate, (Na2S2O3·5 H2O) ina 0,1 M solution .
7.2.3. 10 % (m/v) solutionof iron(III) ammon ium sulphate (Fe2(SO4)3·
(NH4)2SO4· 24H2O).
7.2.4. 0,01 M solutionof 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) an d 1 ml of the iron(III) ammo-
nium 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) un til the violet coloration
obtained has the same intensity as that of the conical flask
containing the wine distillate.
Letn″ml be the volume of solutionadded from the burette.
7.3.3. Correction to the volatile acidity
Subtract the volume 0,1 × n/tripleprimeml from the volume nml of 0,1 M
sodium hydroxide solutionused to titrate the acidity of the distillate
during the determination of volatile acidity.1990R2676 — EN — 18.03.2003 — 007.001 — 86
▼B
15.FIXEDACIDITY
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:
— milliequivalents per litre,
— grams of tartaric acid per litre.1990R2676 — EN — 18.03.2003 — 007.001 — 87
▼B
16.TARTARICACID
1. PRINCIPLE OF METHODS1.1.Reference method
Tartaric acid is precipitated inthe form of calcium (±)tartrate an d
determined gravimetrically. This determination may be completed
by a volumetric procedure for comparison. The conditions for preci-
pitation (pH, total volume used, concentrations of precipitating ions)
are such that precipitationof the calcium (±)tartrate is complete
whereas the calcium D(−) tartrate remains in solution.
Whenmesotartaric acid has beenadded to the win e, which causes
the precipitationof the calcium (±)tartrate to be in complete, it must
first be hydrolysed.
1.2.Usualmethod
The tartaric acid, separated using an ion exchange column, is deter-
mined colorimetrically in the eluate by measurement of the red
colour produced onreactionwith van adic acid. The eluate also
contains lactic and malic acids which do not interfere.
2. REFERENCE METHOD2.1.Gravimetric method
2.1.1. Reagents
2.1.1.1. Calcium acetate solution containing 10 g of calcium per litre:
calcium carbonate (CaCO
3)…........................ 25 g
acetic acid glacial (CH3COOH) ( ρ20= 1,05 g/ml) …... 40 ml
water to …........................................... 1 litre
2.1.1.2. Calcium (±)tartrate, crystallized: CaC4O6H4·4 H2O:
Place 20 ml of L(+) tartaric acid solution(5 g/l) in to 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 ºC. Using the quantities of reagent indicated above, about
340 mg of crystallized calcium (±)tartrate is obtained.
Store ina stoppered bottle.
2.1.1.3. Precipitationsolution(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).1990R2676 — EN — 18.03.2003 — 007.001 — 88
▼B
Filter the liquid and precipitate through a weighed sintered glass
crucible of porosity No 4 fitted ona cleanvacuum flask. Rin se the
vessel inwhich precipitationtook place with the filtrate to en sure
that all precipitate is transferred.
Dry to constant weight in an oven at 70 ºC. Weigh. Let pbe the
weight of crystallized calcium (±)tartrate (CaC4O6H4·4 H2O)
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 inthe win e.
The quantity of tartaric acid per litre of wine, expressed in milliequi-
valents 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.Comparativevolumetric analysis
2.2.1. Reagents
2.2.1.1. Hydrochloric acid (HCl) (1:5 v/v) ( ρ
20= 1,18 to 1,19 g/ml)
2.2.1.2. EDTA solution, 0,05 M:
EDTA (ethylenediaminetetraacetic acid disodium
salt:
(C10H14N2O8Na2·2 H2O) ….......................... 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·3 H2O)… ............... 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 onthe vacuum flask an d 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.1990R2676 — EN — 18.03.2003 — 007.001 — 89
▼B
Add 5 ml of 40 % sodium hydroxide solution(2.2.1.3) an d 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 milliequi-
valents 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.
3. USUAL METHOD
3.1.Reagents
3.1.1. For preliminary treatment of the wine
3.1.1.1. Acetic acid (CH3COOH; ρ20= 1,05 g/ml), diluted 30 % (v/v).
3.1.1.2. A strongly basic-ion exchange resin (e.g. anion exchange resin of
Merck 20-50 mesh basicity strength III) in acetate form. Prepare a
suspension of the ion exchange resin (about100 g) in 200 ml 30 %
acetic acid (3.1.1.1). Leave incon tact for at least 24 hours before
use. Keep the ionexchan ge resininthe 30 % acetic acid for later
determinations.
3.1.1.3. Acetic acid (CH3COOH; ρ20= 1,05 g/ml), diluted 0,5 % (v/v).
3.1.1.4. Sodium sulphate solution7,1 g per 100 ml (0,5 M)
Dissolve 71 g of anhydrous sodium sulphate (Na2SO4) indistilled
water and make up to 1 000 ml with distilled water.
3.1.2. For the determination of tartaric acid
3.1.2.1. Sodium acetate solution(CH3CO Na) 27 % (m/v):
Dissolve 270 g of anhydrous sodium acetate (CH3COONa) in
distilled water and make up to 1 000 ml.
3.1.2.2. Vanadic reagent:
Dissolve 10 g of ammonium metavanadate (NH4VO3) in150 ml of
1 M sodium hydroxide solution(3.1.2.10). Tran sfer this solutionto
a 500 ml volumetric flask and add 200 ml of the 27 % sodium
acetate solution(3.1.2.1). Make up to 500 ml with distilled water.
3.1.2.3. 1 M H2SO4solution
3.1.2.4. 0,5 M H2SO4solution
3.1.2.5. 0,05 M H2SO4solution
3.1.2.6. 0,05 M periodic acid solution:
Introduce 10,696 g of sodium periodate, NaIO4, and 50 ml of 0,5 M
sulphuric acid (3.1.2.4) into a 1 000 ml volumetric flask and make
up to 1 000 ml with distilled water.
3.1.2.7. Glycerol solution, 10 % (m/v):
Place 10 g of glycerol, C3H8O3, into a 100 ml volumetric flask and
make up to 100 ml with distilled water.
3.1.2.8. Sodium sulphate solution, 7,1 g per 100 ml (see 3.1.1.4).
3.1.2.9. Tartaric acid solution, 1 g/l:
Introduce 0,5 g of tartaric acid and 6,66 ml of 1 M sodium hydro-
xide solution (3.1.2.10) into a 500 ml volumetric flask and make up
to 500 ml with the 7,1 % sodium sulphate solution(3.1.1.4).
3.1.2.10. Sodium hydroxide solution, NaOH, 1 M.1990R2676 — EN — 18.03.2003 — 007.001 — 90
▼B
3.2.Apparatus
3.2.1. Glass column of 10 to 11 mm internal diameter and approximately
300 mm long fitted with a drain tap.
3.2.2. Spectrophotometer enabling absorbence measurements to be made at
a wavelength of 490 nm, having cells with a 10 mm optical path.
3.3.Procedure
3.3.1. Preparation of the ion exchange column
Place a glass wool plug inthe glass columnabove the draintap
(3.2.1). Soak this plug with distilled water. Pour into the column
10 ml of the suspension of ion exchange resin in acetateform
(3.1.1.2). Allow to settle. Place a plug of glass wool ontop of the
resin (to prevent it being disturbed during subsequent washings).
The ion exchange resin must only be used for one operation.
3.3.2. Separation of organic acids
With the tap open, allow the acetic acid solution to flow down the
columnto withinapproximately 2 to 3 mm of the upper glass wool
plug.
Add 10 ml of the 0,5 % acetic acid solution(3.1.1.3) an d allow the
liquid to drainagaindownto the same level as the previous opera-
tion. Repeat this washing operation four more times.
After the last wash, close the tap, and pour 10 ml of the wine or
must on to the ion exchange resin. Allow it to flow a drop at a
time (average rate one drop per second) and stop the flow just above
the ion exchange resin. Once more add 10 ml of the 0,5 % acetic
acid solution(3.1.1.3) to the column , allow to drainat the same
rate as previously and subsequently wash seven times in the same
way using 10 ml of water each time. During the last washing, close
the tap as soonas the liquid level is just above the upper glass wool
plug.
Elute the acids absorbed onthe ionexchan ge resinwith a 7,1 %
sodium sulphate solution(3.1.1.4). Collect the eluate ina 100 ml
graduated flask up to the calibrationmark.
3.3.3. Determination of tartaric acid
3.3.3.1. Wines with no added mesotartaric acid
Place 20 ml of eluate intwo con ical flasks aandb.
Use flask afor the determination and flask b, inwhich the tartaric
acid has been destroyed by periodic acid, for the blank experiment.
Introduce into flask a:
— 2 ml of M H2SO4(3.1.2.3),
— 5 ml of 0,05 M H2SO4(3.1.2.5),
— 1 ml of 10 % glycerol (3.1.2.7).
Introduce into flask b:
— 2 ml of M H2SO4(3.1.2.3),
— 5 ml of 0,05 M periodic acid solution(3.1.2.6).
Wait 15 minutes; add1 ml of 10 % glycerol (3.1.2.7) to destroy the excess periodic acid.Wait two minutes.Then, while stirring, pipette 5 ml of the vanadic reagent (3.1.2.2)
first into flask band then immediately afterwards into flask a.
Immediately start a timer and pour the contents of flasks aandb
into the spectrophotometer cells. After 90 seconds, record the
absorbence at 490 nm of the liquid from flask a(determination)
with respect to liquid b (blank).
Solutions containing high levels of tartaric acid that give absorben-
cies that are above the top standard should be diluted with 7,1 %
sodium sulphate and then measured as before.1990R2676 — EN — 18.03.2003 — 007.001 — 91
▼B
3.3.3.2. 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 described for the reference method.
After cooling, the contents of the conical flask are poured into the
top of the ion exchange column, followed by rinsing water (5 ml,
twice). Continue as indicated above.
The mesotartaric acid is included as tartaric acid in the final result.
3.3.4. Plotting the calibration curve
Pipette 10, 20, 30, 40 and 50 ml of the 1 g/l tartaric acid solution
(3.1.2.9) into graduated 100 ml flasks and make up to 100 ml with
the 7,1 % sodium sulphate solution (3.1.1.4). The concentrations of
these solutions correspond to wine eluates containing 1, 2, 3, 4 and
5 g/l of tartaric acid.
Introduce into two conical flasks aandb20 ml of these solutions
and continue as described above for the wine eluate.
A graph of the absorbences of these solutions as a function of their
tartaric acid concentration is a straight line curving slightly inwards
towards the origin. If necessary, draw this part of the curve more
carefully using determinations on solutions of accurately known
concentrations below 1,0 g/l.
3.3.5. Expression of results
The absorbence measured for the eluate is found on the calibration
curve and gives the tartaric acid concentration in the wine in grams
per litre.
The result is expressed to one decimal place.1990R2676 — EN — 18.03.2003 — 007.001 — 92
▼B
17.CITRICACID
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 dehy-
drogenase (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+inthese reaction s 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 solutionpH 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 (ZnCl280 mg in
100 ml H2O) solutionan d make up to 100 ml with doubly distilled
water.
2.2. Reduced nicotinamide adenine dinucleotide (NADH) solution
(approximately 6 × 10−3M): dissolve 30 mg NADH and 60 mg
NaHCO3in6 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 ºC.
2.4. Citrate-lyase (CL, 5 mg protein/ml): dissolve 168 mg lyophilisate in
1 ml ice-cold water. This solutionremain s stable for at least a week
at 4 ºC 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. calibra-
tion curves are not used but standardization is made by
consideration of the extinction coefficient of NADH), the wave-
length 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.1990R2676 — EN — 18.03.2003 — 007.001 — 93
▼B
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 concentra-
tion lies between 20 and 400 mg/l (i.e. between 5 and 80 µg of
citrate inthe 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:
Reference cell
(ml)Sample cell
(ml)
Solution2.1 1,00 1,00
Solution2.2 0,10 0,10
Sample to be measured - 0,20
Doubly distilled water 2,00 1,80
Solution2.3 0,02 0,02
Mix, and after about five minutes read the absorbence of the solu-
tions in the reference and sample cells ( A1).
Add:
Solution2.4 0,02 ml 0,02 ml
Mix; wait until the reaction is completed (about five minutes) and
read the absorbences of the solutions in the reference and sample
cells (A2).
Calculate the absorbence difference ( A2−A1) for the reference and
sample cells, ∆ARand∆AS.
Finally, calculate the difference between those differences:∆A=∆A
s−∆AR
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.Methodofcalculation
The general formula for calculating the concentration in mg/l is:
C¼V/C2M
ε/C2d/C2ν/C2∆A
whereV= volume of test solutioninml (here 3,14 ml)
ν = volume of the sample inml (here 0,2 ml)
M = molecular mass of the substance to be determined1990R2676 — EN — 18.03.2003 — 007.001 — 94
▼B
(here, for anhydrous citric acid, M= 192,1)
d = optical path inthe cell incm (here, 1 cm)
ε = absorption coefficient of NADH, (at 340 nm, ε
= 6,3 mmol−1×l×c m−1),
so that
C= 479 × ∆A
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor.
Note:At 334 nm: C= 488 × ∆A(= 6,2 m mol
−1×l×c m−1).
At 365 nm: C= 887 × ∆A(= 3,4 m mol−1×l×c m−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.1990R2676 — EN — 18.03.2003 — 007.001 — 95
▼B
18.LACTICACID
1. PRINCIPLE OF THE METHOD
Total lactic acid (L-lactate and D-lactate) is oxidized by nicotina-
mide adenine dinucleotide (NAD) to pyruvate in a reaction
catalysed by L-lactate dehydrogenase (L-LDH) and D-lactate dehy-
drogenase (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 formationof pyruvate.
In the presence of L-glutamate, the pyruvate is transformed into L-
alanine in a reaction catalysed by glutamate pyruvate transaminase(GPT):
(1)L-lactate + NAD
+
pyruvate + NADH + H+
(2)D-lactate + NAD+
pyruvate + NADH + H+
(3)Pyruvate + L-glutamate
 L-alanine + α-ketogluta-
rate
The amount of NADH formed, measured by the increase in absorb-
ence 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.Usualmethod
The lactic acid, separated by passage through anionexchan ge 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 solutionwill remainstable for at least 12 weeks at 4 ºC.
2.1.2. Nicotinamide adenine dinucleotide (NAD) solution, approximately
40 × 10−3M: dissolve 900 mg of NAD in30 ml of doubly distilled
water. This solutionwill remainstable for at least four weeks at 4 ºC.
2.1.3. Glutamate pyruvate transaminase (GPT) suspension, 20 mg/ml. The
suspension remains stable for at least a year at 4 ºC.
2.1.4. L-lactate dehydrogenase (L-LDH) suspension, 5 mg/ml. This suspen-
sionremain s stable for at least a year at 4 ºC.
2.1.5. D-lactate dehydrogenase (D-LDH) suspension, 5 mg/ml. This
suspension remains stable for at least a year at 4 ºC.
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.1990R2676 — EN — 18.03.2003 — 007.001 — 96
▼B
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. calibra-
tion curves are not used, but standardization is made by
consideration of the extinction coefficient of NADH), the wave-
length 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.Preparationofthesample
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:
— 100 mg/l and 1 g/l, dilute 1/10 with doubly distilled water,
— 1 g/l and 2,5 g/l, dilute 1/25 with doubly distilled water,
— 2,5 g/l and 5 g/l, dilute 1/50 with doubly distilled water.
2.4.Procedure
2.4.1. Determination of total lactic acid
The buffer solutionmust be at a temperature between20 an d 25 ºC
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
inthe optical path) or with water as the stan dard.
Place the following in the cells having 1 cm optical paths:
Reference
cell
(ml)Sample
cell
(ml)
Solution2.1.1 1,00 1,00
Solution2.1.2 0,20 0,20
Doubly distilled water 1,00 0,80
Suspension 2.1.3 0,02 0,02
Sample to be measured — 0,20
Mix using a glass stirrer or a rod of synthetic material with a flat-
tened end; after about five minutes, measure the absorbences of the
solutions in the reference and sample cells ( A1).
Add 0,02 ml of solution2.1.4 an d 0,05 ml of solution2.1.5, homo-
genize, wait for the reaction to be completed (about 30 minutes) and
measure the absorbences of the solutions in the reference and sample
cells (A2).
Calculate the differences ( A2−A1) inthe absorben ces of the solu-
tions in the reference and sample cells, ∆ARand∆AS.
Finally, calculate the difference between those differences:
∆A=∆AS−∆AR
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 A1and 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 ( A2).1990R2676 — EN — 18.03.2003 — 007.001 — 97
▼B
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 ( A3).
Calculate the differences ( A2−A1) for L-lactic acid and ( A3−A2) for
D-lactic acid between the absorbences of the solutions in the refer-
ence and sample cells, ∆ARand∆AS.
Finally, calculate the difference between those differences:∆A=∆A
S−∆AR
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.Expressionofresults
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:
C¼V/C2M
ε/C2d/C2ν/C21000/C2∆A
where
V= volume of test solutioninml (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 inml (here 0,2 ml)
M= molecular mass of the substance to be determined (here, for
DL-lactic acid, M= 90,08)
d= optical path inthe cell incm (here, 1 cm)
ε= absorption coefficient of NADH, (at 340 nm, ε= 6,3 mmol−1
×l×c m−1).
2.5.1.1. Total lactic acid and D-lactic acid
C= 0,164 × ∆A
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor.
Note:Measurement at 334 nm: C= 0,167 × ∆A,(ε= 6,2 m mol
−1×l×
cm−1).
Measurement at 365 nm: C= 0,303 × ∆A,(ε= 3,4 m mol−1×l×
cm−1).
2.5.1.2. L-lactic acid
C= 0,160 × ∆A
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor.
Note:Measurement at 334 nm: C= 0,163 ∆A,(ε= 6,2 m mol
−1×l×c m−1).
Measurement at 365 nm: C= 0,297 ∆A,(ε= 3,4 m mol−1×l×c m−1).
2.5.2. Repeatability (r)
r= 0,02 + 0,07 xig/l
xiis the lactic acid concentration in the sample in g/l.
2.5.3. Reproducibility (R)
R= 0,05 + 0,125 xig/l
xiis the lactic acid concentration in the sample in g/l.1990R2676 — EN — 18.03.2003 — 007.001 — 98
▼B
3. USUAL METHOD
3.1.1. For preliminary treatment of the wine
Refer to the chapter ‘Tartaric acid’, usual method, section3.1.1.
3.1.2. For the determination of lactic acid
3.1.2.1. 0,1 M solutionof cerium (IV) sulphate in0,35 M sulphuric acid:
Keeping the solution cool, dissolve 40,431 g of cerium (IV)
sulphate, Ce(SO4)2·4 H2O, in350 ml of anaccurately measured
1 M solutionof sulphuric acid (3.1.2.4). Make up to 1 000 ml with
distilled water.
3.1.2.2. 2,5 M solutionof sodium hydroxide (NaOH).
3.1.2.3. Sodium acetate solution, 270 g per litre (prepared from dry sodium
acetate, CH3COONa).
3.1.2.4. 1 M sulphuric acid, H2SO4.
3.1.2.5. 2 % (m/v) sodium nitroprusside solution (Na2FeNO(CN)5·2 H2O)
Keep inthe dark ina well stoppered bottle.
Do not keep the solution longer than eight hours.
3.1.2.6. 10 % (v/v) solutionof piperidin e (C5H11N).
3.1.2.7. 1 M solutionof lactic acid:
100 ml of lactic acid (C3H6O3) are diluted in400 ml of water. This
solution is heated in an evaporating dish over a boiling water bath
for four hours, topping up the volume occasionally with distilled
water. After cooling, make up to a litre. Titrate the lactic acid in
10 ml of this solutionwith 1 M sodium hydroxide solution
(3.1.2.8). Adjust the solutionto 1 M lactic acid (90 g).
3.1.2.8. 1 M sodium hydroxide solution(NaOH).
3.2.Apparatus
3.2.1. Glass column of 10 to 11 mm internal diameter and approximately
300 mm long fitted with a drain tap to regulate the flow.
3.2.2. Constant temperature water bath at 65 ºC.
3.2.3. Spectrophotometer enabling absorbence measurements to be made at
a wavelength of 570 nm having cells with a 1 cm optical path.
3.3.Procedure
3.3.1. Preparation of the ion exchange column
See the chapter ‘Tartaric acid’, usual method, section3.3.1.
3.3.2. Separation of organic acids
See the chapter ‘Tartaric acid’, usual method, section3.3.2.
3.3.3. Determination of lactic acid
Place 10 ml of the eluate ina 50 ml glass test tube fitted with a
ground glass stopper, and add 10 ml of cerium sulphate solution
(3.1.2.1). Stir; place the tube in a constant temperature water bath
at 65 ºC for exactly 10 minutes. At the time when it is immersed
in the bath, remove the glass stopper for a few seconds to compen-
sate for the rise in pressure due to the heating, then insert the stopper
so as to seal the tube tightly and avoid losing any of the ethanal
(acetaldehyde) formed. Remove the tube from the bath, and cool it
under running water to bring it to a temperature of around 20 ºC.
Add 5 ml of the 2,5 M sodium hydroxide solution(3.1.2.2), mix
and filter.
Take 15 ml of the filtrate and pour it into a 50 ml stoppered flask
already containing a homogeneous mixture of 5 ml of 27 % sodium
acetate solution(3.1.2.3) an d 2 ml of 1 M sulphuric acid (3.1.2.4).
Add 5 ml of sodium nitroprusside solution (3.1.2.5), mix, then add
5 ml of the piperidine solution (3.1.2.6), mix rapidly and introduce
this solutionimmediately in to the spectrophotometer cell. The
colourationproduced varies from greento violet an d is measured at
570 nm with respect to air (no cell in the optical path): it increases
and then rapidly decreases.1990R2676 — EN — 18.03.2003 — 007.001 — 99
▼B
Follow the corresponding variation in absorbence and choose the
maximum value as the definitive value.
If the eluate is too rich in lactic acid and the absorbence too high,
dilute the eluate with the 7,1 % sodium sulphate solution(3.1.1)
and proceed with the measurement on the diluted solution.
3.3.4. Plotting the calibration curve
Pipette 10 ml of the 1 M lactic acid solution(3.1.2.7) an d 10 ml of
the titrated 1 M sodium hydroxide solution(3.1.2.8) in to a 1 000-ml
graduated flask and make up to the mark with 7,1 % sodium
sulphate solution. Take 5, 10, 15, 20 and 25 ml of this solution and
introduce them into 100 ml graduated flasks. Make up to the refer-
ence mark with 7,1 % (3.1.1) sodium sulphate solution. Take 10 ml
of each of the resultant solutions and determine their absorbences
according to the procedure described in 3.3.3 for the eluate.
The concentrations of these solutions correspond to wine eluates
containing 0,45, 0,9, 1,35, 1,80 and 2,25 g/l of lactic acid.
A graph of the absorbences of these solutions as a function of their
lactic acid concentration is a straight line.
3.4.Expressionofresults
The absorbence measured for the eluate is found on the calibration
curve and gives the lactic acid concentration in the wine in grams
per litre.
The result is expressed to one decimal place.
Note:Wines containing more than 250 mg/l of sulphur dioxide may have
an ethanalsulphonic acid content which is included with that of the
lactic acid. Inthis case, the result must be corrected by anamoun t
given by the following additional operation: 15 ml of the eluate is
mixed ina stoppered test tube with 5 ml of 27 % sodium acetate
(3.1.2.3) and 2 ml of 0,775 H
2SO4(775 ml of 1 M H2SO4is made
up to 100 ml with distilled water). Then, as in the determination of
lactic acid, add 5 ml of 2 % sodium nitroprusside (3.1.2.5) and 5 ml
of 10 % (3.1.1.6) piperidine. After mixing, measure the absorbence
under the same conditions as those described for the determination
of lactic acid. Locate this absorbence on the calibration curve to
obtainthe apparen t value Bof the lactic acid ingrams per litre due
to the ethanalsulphonic acid. If L′is the apparent concentration of
the lactic acid in the wine in grams per litre, the real concentration
Lof lactic acid is givenby:
L=L′−B× 0,4 (g/l)1990R2676 — EN — 18.03.2003 — 007.001 — 100
▼B
19.L-MALICACID
1. PRINCIPLE OF THE METHOD
L-malic acid (L-malate) is oxidized by nicotinamide adenine dinu-
cleotide (NAD) to oxaloacetate ina reactioncatalysed 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 formationof oxaloace-
tate. In the presence of L-glutamate, the oxaloacetate is transformed
into L-aspartate in a reaction catalysed by glutamate oxaloacetate
transaminase (GOT):
(1)L-malate + NAD+
oxaloacetate + NADH + H+
(2)Oxaloacetate + L-glutamate
 L-aspartate + α-
ketoglutarate
The amount of NADH formed, measured by the increase in absorb-
ence 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 solutionwill remainstable for at least 12 weeks at 4 ºC.
2.2. Nicotinamide adenine dinucleotide (NAD) solution, approximately
47 × 10−3M:dissolve 420 mg of NAD in12 ml of doubly distilled
water. This solutionwill remainstable for at least four weeks at 4 ºC.
2.3. Glutamate oxaloacetate transaminase (GOT) suspension, 2 mg/ml.
The suspension remains stable for at least a year at 4 ºC.
2.4. L-malate dehydrogenase (L-MDH) solution, 5 mg/ml. This solution
remains stable for at least a year at 4 ºC.
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. cali-
bration curves are not used, but standardization is made by
consideration of the extinction coefficient of NADH), the wave-
length 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 concentrationis 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 between3 an d 35 µg).1990R2676 — EN — 18.03.2003 — 007.001 — 101
▼B
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 insuch a way
that the total volumes inthe 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
inthe optical path) or with water as the stan dard.
Place the following in the cells having 1 cm optical paths:
Reference
cell
(ml)Sample
cell
(ml)
Solution2.1 1,00 1,00
Solution2.2 0,20 0,20
Doubly distilled water 1,00 0,90
Suspension 2.3 0,01 0,01
Sample to be measured - 0,10
Mix; after about three minutes, measure the absorbences of the solu-
tions in the reference and sample cells ( A1).
Add:
Solution2.4 0,01 ml 0,01 ml
Mix; wait for the reactionto be completed (about 5 to 10 min utes)
and measure the absorbences of the solutions in the reference and
sample cells ( A2).
Calculate the differences ( A2−A1) inthe absorben ces of the solu-
tions in the reference and sample cells, ∆ARand∆AS.
Finally, calculate the difference between those differences: ∆A=
∆AS−∆AR
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.Methodofcalculation
The general formula for calculating the concentration in g/l is:
C¼V/C2M
ε/C2d/C2ν/C21000/C2∆A
where
V= volume of test solutioninml (here 2,22 ml)
ν= volume of the sample inml (here 0,1 ml)
M= molecular mass of the substance to be determined (here, for
L-malic acid, M= 134,09)
d= optical path inthe cell incm (here, 1 cm)
ε= absorption coefficient of NADH, (at 340 nm, ε= 6,3 m mol−1
×l×c m−1),
so that for L-malate:C= 0,473 × ∆Ag/l1990R2676 — EN — 18.03.2003 — 007.001 — 102
▼B
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor.
Note:
Measurement at 334 nm: C= 0,482 × ∆A
Measurement at 365 nm: C= 0,876 × ∆A
6.2.Repeatability(r)
r= 0,03 + 0,034 xi
xiis the malic acid concentration in the sample in g/l.
6.3.Reproducibility(R)
R= 0,05 + 0,071 xi
xiis the malic acid concentration in the sample in g/l.1990R2676 — EN — 18.03.2003 — 007.001 — 103
▼M7
20.D-MALICACID
(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
carbondioxide.
D-malate þNADþ/harpoonleftrightD-MDHpyruvate þCO2þNADH þHþ
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:
(a) Bottle 1 with about 30 ml of solution consisting of Hepes buffer
[N-(2-hydroxyethyl)piperazine-N ′-2-ethanesulfonicacid]pH=9,0
and stabilisers;
(b) Bottle 2 with about 210 mg of NAD lyophilisate;
(c) Three bottles 3 with D-MDH lyophilisate, about 8 U each.
Preparationofthesolutions
1. Use content of bottle 1 undiluted. Before using bring solution to
20 to 25 ºC.
2. Dissolve content of bottle 2 in 4 ml double-distilled water.3. Dissolve content of one of bottles 3 in 0,6 ml double-distilled
water. Before using bring solution to 20 to 25 ºC.
Stabilityofthesolutions
The content of bottle 1 is stable for at least one year if stored
at +4 ºC; solution2 is stable for three weeks if stored at +4 ºC, an d
for two months if stored at − 20 ºC; solution 3 is stable for five days
if stored at +4 ºC.
3. APPARATUS
3.1. A spectrophotometer permitting measurement to be made at 340 nm,
the wavelength at which NADH absorption 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 absorbance measurements are involved (i.e. no
set of calibration solutions but reference to the extinction coefficient
of NADH), the wavelength scales and spectral absorbance of the
apparatus must be checked.
3.2. Glass cuvettes with optical path lengths of 1 cm (if preferred dispo-
sable cuvettes may be used).
3.3. Micropipettes for pipetting volumes in the range 0,01 to 2 ml.
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
50µg. 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, respec-
tively (depending on the apparatus used).1990R2676 — EN — 18.03.2003 — 007.001 — 104
▼M7
Dilutiontable:
Estimated quantity of D-malate/litre
Dilutionwith
waterDilutionfactor F Measured at:
340 or 334 nm 365 nm
< 0,3 g < 0,5 g — 1
0,3 - 3,0 g 0,5 - 5,0 g 1 + 9 10
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:
Reference Test
Solution1 1,00 ml 1,00 ml
Solution2 0,10 ml 0,10 ml
Double-distilled
water1,80 ml 1,70 ml
Sample for measure-
ment— 0,10 ml
Mix, and after about six minutes measure the absorbance of the
reference and test solutions (A1).
Add:
Reference Test
Solution3 0,05 ml 0,05 ml
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
(∆ΑT) and test ( ∆ΑE) solutions. Finally, calculate the difference
betweenthose differen ces: ∆Α=∆ΑE−∆ΑT.
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:
C¼
V/C2PM
ε/C2d/C2ν/C2∆A
where:
V = volume of test solutioninml (2,95 ml)
ν= volume of the sample inml (0,1 ml)
PM = molecular mass of the substance to be determined (for D-
malic acid, PM = 134,09)1990R2676 — EN — 18.03.2003 — 007.001 — 105
▼M7
d = optical path of the cuvette incm (1 cm)
ε= absorptioncoefficien t of NADH:
at 340 nm = 6,3 (1 mmol−1cm−1)
at 365 nm = 3,4 (1 mmol−1cm−1)
at 334 nm = 6,18 (1 mmol−1cm−1).
If the sample was diluted during its preparation, multiply the result
by the dilutionfactor.
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 interlabora-
tory 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 repeat-
ability value r inmore than5 % 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.
8. DOSAGE OF D-MALIC ACID (D(+)-MALIC ACID) IN WINES
WITH LOW LEVELS
8.1.Fieldofapplication
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 wave-
length 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.Preparationofthesample
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.1990R2676 — EN — 18.03.2003 — 007.001 — 106
▼M8
▼M8
8.7.Internalvalidation
The table below summarises the internal validation file on the
method for determining the dosage of D(+)-malic acid after the addi-
tionof 50 mg/l of the isomer.
Working range 0 mg to 70 mg of D-malic acid per litre.
Withinthese limits, the method is lin ear with a correlation
coefficient of between 0,990 and 0,994
Limit of quantification 24,4 mg/lLimit of detection8,3 mg/lSensitivity 0,0015 abs/mg/l
Recovery ratio 87,5 to 115,0 % for white wines and 75 to 105 % for red
wines
Repeatability = 12,4 mg/l for white wines (according to the OIV
method = 12,5 mg/l)
= 12,6 mg/l for red wines (according to OIV
method = 12,7 mg/l)
Coefficient of variation 4,2 % to 7,6 % (white wines and red wines)Intralaboratory variability CV=7,4 % (s = 4,4 mg/l; mean = 59,3 mg/l)1990R2676 — EN — 18.03.2003 — 007.001 — 107
▼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’.
Whenside reactionoccurs, the absorban ce of the solutionshould be measured at
regular intervals (every two to five minutes) after the required time for the stan-
dard 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 extrapo-
lated at that time (Af − Ai).
Figure 1. Side reaction1990R2676 — EN — 18.03.2003 — 007.001 — 108
▼M7
Appendix B
Statisticalresultsofinterlaboratory trial
Year of the interlaboratory trial: 1995
Number of laboratories: 8
Number of samples: 5 with additionof D-malic acid
Sample ABCDE
Number of laboratories retained after elimination of
laboratories presenting aberrant results 7 8 7 8 7
Number of laboratories presenting aberrant results 1 — 1 — 1
Number of results accepted 35 41 35 41 36
Average value ( χ-) (mg/l) 161,7 65,9 33,1 106,9 111,0
Standard deviation of repeatability (sr) (mg/l) 4,53 4,24 1,93 4,36 4,47
Relative standard deviation of repeatability (RSDr) (%) 2,8 6,4 5,8 4,1 4,00
Repeatability limit (r) (mg/l) 12,7 11,9 5,4 12,2 12,5Standard deviation of reproducibility (s
R) (mg/l) 9,26 7,24 5,89 6,36 6,08
Relative standard deviation of reproducibility (RSDR)
(%) 5,7 11 17,8 5,9 5,5
Reproducibility limit (R) (mg/l) 25,9 20,3 16,5 17,8 17,0
Types of samples:
A: red wine; B: red wine; C: white wine; D: white wine; E: white wine;1990R2676 — EN — 18.03.2003 — 007.001 — 109
▼B
21.TOTALMALICACID
1. PRINCIPLE
Malic acid, separated by means of an anion exchange column, is
determined colorimetrically in the eluent by measuring the yellow
colorationit forms with chromotropic acid inthe presen ce 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 draintap.
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 ºC.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 solution10 % (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, (C
10H6Na2O8S2.2H20) in10 ml
distilled water.
3.9. DL-Malic acid solution0,5 g/l.
Dissolve 250 g malic acid (C4H6O5) insodium sulphate solution
(10 %), make up to 500 ml with sodium sulphate solution(10 %)
(3.5).
4. PROCEDURE
4.1.Preparationofionexchangeresin
Place a plug of cottonwool impregn ated with distilled water at the
bottom of the column(35 × 250 mm) above the tap. Pour a suspen -
sion 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 columnwith sodium hydroxide
solution(5 %), allow to drainto within2 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 1000 ml of distilled
water. Refill the columnwith acetic (30 %) acid, allow to drainto
within2 to 3 mm from the top of the columnan d repeat with two
further washings of acetic acid (30 %). Leave for at least 24 hours
before use. Keep the ionexchan ge resininacetic acid (30 %) for the
subsequent analyses.
4.2.Preparationofionexchangecolumn
Place a plug of cottonwool at the bottom of the column
(11 × 300 mm) above the tap. Pour inthe suspen sionof 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 %).1990R2676 — EN — 18.03.2003 — 007.001 — 110
▼B
4.3.SeparationofDL-malicacid
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 columnwith 50 ml acetic acid (0,5 %) thenwith 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 onthe exchan ge resinwith a 10 % sodium
sulphate solution(3.5). Collect the eluate ina 100 ml volumetric
flask.
The column can be regenerated using the procedure desribed in
(4.1).
4.4.Determinationofmalicacid
Label two wide necked 30-ml tubes (fitted with ground glass stop-
pers) 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 ina boilin g water bath for
exactly 10 minutes. Cool the tubes in the dark at 20 ºC for exactly
90 minutes. Immediately measure the absorbence relative to the
control at 420 nm in a 10 mm cell.
4.5.Plottingthecalibrationcurve
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 solu-
tion(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 cali-
bration 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 extrapolationof the measured absorben ce value to give the corre-
sponding 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.1990R2676 — EN — 18.03.2003 — 007.001 — 111
▼B
22.SORBICACID
1. PRINCIPLE OF METHODS
1.1.Determinationbyultraviolet absorptionspectrophotometry
Sorbic acid ( trans, trans, 2,4-hexadienoic acid) extracted by steam
distillation is determined in the wine distillate by ultraviolet absorp-
tion spectrophotometry. Substances that interfere in the ultraviolet
are removed by evaporation to dryness using a lightly alkali, calcium
hydroxide. Thinlayer chromatography is used for con firmationof
levels (1 mg/l) less than20 mg/l.
1.2.Determinationbygaschromatography
Sorbic acid extracted inethyl ether is determin ed by gas chromato-
graphy with an internal standard.
1.3.Identificationoftracesbythin-layerchromatography
Sorbic acid extracted inethyl ether is separated by thinlayer chro-
matography and its concentration is evaluated semi-quantitatively.
2. DETERMINATION BY ULTRAVIOLET ABSORPTION SPEC-
TROPHOTOMETRY
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, inapproximately 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, inwater an d
make up to 1 000 ml with water.
2.2.Apparatus
2.2.1. Steam distillationapparatus (see chapter ‘Volatile acidity’).
2.2.2. Water bath at 100 ºC.
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 inthe flask of the steam distillationapparatus 10 ml of win e
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 dilutionof the referen ce 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 spectrophotometerat
256 nm using that of distilled water as a blank. Plot a curve showing
the variation of absorbence as a function of concentration. The varia-
tionis lin ear.
2.3.3. Determination
Place 5 ml of the distillate inanevaporatin g 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 inseveral ml of distilled water, tran sfer comple-
tely to a 20 ml volumetric flask and make up to the mark with
rinsing water. Measure the absorbence at 256 nm using the spectro-
photometer against a blank consisting of a solution obtained by
diluting 1 ml of calcium hydroxide solution (2.1.2) to 20 ml with
water.1990R2676 — EN — 18.03.2003 — 007.001 — 112
▼B
Plot the value of the measured absorbence on the calibration curve
and from this find the concentration Cof sorbic acid inthe 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.Expressionofresults
2.4.1. Calculation
The sorbic acid concentration in the wine expressed in mg per litre
is givenby 100 × C
whereC= concentration of sorbic acid in the solution analysed by spec-
trophotometry expressed inmg per litre.
3. DETERMINATION BY GAS CHROMATOGRAPHY3.1.Reagents
3.1.1. Ethyl ether, (C
2H5)2O, distilled just before use.
3.1.2. Internal reference solution: solution of undecanoic acid, C11H22O2,i n
95 % vol ethanol at a strength of 1 g/l.
3.1.3. Aqueous solutionof 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 × ⅛inch) 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:
Oventemperature: 175 ºC.Temperature of the injector and detector: 230 ºC.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 givenas anexample.
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. Treat1990R2676 — EN — 18.03.2003 — 007.001 — 113
▼B
20 ml of the sample prepared inthis way accordin g to the procedure
described in3.3.1.
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 stan-
dard. Measure the height (or area) of each of the recorded peaks.
3.4.Expressionofresults
3.4.1. Calculation
The concentration of sorbic acid in the analysed wine, expressed in
mg per litre, is givenby:
100/C2h
H/C2I
i
where
H= height of the sorbic acid peak inthe referen ce solution
h= height of the sorbic acid peak inthe sample for an alysis
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 thinlayer chromatography coated
with polyamide gel (0,15 mm thick) with the additionof a fluores-
cent indicator.
4.2.2. Cell for thinlayer 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 repeat-
edly 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.1990R2676 — EN — 18.03.2003 — 007.001 — 114
▼B
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 refer-
ence 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) inthe chromatograph tan k 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.Expressionofresults
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 solu-
tionto be an alysed.
Concentrations above 10 mg per litre could be determined with the
depositionof less than5 µl of the solutionto be an alysed (measured
out using a microsyringe).1990R2676 — EN — 18.03.2003 — 007.001 — 115
▼B
23.L-ASCORBICACID
1. PRINCIPLE OF METHODS
The methods proposed enable the L-ascorbic acid and dehydroas-
corbic acid present in wines or musts to be determined.
1.1.Reference method(fluorimetry)
The L-ascorbic acid is oxidized onactivated carbonin to dehydroas-
corbic 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
formationof a boric acid/dehydroascorbic acid complex) an d the
fluorimetric determination to be deduced.
1.2.Usualmethod(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, C
6H10Cl2N2, 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 solutionof boric acid an d sodium acetate:
dissolve 3 g of boric acid, H3BO3, in100 ml of sodium acetate solu-
tion(2.1.2). This solutionmust 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 ina desiccator protected again st light, in
50 ml of acetic acid solution(2.1.4).
2.1.6. Very pure analytical grade activated carbon (1)
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 carbontreated inthis way ina 2-litre con ical flask, add 1
litre of water, shake and filter using a sintered glass filter of porosity
3. Repeat this operationtwo more times. Place the residue inan
oven controlled to 115 ± 5 ºC 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 exci-
tation 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.1990R2676 — EN — 18.03.2003 — 007.001 — 116
(1) One of the trade names is ‘Norite’.
▼B
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 grad-
uated flask with the 56 % acetic acid solution(2.1.4) inorder 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,
inthe first, 5 ml of the mixed solutionof boric acid an d 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 stan d 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 orthophe-
nylenediamine solution (2.1.1), stir; leave the reaction to proceed for
30 minutes until the solution darkens and then make the spectro-
fluorimetric 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 an d 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 inthe first series (corre-
sponding 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
thenmake the spectrofluorimetric measuremen ts.
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 inten-
sity 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 deter-
mination and thus deduce the concentration CL-ascorbic acid +
dehydroascorbic acid inthe solutionto be an alysed.
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 ,whereFis the
dilutionfactor.
3. USUAL METHOD (colorimetric method)
3.1.Reagents
3.1.1. Metaphosphoric acid, 30 % (m/v) solution.
take 30 g of metaphosphoric acid, (HPO3)n, previously crushed ina
mortar. Wash rapidly by immersion in distilled water and stirring.
Dissolve the washed acid indistilled water by shakin g ina 100 ml
graduated flask and make up to the reference mark. The resultant
solution will have a concentration of approximately 30 % (m/v) of
metaphosphoric acid.
3.1.2. 3 % (m/v) metaphosphoric acid solutionprepared just before use by
1:10 dilutionwith distilled water of solution3.1.1.1990R2676 — EN — 18.03.2003 — 007.001 — 117
▼B
3.1.3. 1 % (m/v) metaphosphoric acid solutionprepared just before use by
1:30 dilutionwith distilled water of solution3.1.1.
3.1.4. Polyamide suspension:
Make a suspension of 10 g of polyamide for chromatography with
60 ml of distilled water. Allow to stand for two hours. The quantity
prepared is enough for four determinations.
3.1.5. Thiourea, (NH2)2CS.
3.1.6. Iodine solution, I2, 0,05 M.
3.1.7. 2,4-Dinitrophenylhydrazine solution, 6 % (m/v):
Make a suspension of 6 g of 2,4-dinitrophenylhydrazine (C6H6N4O4)
in50 ml of glacial acetic acid ( ρ20= 1,05 g/ml) and add 50 ml of
sulphuric acid ( ρ20= 1,84 g/ml). The 2,4-dinitrophenylhydrazine is
dissolved by stirring.
3.1.8. Ethyl acetate (C4H8O2) with the additionof 2 % (v/v) glacial acetic
acid (3.1.12).
3.1.9. Chloroform, CHCl3.
3.1.10. Aqueous starch solution, 0,5 % (m/v).3.1.11. Mobile phase:
ethyl acetate …........................................... 50 vol
chloroform … ............................................. 60vol
glacial acetic acid … ...................................... 5vol
Leave the solvent mixture undisturbed for 12 hours before use.
3.1.12. Glacial acetic acid, CH
3COOH, ( ρ20= 1,05 g/ml).
3.1.13. L-ascorbic acid solution, 0,1 g per 100 ml of 1 % metaphosphoric
acid solution(3.1.3).
3.2.Apparatus
3.2.1. Laboratory centrifuge having 50 ml centrifuge tubes with ground
glass stoppers.
3.2.2. Cool waterbath, temperature thermostatically controlled between 5
and 10 ºC.
3.2.3. Waterbath temperature thermostatically controlled to 20 ºC.3.2.4. Precoated plates for thinlayer chromatography, 20 × 20 cm, coated
with silica gel G (thickness 0,25 or 0,3 mm).
3.2.5. Chromatography tank.
3.2.6. Micropipette capable of delivering 0,2 ml volumes.3.2.7. Spectrophotometer capable of making absorbence measurements at
500 nm equipped with cells having a 1 cm optical path.
3.3.Procedure
3.3.1. Oxidation of the L-ascorbic acid to dehydroascorbic acid
Place 50 ml of the wine in a 100 ml volumetric flask, add 15 ml of
the polyamide suspension (3.1.4) and make up to the mark with 3 %
metaphosphoric acid solution (3.1.2). Allow to stand for one hour,
shaking at frequent intervals. Filter through a fluted filter. Collect
20 ml of the filtrate in a centrifuge tube, add 1 ml of 0,05 M iodine
solution (3.1.6). Homogenize by shaking the stoppered centrifuge
tube and, after 1 minute, reduce the excess iodine by the addition
of approximately 25 mg of thiourea.
3.3.2. Formation and extraction of the bis(2,4-dinitrophenylhydrazone)
derivative of diketogulonic acid
Place the tube into a waterbath with the temperature maintained to
between 5 and 10 ºC; add 4 ml of the 2,4-dinitrophenylhydrazine
solution (3.1.7). Mix the contents carefully, avoiding any wetting of
the glass stopper. Seal the tube completely and place in a waterbath
at 20 ºC for approximately 16 hours (overnight).1990R2676 — EN — 18.03.2003 — 007.001 — 118
▼B
Introduce 15 ml of ethyl acetate (3.1.8) into the centrifuge tube.
Close the tube with its ground glass stopper and shake vigorously
for 30 seconds. Centrifuge for five minutes using a centrifugal force
of 350 to 400 g. Pipette 10 ml of the ethyl acetate extract into a
conical flask with a ground glass stopper. Remove stopper and add
a further 5 ml of ethyl acetate (3.1.8) to the centrifuge tube, again
shake for 30 seconds and centrifuge for five minutes at the same
speed. Pipette 5 ml of the ethyl acetate extract into the conical flask
containing the 10 ml from the first extraction. Mix.
3.3.3. Separation of the bis(2,4-dinitrophenylhydrazone) by chromato-
graphy; this is to be carried out in the 2 hours following the
extraction (3.3.2).
Apply 0,2 ml of the ethyl acetate extract to the whole of a starting
line situated 2 cm from the edge of the plate, leaving a margin of
2 cm at the sides of the plate. Place the mobile phase (3.1.11) in
the chromatography tank to a depth of 1 cm and allow the atmo-
sphere to become saturated with solvent vapours. Introduce the
plate, allowing the solvent to run to the top edge of the plate.
Dry the plate for one hour under a ventilated hood. Hold the plate in
a vertical positionabove a sheet of glazed paper an d with a spatula
scrape off (at right angles to the direction of the run) the red-
coloured zone (characteristic of the 2,4-dinitrophenylhydrazone).
Transfer the powdered product obtained, without loss, to a conical
flask fitted with a ground glass stopper, and add 4 ml of acetic acid
(3.1.12). Allow to stand for 30 minutes, stirring frequently. Filter
through a fluted filter paper directly into a spectrophotometric cell
(3.2.7). The filtrate obtained must be perfectly clear. Measure the
absorbence of this solution at 500 nm using acetic acid (3.1.12) in
the reference cell to provide a zero for the measurements.
3.3.4. Preparation of the calibration curve
Place 5, 10 and 15 ml of the L-ascorbic acid solution (3.1.13) in
three 100 ml volumetric flasks and make up to the mark with the
1 % metaphosphoric acid solution(3.1.3). The solution s thus
obtained have concentrations of 50, 100 and 150 mg of ascorbic
acid per litre respectively.
Treat each of these solutions according to the procedure described in
3.3.1, 3.3.2 and 3.3.3. Plot the calibration curve, which should be a
straight line passing through the origin.
3.3.5. Expression of results
The concentration of L-ascorbic acid + dehydroascorbic acid in the
wine is expressed in milligrams per litre.
3.3.5.1. Calculation
Find the value of the absorbence measured in 3.3.3 on the straight
calibration curve and thus determine the concentration of L-ascorbic
acid + dehydroascorbic acid inthe solutionto be an alysed.
Note:
If the concentration of L-ascorbic acid + dehydroascorbic acid is
higher than150 mg/litre, reduce the volume of the test sample to
25, 20 or 10 ml of wine and multiply the result obtained by the dilu-
tionfactor F.1990R2676 — EN — 18.03.2003 — 007.001 — 119
▼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 elec-
trode.
2. APPARATUS
2.1. pH meter with a scale calibrated in pH units and enabling measure-
ments to be made to at least ±0,05 pH unit.
2.2.Electrodes:
2.2.1. Glass electrode, kept indistilled water.
2.2.2. Calomel-saturated potassium chloride reference electrode, kept in a
saturated solutionof potassium chloride.
2.2.3. Or a combined electrode, kept in distilled water.
3. REAGENTS
3.1.Buffersolutions
3.1.1. Saturated solution of potassium hydrogen tartrate, containing at least
5,7 g of potassium hydrogentartrate per litre (C4H5KO6) at 20 ºC.
(This solution may be kept for up to two months by adding 0,1 g
of thymol per 200 ml.)
pH/C144/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C1463,57 at 20 ºC
3,56 at 25 ºC
3,55 at 30 ºC
3.1.2. Solution of potassium hydrogen phthalate, 0,05 M, containing
10,211 g of potassium hydrogenphthalate (C
8H5KO4) per litre at
20 ºC. (Maximum keeping period, two months.)
pH/C144/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C1463,999 at 15 ºC
4,003 at 20 ºC
4,008 at 25 ºC
4,015 at 30 ºC
3.1.3. Solution containing:
monopotassium phosphate, KH
2PO4…............. 3,402g
dipotassium phosphate, K2HPO4…................. 4,354g
water to …........................................ 1000 ml
(maximum keeping period, two months)
pH/C144/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C145/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C1466,90 at 15 ºC
6,88 at 20ºC6,86 at 25 ºC
6,85 at 30 ºC
Note:
Commerical reference buffer solutions may also be used.1990R2676 — EN — 18.03.2003 — 007.001 — 120
▼B
4. PROCEDURE
4.1.Preparationofthesampleforanalysis
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 º Brix).
IfPis the percentage concentration (m/m) of total sugars in the
rectified concentrated must, weigh a mass of
2500
P
and make up to 100 g with water. The water used must have a
conductivity below 2 microsiemens per cm.
4.2.Zeroingoftheapparatus
Zeroing is carried out before any measurement is made, according to
the instructions provided with the apparatus used.
4.3.CalibrationofthepHmeter
Calibrate the pH meter at 20 ºC using buffer solutions of pH 6,88
and 3,57 at 20 ºC.
Use the buffer solutionof pH 4,00 at 20 ºC 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 ºC and as close as possible to
20 ºC. Read the pH value directly off the scale.
Carry out at least two determinations on the same sample.
The fin al result is takento be the arithmetic meanof two determin a-
tions.
5. EXPRESSION OF RESULTS
The pH of the must, the wine or the 25 % (m/m) (25 º Brix) solution
of rectified concentrated must is quoted to two decimal places.1990R2676 — EN — 18.03.2003 — 007.001 — 121
▼B
25.SULPHURDIOXIDE
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 andtemperature:
H
2SO3
H++ HSO3−
H2SO3represents 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 DIOXIDE2.1.Principle ofthemethods
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 titrationwith a stan dard solutionof sodium hydroxide. Free
sulphur dioxide is purged from the wine by entrainment at low
temperature (10 ºC).
Total sulphur dioxide is purged from the wine by entrainment at
high temperature (approximately 100 ºC).
2.1.1.2. For rectified concentrated musts
Total sulphur dioxide is extracted from the previously diluted recti-
fied concentrated must by entrainment at high temperature
(approximately 100 ºC).
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
titrationafter alkalin e hydrolysis. Whenadded 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.1990R2676 — EN — 18.03.2003 — 007.001 — 122
▼B
Thedimensionsgiven are in millimetres. The internal diameters
ofthefourconcentrictubesformingthecondenserare45,34,27
and10mm.
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 reductionproduced 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 % (H
3PO4,ρ20= 1,71 g/ml).
2.2.2.2. Hydrogenperoxide 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 ºC for
two days before the determination.
Place 2 to 3 ml of hydrogenperoxide solution(2.2.2.2) an d two
drops of the indicator reagent in the bubbler B and neutralize the
hydrogenperoxide solutionwith the 0,01 M sodium hydroxide solu-
tion (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 nml
be the volume used.1990R2676 — EN — 18.03.2003 — 007.001 — 123
▼B
2.2.3.2. Expressionof results
The liberated sulphur dioxide is expressed inmg/l to the n earest
whole number.
2.2.3.2.1. Calculation
The free sulphur dioxide inmilligrams per litre is 6,4 n.
2.2.3.3. Determination of total sulphur dioxide2.2.3.3.1. 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’, section5.1.2. In troduce 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 SO
2per litre, place 50 ml of the sample and 15 ml of phos-
phoric acid (2.2.2.1) in the 250-ml flask A of the entrainment
apparatus. Connect the flask to the apparatus.
However, until ►M331 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 SO2per 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 hydrogenperoxide solution(2.2.2.2) inthe
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 ona 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).
Letnml be the volume used.
2.2.3.4. Expressionof 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 inmilligrams per litre:
— samples low insulphur dioxide (50 ml test sample): 6,4 n
— other samples (20 ml test sample): 16 n
For rectified concentrated musts:Total sulphur dioxide inmilligrams per kilogram of total sugars
(50 ml prepared test sample (2.2.3.3.1)):
1600 /C2n
P
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.1990R2676 — EN — 18.03.2003 — 007.001 — 124
▼B
20ml test sample > 50 mg/l; R= 15 mg/l.
2.3.Rapidmethodofdetermination
2.3.1. Reagents
2.3.1.1. EDTA Complexone III: disodium salt of ethene diamine tetra-acetic
acid (C10H14N2O8Na2·2 H2O)
2.3.1.2. Sodium hydroxide solution, NaOH, 4 M (160 g/l).
2.3.1.3. Sulphuric acid, H2SO4(ρ20= 1,84 g/ml) (1:10 v/v).
2.3.1.4. Starch solution5 g/l:
Mix 5 g starch with about 500 ml of water. Bring to the boil stirring
continuously and keep boiling for 10 minutes. Add 200 g of sodium
chloride (NaCl). Make up to one litre after cooling.
2.3.1.5. 0,025 M iodine solution, I2.
2.3.2. Apparatus
2.3.2.1. 500 ml conical flasks.
2.3.2.2. Burette.
2.3.2.5. 1, 2, 5 and 50 ml pipettes.2.3.3. Procedure
2.3.3.1. Free sulphur dioxide
Place ina 500 ml con ical flask:
— 50 ml wine,
— 5 ml starch solution(2.3.1.4),— 30 mg EDTA Complexone III (2.3.1.1),— 3 ml of 1/10 (v/v) H
2SO4(2.3.1.3).
Titrate immediately with 0,025 M iodine (2.3.1.5) until the blue
colorationpersists clearly for 10 to 15 secon ds. Let nml be the
volume of iodine used.
2.3.3.2. Sulphur dioxide
Add 8 ml of 4 M sodium hydroxide solution(2.3.1.2), shake the
mixture once and allow to stand for five minutes. Add, with
vigorous stirring and in one operation, the contents of a small beaker
inwhich 10 ml of 1:10 v/v sulphuric acid (2.3.1.3) have been
placed. Titrate immediately with 0,025 M iodine (2.3.1.5); let the
volume used be n′ml.
Add 20 ml of 4 M sodium hydroxide solution(2.3.1.2), shake on ce
and allow to stand for five minutes. Dilute with 200 ml of ice-cold
water.
Add, with vigorous stirring and in one operation, the contents of a
test tube inwhich 30 ml of 1:10 v/v sulphuric acid (2.3.1.3) have
previously beenplaced. Titrate the free sulphur dioxide immediately
with 0,025 M iodine (2.3.1.5), and let the volume used be n″ml.
2.3.4. Expression of results
2.3.4.1. Calculation:
Free sulphur dioxide inmilligrams per litre: 32 n.
Total sulphur dioxide inmilligrams per litre: 32 ( n+n′+n″).
Notes:(1) For red wines with low SO
2concentrations, it is worth using
iodine more dilute than 0,025 M (e.g. 0,01 M. Then replace the
coefficient 32 by 12,8 in the above formulae).
(2) For red wines, it is useful to illuminate from below with a beam
of yellow light from an ordinary electric light bulb shining
through a solutionof potassium chromate or from a sodium
vapour lamp. The determination should be carried out in a dark
room and the transparency of the wine observed: it becomes
opaque when the end-point is reached.
(3) If the quantity of sulphur dioxide (H2SO3) is close to, or
exceeds, the legal limit, the total sulphur dioxide should be
determined by the reference method.1990R2676 — EN — 18.03.2003 — 007.001 — 125
▼B
(4) If the determination of free sulphur dioxide is particularly
required, carry out the analysis on a sample kept under anae-
robic conditions for two days at 20 ºC before analysis. Carry
out the determination at 20 ºC.
(5) Because certain substances are oxidized by iodine in an acid
medium, the quantity of iodine used in this way must be evalu-
ated for more accurate determinations. To achieve this, the free
sulphur dioxide should be combined with excess ethanol or
propanal before carrying out the iodine titration. Add 5 ml of
7 g/l ethanol solution (C2H4O) or 5 ml of a 10 g/l propanal solu-
tion(C3H6O) to 50 ml of wine in a 300 ml conical flask.
Put a stopper on the flask and leave standing for at least 30
minutes. Add 3 ml of 1:10 v/v sulphuric acid (2.3.1.3) and suffi-
cient 0,025 M iodine (2.3.1.5) to cause the starch to change
colour. Let n/tripleprimeml be the volume of iodine used. This must be
subtracted from n(free sulphur dioxide) and from n+n′+n″
(total sulphur dioxide).
n/tripleprimeis generally small, from 0,2 to 0,3 ml 0,025 M iodine. If
ascorbic acid has beenadded to the win e, n/tripleprimewill be much
higher and it is possible, at least approximately, to measure the
amount of this substance from the value of n/tripleprime, giventhat 1 ml
of 0,025 M iodine will oxidize 4,4 mg of ascorbic acid. By
determining n/tripleprimeit is possible to detect quite easily the presence
of residual ascorbic acid inamoun ts greater than20 mg/l in
wines to which it has been added.
3. MOLECULAR SULPHUR DIOXIDE
3.1.Principle ofthemethod
The percentage of molecular sulphur dioxide, H2SO3, infree sulphur
dioxide is calculated as a function of pH, alcoholic strength and
temperature.
For a given temperature and alcoholic strength:
H2SO3
H++ HSO3−,
(1) H2SO3 ½/C138 ¼L
10pH/C0pKM ðÞ þ 1
with
pKM=pKT/C0Aﬃﬃ ﬃ
Ip
1þBﬃﬃ ﬃ
Ip
L=[ H2SO3] + [HSO3−]
whereI = ionic strength,
AandB= coefficients varying with temperature and alcoholic
strength,
K
T= thermodynamic dissociation constant: values of p KTare
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 pKMfor various temperatures and alcoholic strengths.
The molecular sulphur dioxide content calculated using expression
(1) is giveninTable 3 for various values of pH, temperature an d
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 TºC. 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.1990R2676 — EN — 18.03.2003 — 007.001 — 126
▼B
TABLE 1
Valuesofthethermodynamicdissociationconstantp KT
Alcohol strength(%
vol)Temperature (ºC)
20 25 30 35 40
0 1,798 2,000 2,219 2,334 2,493
5 1,897 2,098 2,299 2,397 2,527
10 1,997 2,198 2,394 2,488 2,606
15 2,099 2,301 2,503 2,607 2,728
20 2,203 2,406 2,628 2,754 2,895
TABLE 2
Valuesofthemixeddissociationconstantp KM(I=0,038)
Alcohol strength(%
vol)Temperature (ºC)
20 25 30 35 40
0 1,723 1,925 2,143 2,257 2,416
5 1,819 2,020 2,220 2,317 2,446
10 1,916 2,116 2,311 2,405 2,522
15 2,014 2,216 2,417 2,520 2,640
20 2,114 2,317 2,538 2,663 2,803
TABLE 3
Molecular sulphurdioxideasapercentageoffree sulphurdioxide
Molecular SO2/
free SO2(%)T=2 0º C
I= 0,038
pHAlcoholic strength (% vol)
0 5 10 15 20
2,8 7,73 9,46 11,55 14,07 17,09
2,9 6,24 7,66 9,40 11,51 14,07
3,0 5,02 6,18 7,61 9,36 11,51
3,1 4,03 4,98 6,14 7,58 9,36
3,2 3,22 3,99 4,94 6,12 7,58
3,3 2,58 3,20 3,98 4,92 6,12
3,4 2,06 2,56 3,18 3,95 4,92
3,5 1,64 2,04 2,54 3,16 3,95
3,6 1,31 1,63 2,03 2,53 3,16
3,7 1,04 1,30 1,62 2,02 2,53
3,8 0,83 1,03 1,29 1,61 2,02
T=2 5º C
2,8 11,47 14,23 17,15 20,67 24,75
2,9 9,58 11,65 14,12 17,15 22,71
3,0 7,76 9,48 11,55 14,12 17,18
3,1 6,27 7,68 9,40 11,55 14,15
3,2 5,04 6,20 7,61 9,40 11,58
3,3 4,05 4,99 6,14 7,61 9,42
3,4 3,24 4,00 4,94 6,14 7,63
3,5 2,60 3,20 3,97 4,94 6,16
3,6 2,07 2,56 3,18 3,97 4,55
3,7 1,65 2,05 2,54 3,18 3,98
3,8 1,32 1,63 2,03 2,54 3,181990R2676 — EN — 18.03.2003 — 007.001 — 127
▼B
Molecular SO2/
free SO2(%)T=2 0º C
I= 0,038
pHAlcoholic strength (% vol)
0 5 10 15 20
T=3 0º C
2,8 18,05 20,83 24,49 29,28 35,36
2,9 14,89 17,28 20,48 24,75 30,29
3,0 12,20 14,23 16,98 20,71 25,66
3,1 9,94 11,65 13,98 17,18 21,52
3,2 8,06 9,48 11,44 14,15 17,88
3,3 6,51 7,68 9,30 11,58 14,75
3,4 5,24 6,20 7,53 9,42 12,08
3,5 4,21 4,99 6,08 7,63 9,84
3,6 3,37 4,00 4,89 6,16 7,98
3,7 2,69 3,21 3,92 4,95 6,44
3,8 2,16 2,56 3,14 3,98 5,19
T=3 5º C
2,8 22,27 24,75 28,71 34,42 42,18
2,9 18,53 20,71 24,24 29,42 36,69
3,0 15,31 17,18 20,26 24,88 31,52
3,1 12,55 14,15 16,79 20,83 26,77
3,2 10,24 11,58 13,82 17,28 22,51
3,3 8,31 9,42 11,30 14,23 18,74
3,4 6,71 7,63 9,19 11,65 15,49
3,5 5,44 6,16 7,44 9,48 12,71
3,6 4,34 4,95 6,00 7,68 10,36
3,7 3,48 3,98 4,88 6,20 8,41
3,8 2,78 3,18 3,87 4,99 6,80
T=4 0º C
2,8 29,23 30,68 34,52 40,89 50,14
2,9 24,70 26,01 29,52 35,47 44,74
3,0 20,67 21,83 24,96 30,39 38,85
3,1 17,15 18,16 20,90 25,75 33,54
3,2 14,12 14,98 17,35 21,60 28,62
3,3 11,55 12,28 14,29 17,96 24,15
3,4 9,40 10,00 11,70 14,81 20,19
3,5 7,61 8,11 9,52 12,13 16,73
3,6 6,14 6,56 7,71 9,88 13,77
3,7 4,94 5,28 6,22 8,01 11,25
3,8 3,97 4,24 5,01 6,47 9,151990R2676 — EN — 18.03.2003 — 007.001 — 128
▼B
25.SODIUM
1. PRINCIPLE OF THE METHODS
1.1.Reference method:atomicabsorptionspectrophotometry
Sodium is determined directly in the wine by atomic absorption
spectrophotometry after the additionof anion izationsuppression
agent (caesium chloride) to prevent ionization of sodium.
1.2.Usualmethod:flamephotometry
Sodium is determined directly in diluted wine (at least 1:10) by
flame photometry.
2. REFERENCE METHOD2.1.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 anhy-
drous sodium chloride, NaCl, in distilled water and making up to a
volume of 1 litre.
Keep this solutionina polyethylen e bottle.
2.1.2. Matrix (model) solution:
citric acid, C
6H8O7·H2O….......................... 3,5 g
sucrose, C12H22O11… ................................. 1,5 g
glycerol, C3H8O3… .................................. 5,0 g
anhydrous calcium chloride, CaCl2…................ 50 mg
anhydrous magnesium chloride, MgCl2… ............ 50 mg
absolute alcohol, C2H5OH… ......................... 50 ml
de-ionized water to …............................... 500 ml
2.1.3. Caesium chloride solution containing 5 % caesium:
dissolve 6,330 g of caesium chloride, CsCl, in100 ml of distilled
water.
2.2.Apparatus
2.2.1. Atomic absorptionspectrophotometer equipped with anair-acetylen e
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) an d make up to the mark with
distilled water.
2.3.2. Calibration
Place 5,0 ml of the matrix solutionineach on e 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 an d
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). Aspirate1990R2676 — EN — 18.03.2003 — 007.001 — 129
▼B
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.Expressionofresults
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 Cinmilligrams per litre.
The sodium concentration in milligrams per litre of the wine will
thenbe 20 C, expressed to the nearest whole number.
2.4.2. Repeatability (r)
r= 1 + 0,024 ximg/l.
xi= concentration of sodium in the sample in mg/l.
2.4.3. Reproducibility (R)
R= 2,5 + 0,05 ximg/l.
xi= concentration of sodium in the sample in mg/l.
3. USUAL METHOD
3.1.Reagents
3.1.1. Reference solution containing 20 mg sodium per litre
Absolute alcohol (C2H5OH)…..................... 10 ml
Citric acid (C6H8O7·H2O)…...................... 700 mg
Sucrose (C12H22O11)…............................. 300 mg
Glycerol (C3H8O3)…............................... 1000 mg
Potassium hydrogentartrate (C4H5KO6) ….......... 481,3 mg
Anhydrous calcium chloride (CaCl2)…............. 10 mg
Anhydrous magnesium chloride (MgCl2)…........ 10 mg
Dry sodium chloride (NaCl) … ..................... 50,84 mg
Water to …........................................ 1 litre
3.1.2. Dilution solution
Absolute alcohol (C2H5OH)…..................... 10 ml
Citric acid (C6H8O7·H2O)…...................... 700 mg
Sucrose (C12H22O11)…............................. 300 mg
Glycerol (C3H8O3)…............................... 1000 mg
Potassium hydrogentartrate (C4H5KO6) ….......... 481,3 mg
Anhydrous calcium chloride (CaCl2)…............. 10 mg
Anhydrous magnesium chloride (MgCl2)…........ 10 mg
Water to …........................................ 1 litre
To prepare solution3.1.1 an d 3.1.2 dissolve the potassium hydrogen
tartrate inapproximately 500 ml of very hot distilled water, mix this
solutionwith 400 ml of distilled water inwhich the other chemicals
have already been dissolved, and make up to one litre.
Preserve the solutions in polyethylene bottles by adding two drops of
allyl isothiocyanate.
3.2.Apparatus
3.2.1. Flame photometer supplied by anair-butan e mixture.1990R2676 — EN — 18.03.2003 — 007.001 — 130
▼B
3.3.Procedure
3.3.1. Calibration
Place 5, 10, 15, 20 and 25 ml of the reference solution (3.1.1) in
100-ml volumetric flasks and make up to 100 ml with the dilution
solution (3.1.2). Solutions containing 1, 2, 3, 4 and 5 mg of sodiumper litre respectively are obtained in this way.
3.3.2. Determination
Make the measurements at 589,0 nm. Carry out the adjustment to
100 % transmission using distilled water. Successively aspirate the
standard solutions (3.3.1) directly into the burner of the photometer,
followed by the wine diluted by 1:10 with distilled water and note
the readings of percentage transmission. If necessary, the wine
already diluted 1:10 may be further diluted with the dilutionsolution
(3.1.2).
3.4.Expressionofresults
3.4.1. Method of calculation
Plot a graph showing the variations in percentage transmission with
sodium concentration in the standard solutions. Record the transmis-
sion obtained with the sample of diluted wine from this graph and
determine the corresponding sodium concentration C.
The sodium concentration in mg of sodium per litre will be F×C
whereFis the dilutionfactor.
3.4.2. Repeatability (r)
r= 1,4 mg/l (except for liqueur wine)
r= 2,0 mg/l for liqueur wine.
3.4.3. Reproducibility (R)
R= 4,7 + 0,08 x
img/l.
xi= sodium concentration in the sample in mg/l.1990R2676 — EN — 18.03.2003 — 007.001 — 131
▼B
27.POTASSIUM
1. PRINCIPLE OF THE METHODS1.1.Reference method
Potassium is determined directly in the diluted wine by atomic
absorptionspectrophotometry after the additionof anion ization
suppression agent (caesium chloride) to prevent ionization of potas-
sium.
1.2.Usualmethod
Potassium is determined directly in the diluted wine by flame photo-
metry.
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 solutionmay be prepared by dissolvin g 4,813 g of potas-
sium hydrogentartrate (C
4H5KO6) in distilled water and making up
the volume to 1 litre.
2.1.2. Matrix (model) solution:
citric acid (C6H8O7·H2O)… ......................... 3,5 g
sucrose (C12H22O11)… ................................ 1,5 g
glycerol (C3H8O3)… ................................. 5,0 g
anhydrous calcium chloride (CaCl2)…............... 50 mg
anhydrous magnesium chloride (MgCl2)… ........... 50 mg
absolute alcohol (C2H5OH)… ........................ 50 ml
water to ….......................................... 500 ml
2.1.3. Caesium chloride solution containing 5 % caesium:
dissolve 6,33 g of caesium chloride, CsCl, in100 ml of distilled
water.
2.2.Apparatus
2.2.1. Atomic absorptionspectrophotometer, equipped with anair-acety-
lene 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 spectrophot-1990R2676 — EN — 18.03.2003 — 007.001 — 132
▼B
ometer, followed in succession by the standard solutions (2.3.2).
Read off the absorbences. Repeat each measurement.
2.4.Expressionofresults
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 concentra-
tionCinmilligrams per litre.
The potassium concentration expressed in milligrams per litre of the
wine to the nearest whole number will then be F×C, whereFis the
dilutionfactor (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
Inmilliequivalen ts per litre: 0,0256 × F×C.
Inmg potassium hydrogentartrate per litre: 4,813 × F×C.
3. USUAL METHOD: FLAME PHOTOMETRY
3.1.Reagents
3.1.1. Reference solution containing 100 mg potassium per litre
Absolute alcohol (C2H5OH)…..................... 10 ml
Citric acid (C6H8O7×H2O)…..................... 700 mg
Sucrose (C12H22O11)…............................. 300 mg
Glycerol (C3H8O3)…............................... 1000 mg
Sodium chloride (NaCl) …......................... 50,8 mg
Anhydrous calcium chloride (CaCl2)…............. 10 mg
Anhydrous magnesium chloride (MgCl2)…........ 10 mg
Dry potassium hydrogentartrate (C4H5KO6) …..... 481,3 mg
Water to …........................................ 1 000 ml
Dissolve the potassium hydrogentartrate in500 ml of very hot
distilled water, mix this solutionwith 400 ml of distilled water in
which the other chemicals have already beendissolved, an d make
up to one litre.
3.1.2. Dilution solution
Absolute alcohol (C2H5OH)…..................... 10 ml
Citric acid (C6H8O7·H2O… ....................... 700 mg
Sucrose (C12H22O11)…............................. 300 mg
Glycerol (C3H8O3)…............................... 1000 mg
Sodium chloride (NaCl) …......................... 50,8 mg
Anhydrous calcium chloride (CaCl2)…............. 10 mg
Anhydrous magnesium chloride (MgCl2)…........ 10 mg
Tartaric acid (C4H6O6)….......................... 383 mg
Water to …........................................ 1 000 ml
Preserve the solutions in polyethylene bottles by adding two drops of
allyl isothiocyanate.1990R2676 — EN — 18.03.2003 — 007.001 — 133
▼B
3.2.Apparatus
3.2.1. Flame photometer supplied by anair-butan e mixture.
3.3.Procedure
3.3.1. Calibration
Place 25, 50, 75 and 100 ml of the reference solution (3.1.1) into a
set of 100 ml volumetric flasks and make up to 100 ml with thedilution solution (3.1.2). Solutions containing 25, 50, 75 and 100 mg
of potassium per litre respectively are obtained in this way.
3.3.2. Determination
Make the measurements at 766 nm. Carry out the adjustment to
100 % transmission using distilled water. Successively aspirate the
standard solutions (3.3.1) directly into the burner of the photometer,
followed by the wine diluted 1:10 with distilled water and note the
readings. If necessary, the wine already diluted 1:10 may be further
diluted with the dilutionsolution(3.1.2).
3.4.Expressionofresults
3.4.1. Method of calculation
Plot the graph of the variation in percentage transmission as a func-
tion of the potassium concentrations in the standard solutions.
Record the transmission obtained for the sample of diluted wine on
this graph and determine the corresponding potassium concentration
C.
The potassium concentration in mg potassium per litre to the nearest
whole number will be F×C ,whereFis the dilutionfactor.
3.4.2. Repeatability (r)
r= 17 mg/l.
3.4.3. Reproducibility (R)
R= 66 mg/l.
3.4.4. Other ways of expressing results:
Inmilliequivalen ts per litre: 0,0256 × F×C.
Inmg potassium hydrogentartrate per litre: 4,813 × F×C.1990R2676 — EN — 18.03.2003 — 007.001 — 134
▼B
28.MAGNESIUM
1. PRINCIPLE OF THE METHOD
Magnesium is determined directly on wine, suitably diluted, by
atomic absorptionspectrophotometry.
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.Dilutestandardsolutioncontaining5mgmagnesiumperlitre.
Note:Keep the standard magnesium solutions in polyethylene bottles.
3. APPARATUS
3.1. Atomic absorptionspectrophotometer fitted with anair-acetylen e
burner.
3.2. Magnesium hollow cathode lamp.
4. PROCEDURE4.1.Preparationofsample
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 insuccessionby the stan dard solu-
tions (4.2).
Read off the absorbences. Repeat each measurement.
5. EXPRESSION OF RESULTS5.1.Methodofcalculation
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.1990R2676 — EN — 18.03.2003 — 007.001 — 135
▼B
29.CALCIUM
1. PRINCIPLE OF THE METHOD
Calcium is determined directly on wine, suitably diluted, by atomic
absorptionspectrophotometry, after additionof anion ization
suppressionagen t.
2. REAGENTS2.1.Standardsolutioncontaining1gcalcium perlitre
Use a stan dard commercial calcium solution1 g/l. This solutionmay
be prepared by dissolving 2,5 g of calcium carbonate, CaCO
3,i na
quantity of 1:10 (v/v) HCl sufficient to dissolve it completely and
making up to one litre with distilled water.
2.2.Dilutestandardsolutioncontaining50mgcalcium perlitre
Note:Keep the standard calcium solutions in polyethylene bottles.
2.3.Lanthanumchloridesolutioncontaining50glanthanumperlitre
Dissolve 13,369 g of lanthanum chloride, LaCl
3·7 H2O, indistilled
water; add 1 ml of HCl diluted 1:10 (v/v) and make up to 100 ml.
3. APPARATUS
3.1. Atomic absorptionspectrophotometer fitted with anair-acetylen e
burner.
3.2. Calcium hollow cathode lamp.
4. PROCEDURE4.1.Preparationofsample
Place 1 ml of the wine, 2 ml of the lanthanum chloride solution (2.3)
ina 20 ml volumetric flask an d 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 inpolyethylen e 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 RESULTS5.1.Methodofcalculation
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 concentra-1990R2676 — EN — 18.03.2003 — 007.001 — 136
▼B
tionC. 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.1990R2676 — EN — 18.03.2003 — 007.001 — 137
▼B
30.IRON
1. PRINCIPLE OF THE METHODS
Reference methodAfter 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 deter-
mined using the coloration produced by orthophenanthroline.
2. REFERENCE METHOD2.1.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
(FeNH
4(SO4)2· 12H2O) indistilled 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 absorptionspectrophotometer equipped with anair-acetylen e
burner.
2.2.3. Ironhollow 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 ºC). 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 successionby the five standard
solutions (2.3.2). Read off the absorbences. Repeat each measure-
ment.
2.4.Expressionofresults
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 dilutionfactor.
3. USUAL METHOD
3.1.Reagents
3.1.1. Hydrogenperoxide, H
2O2, 30 % (m/v) solution, iron-free.1990R2676 — EN — 18.03.2003 — 007.001 — 138
▼B
3.1.2. 1 M hydrochloric acid, ironfree.
3.1.3. Ammonium hydroxide ( ρ20= 0,92 g/ml).
3.1.4. Pumice stone grains, treated with boiling hydrochloric acid 1:2 dilu-
tionan d washed with distilled water.
3.1.5 Hydroquinone solution, C6H6O2, 2,5 %, acidified with 1 ml concen-
trated sulphuric acid ( ρ20= 1,84 mg/l) per 100 ml of solution. This
solutionmust be kept ina yellow bottle inthe refrigerator an d
discarded at the slightest sign of darkening.
3.1.6. Sodium sulphite solution, Na2SO3, 20 %, prepared from neutral
anhydrous sodium sulphite.
3.1.7. Orthophenanthroline solution, C12H8N2, 0,5 % in96 % vol alcohol.
3.1.8. Ammonium acetate solution, CH3COONH4, 20 % (m/v).
3.1.9. Fe(III) solutionwith 1 g of ironper litre. Use of a commercial solu-
tionis preferred. Altern atively, a 1000 mg/l Fe(III) solutioncanbe
prepared by dissolving 8,6341 g of ferric ammonium sulphate,
(FeNH4(SO4)2· 12H2O), in100 ml of 1 M hydrochloric acid (3.1.2)
and making up the volume to one litre with the 1 M hydrochloric
acid (3.1.2).
3.1.10. Dilute standard iron solution containing 100 milligrams of iron per
litre.
3.2.Apparatus
3.2.1. Kjeldahl flask, 100 ml.3.2.2. Spectrophotometer enabling measurements to be made at a wave-
length of 508 nm.
3.3.Procedure
3.3.1. Digestion
3.3.1.1. For wines with sugar content below 50 g/l:
Introduce 25 ml of the wine, 10 ml of the hydrogen peroxide solu-
tion (3.1.1) and a few grains of pumice (3.1.4) into the 100 ml
Kjeldahl flask. Concentrate the mixture to a volume of 2 to 3 ml.
Allow to cool and add to the residue, taking care not to wet the
walls of the flask, sufficient ammonium hydroxide (3.1.3) to make
the liquid alkaline and precipitate any hydroxides.
After cooling, add 1 M hydrochloric acid (3.1.2) to the alkaline
liquid in sufficient quantity to dissolve the precipitated hydroxides
and transfer the solution obtained to a 100-ml volumetric flask.
Rinse the Kjeldahl flask with 1 M hydrochloric acid (3.1.2) and
add the rinsings to the volumetric flask to make the volume up to
100 ml.
3.3.1.2. For musts and wines with sugar content a bove 50 g/l:
3.3.1.2.1. If the sugar content is between 50 and 200 mg/l, the 25 ml wine
sample is treated with 20 ml of hydrogenperoxide solution(3.1.1).
Proceed as in3.3.1.1.
3.3.1.2.2. If the sugar content is above 200 g/l, the samples of wine or must
should be diluted 1:2 or possibly 1:4 before being treated as in
3.3.1.2.1.
3.3.2. Blank experiment
Carry out a blank experiment with distilled water, using the same
volume of hydrogenperoxide solution(3.1.1) as that used for diges-
tion and following the same procedure as that described in 3.3.1.1.
3.3.3. Determination
Introduce 20 ml of the hydrochloric acid solution produced by the
digestionprocedure (3.3.1.1) an d 20 ml of the hydrochloric acid
solution from the ‘blank experiment’ (3.3.2) into two separate 50 ml
volumetric flasks. Add 2 ml of hydroquinone solution (3.1.5), 2 ml
of sulphite solution (3.1.6) and 1 ml of orthophenanthroline solution
(3.1.7) to each flask. Allow to stand for 15 minutes, during which
time the Fe(III) is reduced to Fe(II). Add 10 ml of ammonium
acetate solution(3.1.8), make up to 50 ml with distilled water an d1990R2676 — EN — 18.03.2003 — 007.001 — 139
▼B
shake the two volumetric flasks. Use the solution originating from
the blank experiment to set the zero on the absorbence scale at
508 nm and measure the absorbence of the solution to be determined
at the same wavelength.
3.3.4. Calibration
Place 0,5, 1, 1,5 and 2 ml of the 100 mg of iron per litre solution
(3.1.10) into a set of 50 ml volumetric flasks and add 20 ml of
distilled water to each. Carry out the procedure described in3.3.3
to measure the absorbence of each of these prepared standard solu-
tions, which contain 50, 100, 150 and 200 micrograms of iron
respectively.
3.4.Expressionofresults
3.4.1. Method of calculation
Plot a graph giving the variations in absorbence as a function of the
iron concentrations in the standard solutions. Record the absorbence
obtained with the solution to be examined and calculate the iron
concentration Cinthe 20 ml hydrochloric acid sample produced by
the digestionprocedure, i.e. in5 ml of the win e sample to be
analysed.
The iron concentration in milligrams per litre of the wine to one
decimal place will be 200 C.
If the wine (or must) has been diluted, the iron concentration in
milligrams per litre of the wine to one decimal place will be 200 ×
F×C, whereFis the dilutionfactor.1990R2676 — EN — 18.03.2003 — 007.001 — 140
▼B
31.COPPER
1. PRINCIPLE OF THE METHOD
The method is based onthe use of atomic absorptionspectrophoto-
metry.
2. APPARATUS
2.1. Platinum dish.2.2. Atomic absorptionspectrophotometer.
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, HNO
3, 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.Preparationofsampleanddeterminationofcopper
If required, prepare a suitably dilute solutionwith 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 stan-
dard solution s prepared in4.2, Carry out induplicate.
5. EXPRESSION OF RESULTS5.1.Methodofcalculation
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 concen-
trationCinmg/l from the calibrationcurve.
IfFis the dilution factor, the concentration of the copper present is
giveninmilligrams per litre by F×C. It is quoted to two decimal
places.
Notes:
(a) Select the solutions for establishing the calibration curve and the
dilutions of the sample appropriate to the sensitivity of the appa-
ratus to be used and the concentration of the copper present in
the sample.
(b) Proceed as follows when very low copper concentrations are
expected inthe sample to be an alysed. Place 100 ml of the1990R2676 — EN — 18.03.2003 — 007.001 — 141
▼B
sample in a platinum dish and evaporate on a waterbath at 100 ºC
until it becomes syrupy. Add 2,5 ml of concentrated nitric acid
(3.2) drop by drop, covering the bottom of the dish completely.
Carefully ash the residue onanelectric hotplate or over a low
flame; thenplace the dish ina muffle furn ace set at 500 ± 25 ºC
and leave for about one hour. After cooling, moisten the ash
with 1 ml of concentrated nitric acid (3.2) while crushing it
with a glass rod; allow the mixture to evaporate and ash again
as before. Place the dish inthe muffle furn ace againfor 15
minutes; repeat the treatment with nitric acid at least three times.
Dissolve the ash by adding 1 ml of concentrated nitric acid (3.2)
and 2 ml of doubly distilled water to the dish and transfer to a
10 ml flask. Wash the dish three times using 2 ml of doubly
distilled water each time. Finally, make up to the volume with
doubly distilled water.1990R2676 — EN — 18.03.2003 — 007.001 — 142
▼B
32.CADMIUM
1. Principle
The cadmium is determined directly in the wine by non-flame
atomic absorptionspectrophotometry.
2. APPARATUS
All the glassware must be washed prior to use in concentrated nitric
acid heated to 70 to 80 ºC and rinsed in double distilled water.
2.1. Atomic absorptionspectrophotometer 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 absorptionmeasure-
ments.
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 ina 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 solutioninsodium 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 solutionan d approximately 20 mg of erio-
chrome 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 inthe preparationof the referen ce solutionaccord-
ingly.
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.Preparationofthesample
Dilute the wine to 1:2 (v/v) with the phosphoric acid solution.
4.2.Preparationofthecalibrationrangeofsolutions
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 ºC for 30 seconds1990R2676 — EN — 18.03.2003 — 007.001 — 143
▼B
Mineralization at 900 ºC for 20 seconds
Atomizationat 2 250 ºC for 2 to 3 secon ds
Nitrogen flow (flushing gas): 6 litres/minuteNote:
At the end of the procedure, increase the temperature to 2 700 ºC to
cleanthe 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 absor-
bences measured. Calculate the mean absorbence value from the
results for the three portions.
5. EXPRESSION OF RESULTS
5.1.Methodofcalculation
Draw the absorbence variation curve as a function of the concentra-
tions 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:
2C1990R2676 — EN — 18.03.2003 — 007.001 — 144
▼B
33.SILVER
1. PRINCIPLE OF THE METHOD
The method is based onthe use of atomic absorptionspectrophoto-
metry after ashing of the sample.
2. APPARATUS
2.1. Platinum dish.2.2. Waterbath, thermostatically controlled to 100 ºC.2.3. Furnace controlled to 500 to 525 ºC.2.4. Atomic absorptionspectrophotometer.2.5. Silver hollow cathode lamp.2.6. Gas supplies: air, acetylene.
3. REAGENTS
3.1. Silver nitrate, AgNO
3.
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 solutionprepared as in3.4 are diluted to 1 000 ml with
dilute nitric acid.
4. PROCEDURE
4.1.Preparationofsample
Place 20 ml of the sample in a platinum dish and evaporate to
dryness over a boiling waterbath. Ash in the furnace at 500 to525 ºC. 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 solu-
tion s prepared in4.2. Carry out induplicate.
5. EXPRESSION OF RESULTS5.1.Methodofcalculation
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 concen-
trationCinmg/l from the calibrationcurve.
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 final1990R2676 — EN — 18.03.2003 — 007.001 — 145
▼B
volume of the liquid to be appropriate to the sensitivity of the appa-
ratus to be used.1990R2676 — EN — 18.03.2003 — 007.001 — 146
▼B
34.ZINC
1. PRINCIPLE OF THE METHOD
After removal of alcohol, zinc is determined directly in the wine by
atomic absorptionspectrophotometry.
2. REAGENTS
The water used inborosilicate glass apparatus must be doubly
distilled or of anequivalen t 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. (ZnSO4×7 H2O)
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 absorptionspectrophotometer equipped with anair-acetylen e
burner.
3.3. Zinc hollow cathode lamp.
4. PROCEDURE4.1.Preparationofsample
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 ºC). 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 insuccessionby the four stan dard
solutions. Read the absorbences. Repeat each measurement.
5. EXPRESSION OF RESULTS5.1.Methodofcalculation
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.1990R2676 — EN — 18.03.2003 — 007.001 — 147
▼B
35.LEAD
1. PRINCIPLE
The lead is determined directly in the wine by non-flame atomic
absorptionspectrophotometry.
2. APPARATUS
All the glassware must be washed prior to use in concentrated nitric
acid heated to 70 to 80 ºC and rinsed in double distilled water.
2.1. Atomic absorptionspectrophotometer 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 absorptionmeasure-
ments.
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 solutionat 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)2inn itric
acid diluted to 1 % (v/v) and made up to one litre. Keep the solution
ina borosilicate glass bottle with a groun d glass stopper.
4. PROCEDURE4.1.Preparationofthesample
Dilute the wine to 1:2 or 1:3 with the phosporic acid solution,
depending on the presumed lead concentration.
4.2.Preparationofthecalibrationrangeofsolutions
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 ºC for 30 secondsMineralization at 900 ºC for 20 seconds
Atomizationat 2 250 ºC for 2 to 3 secon ds
Nitrogen flow (flushing gas): 6 litres/minute.Note:
At the end of the procedure, increase the temperature to 2 700 ºC 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.1990R2676 — EN — 18.03.2003 — 007.001 — 148
▼B
5. EXPRESSION OF RESULTS
5.1.Methodofcalculation
Draw the absorbence variation curve as a function of the concentra-
tions of lead in the calibration range. The variation is linear. Carry
over the meanabsorben ce value of the sample solutiononthe cali-
bration 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 dilutionfactor.1990R2676 — EN — 18.03.2003 — 007.001 — 149
▼B
36.FLUORIDES
1. PRINCIPLE
The fluoride content of the wine, added to a buffer solution, is deter-
mined 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: (1)
E=Eo± S log aF
WhereE = potential of the ion-selective electrode measured against the
reference electrode in the medium being analysed;
o= standard potential of the sensor;
S = slope of the ion-selective electrode (Nernst factor). At 25 ºC
the theoretical slope is equal to 59,2 mV;
aF= 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 solu-
tionfrom the heat of the motor. Stirrin g 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. Precisionpipettes (pipettes graduated inmicrolitres or an y other
equivalent pipettes).
3. REAGENTS
3.1. Stock fluoride solutionof 1 g/l.
Use stan dard commercial quality solutionof 1 g/l. This solutioncan
be prepared by dissolving 2,210 g of sodium fluoride (dried for three
to four hours at 105 ºC) indistilled water. Make up to on e litre with
distilled water. The solutionis kept ina 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 inadvan ce.
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 ( ρ
20= 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 ºC (± 1 ºC) during measurement. (A deviation of
more than1 ºC causes a modificationof about 0,2 mV.)
4.1.Directmethod
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 by1990R2676 — EN — 18.03.2003 — 007.001 — 150
▼B
not more than 0,2 to 0,3 mV/three minutes), read the value of the
potential in mV.
4.2.Theknownadditionsmethod
Stirring continuously, add a known volume of standard fluoride solu-
tion 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:
(a) double or treble the fluoride concentration in the analysis
medium;
(b) the volume of the analysis medium must remain practically
constant (an increase in volume of 1 % or less).
(Condition (b) simplifies the calculations, see 5.)
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:
CF¼Va/C2Ca
Vo/C21
antilog ∆E=S ðÞ /C0 1
CF= fluoride concentration of the analysis medium (mg/l);
Ca= concentration of fluoride added (mg/l) to analysis medium
(Va);
Vo= initial volume of the analysis medium before overloading
(ml);
Va= volume of the overloaded solution(ml);
∆E= difference between potentials E1andE2obtained in 4.1 and
4.2 (mV);
S= slope of the electrode in the analysis solution.
If Vais very close to Vo(see 4.2), the following simplified formula
is applied:
CF¼Ca/C21
antilog ∆E=S ðÞ /C0 1
The value obtained must be multiplied by the dilution factor arising
from additionof the buffer solution .1990R2676 — EN — 18.03.2003 — 007.001 — 151
▼B
37.CARBONDIOXIDE
1. PRINCIPLE OF METHODS1.1.Reference method
1.1.1. Still wines (CO
2over pressure ≤0,5 × 105Pa)(1)
The volume of wine taken from the sample is cooled to around 0 ºC
and mixed with a sufficient quantity of sodium hydroxide to give a
pH of 10 to 11. Titrationis carried out with anacid solutioninthe
presence of carbonic anhydrase. The carbon dioxide content is calcu-
lated 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 decarbona-
tion, the remainder of the bottle is made alkaline to fix all the
carbondioxide inthe form of Na2CO3. Titrationis 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 condi-
tions in decarbonated wine in order to take account of the volume
of sodium hydroxide takenup by the win e acids.
1.2.Usualmethod: sparkling and semi-sparkling wines
Manometric method: the excess pressure of the carbon dioxide is
measured directly inthe bottle usin g anaphrometer.
2. REFERENCE METHOD
2.1.Stillwines (CO2over pressure ≤0,5 × 105Pa)
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, H
2SO4, 0,05 M.
2.1.2.3. Carbonic anhydrase solution, 1 g/l.2.1.3. Procedure
Cool the wine sample to approximately 0 ºC together with the 10 ml
pipette used for sampling.
Place 25 ml of sodium hydroxide solution(2.1.2.1) ina 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 ºC.
Place the beaker on the magnetic stirrer, set up the pH electrode and
stir moderately.
Whenthe liquid has reached room temperature, titrate slowly with
the sulphuric acid solution(2.1.2.2) un til 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 nml be the volume used betweenpH 8,6 an d 4,0.
Remove CO
2from approximately 50 ml of the wine sample by
agitation under vacuum for three minutes, the flask being heated in
a waterbath to about 25 ºC.
Carry out the above procedure on 10 ml of the decarbonated wine.
Letn′ml be the volume used.1990R2676 — EN — 18.03.2003 — 007.001 — 152
(1)1 05pascal (Pa) = 1 bar.
▼B
2.1.4. Expression of results
1 ml of the titrated 0,1 M sodium hydroxide solutioncorrespon ds to
4,4 mg of CO2.
The quantity of CO2ingrams per litre of win e is givenby the
formula:
0,44 (n−n′)
It is quoted to two decimal places.Note:Where wines contain little CO
2(CO2< 1 g/l), the additionof
carbonic anhydrase to catalyse the hydration of CO2is unnecessary.
2.2.Sparklingandsemi-sparklingwines
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, H
2SO4, 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, νml, 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) inthe 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 un til 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 nml be the volume used betweenpH 8,6 an d
4,0.
Remove CO
2from the νml of wine placed on one side for the blank
titration by agitating under vacuum for three minutes, the flask being
heated ina waterbath at about 25 ºC. Remove 10 ml of decarbon ated
wine and add to 30 ml of boiled distilled water, add two to three
drops of sodium hydroxide solution(2.2.2.1) to brin g the pH to 10
to 11. Thenfollow the above procedure. Let n′ml be the volume
of 0,5 M sulphuric acid added.1990R2676 — EN — 18.03.2003 — 007.001 — 153
▼B
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 deter-
mine to within 1 ml the initial volume of wine by making up to the
mark with water, say Vml.
The quantity of CO2ingrams per litre of win e is givenby the
following formula:
0;44n/C0n0ðÞ /C2V/C0νþ20 ðÞ
V/C0ν
The result is quoted to two decimal places.
2.3.Expressionofresults
The excess pressure at 20 ºC (Paph20) expressed inpascals is given
by the formula:
Paph20¼Q
1;951/C210/C050;86/C00;01A ðÞ 1/C00;00144S ðÞ/C0Patm
where:
Q:C O2content in g/l of wine,
A: the alcoholic strength of wine at 20 ºC,
S: the sugar content of the wine in g/l,
Patm: the atmospheric pressure, expressed inpascals.
3. USUAL METHOD: SPARKLING AND SEMI-SPARKLING
WINES
3.1.Apparatus
3.1.1. Aphrometer
The apparatus enabling the excess pressure in bottles of sparkling
and semi-sparkling wine is called an aphrometer. It takes a different
form according to the way in which the bottle is stoppered (metallic
top, cap, cork or plastic stopper, see Figures 1 and 2).
They are graduated inpascals (Pa)(1), although it is more practical
to use 105Pa or the kilopascal (kPa) as the unit.
They fall into various classes. The class of a manometer is the preci-
sion of a reading in relation to a full scale reading expressed as a
percentage (e.g. a 1000 kPa manometer class I means that the
maximum pressure of 1 000 kPa canbe read to within± 10 kPa).
A class I instrument is recommended for accurate measurement.
Aphrometers must be checked regularly (at least once a year).
3.2.Procedure
Measurements must be carried out on bottles whose temperature has
beenstabilized for at least 24 hours.
After having pierced the cap, the cork or the stopper, the bottle must
then be shaken vigorously until the pressure is constant in order to
take the reading.1990R2676 — EN — 18.03.2003 — 007.001 — 154
(1) 1 Pa = 1 N/m2=1 0−5bar.
▼B
Figure 1: Aphrometer for caps
Figure 2: Aphrometer for corks and stop-
pers
3.3.Expressionofresults
The excess pressure at 20 ºC (Paph20) is expressed inpascals (Pa) or
inkilopascals (kPa).
It must be quoted in a form consistent with the precision of the
manometer (e.g. 6,3 × 105Pa or 630 kPa and not 6,33 × 105Pa or
633 kPa for a class I manometer with a full scale reading of
1 000 kPa).
If the temperature at which the mesurement is carried out is different
from 20 ºC, a correctionshould be made by multiplyin g the
measured pressure by the coefficient
Paph20
Papht
giveninTable I. This relates the result to 20 ºC.
4. RELATIONSHIP BETWEEN THE PRESSURE AND THE QUAN-
TITY OF CARBON DIOXIDE CONTAINED IN A SEMI-
SPARKLING WINE(1)
From the excess pressure at 20 ºC (Paph20), the absolute pressure at
20 ºC (Pabs20) is calculated using the formula:
Pabs20= Patm + Paph20
where Patm is the atmospheric pressure expressed inbars.The quantity of carbon dioxide contained in a wine is given by the
following relationships:
— inlitres of CO
2per litre of wine:
0,987 × 10−5Pabs20(0,86 − 0,01 A) (1 − 0,00144 S),1990R2676 — EN — 18.03.2003 — 007.001 — 155
(1) No account is taken of other gases present (O2,N2, etc.) inamoun ts that are too small
to have any effect on the excess pressure.
▼B
— ingrams of CO2per litre of wine:
1,951 × 10−5Pabs20(0,86 − 0,01 A) (1 − 0,00144 S),
whereAis the alcoholic strength of the wine at 20 ºC,
Sis the sugar content of the wine in grams per litre.
Table I
RatiooftheexcesspressurePaph20inasparklingorsemi-sparklingwineat
20ºCtotheexcesspressurePaphtatatemperature
0 1,85 13 1,24
1 1,80 14 1,20
2 1,74 15 1,16
3 1,68 16 1,13
4 1,64 17 1,09
5 1,59 18 1,06
6 1,54 19 1,03
7 1,50 20 1,00
8 1,45 21 0,97
9 1,40 22 0,95
10 1,36 23 0,93
11 1,32 24 0,91
12 1,28 25 0,881990R2676 — EN — 18.03.2003 — 007.001 — 156
▼M7
38.CYANIDEDERIVATIVES
(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 chlora-
mine-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. APPARATUS2.1.Distillationapparatus
Use the distillation apparatus described for determining the alcohol
content of wine
2.2. 500-ml round-bottomed flask with standardised ground joints2.3. Water bath thermostatically controlled at 20 ºC
2.4. Spectrophotometer allowing absorbance to be measured at wave-
length of 590 nm
2.5. Glass cells or single-use cells with optical paths of 20 mm
3. REAGENTS
3.1. Phosphoric acid (H
3PO4) at 25 % (m/v)
3.2. Chloramine-T solution (C7H7ClNNa O2S, 3H2O) 3 % (m/v)
3.3. Solutionof 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) solutionat 10 % (m/v)
3.6. Silver nitrate solution (AgNO3), 0,1 M
4. PROCEDURE4.1.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 imme-
diately 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 iniced water. Collect 30 to 35 ml of distil-
late (or aroun d 45 ml liquid intotal) inthe calibrated flask.
Flush the tapering tube of the condenser with a few millilitres of
distilled water, bring distillate to 20 ºC 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-barbi-
turic 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 titrationofthepotassiumcyanide
Dissolve around 0,2 g KCN (3.4), carefully measured, in 100 ml
distilled water ina 300-ml calibrated flask. Add 0,2 ml of potassium1990R2676 — EN — 18.03.2003 — 007.001 — 157
▼M7
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.Standardcurve
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 /approxorequal72,3 ml KCN). Dilute the
solutionto 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 cali-
bration 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 solu-
tions 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.Methodofcalculation
Read off the hydrocyanic acid content on the calibration curve. If the
sample has beendiluted, multiply the result by the dilutionfactor.
Repeatability (r) and reproducibility (R)White wine = r = 3,1 µg/l or approximately 6 %· x
i
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.1990R2676 — EN — 18.03.2003 — 007.001 — 158
▼B
39.ALLYLISOTHIOCYANATE
1. PRINCIPLE OF THE METHOD
Any allyl isothiocyanate present in the wine is collected by distilla-
tion 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 ºC).
3. APPARATUS
3.1. Distillationapparatus as showninthe 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 dispersionrods 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 ºC 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:
— injector temperature: 200 ºC,
— columntemperature: 130 ºC,— helium carrier gas flow rate: 20 ml per minute.
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 chromato-
graph. 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.1990R2676 — EN — 18.03.2003 — 007.001 — 159
▼B
Apparatus for distillation under a current of nitrogen
1990R2676 — EN — 18.03.2003 — 007.001 — 160
▼B
40.CHROMATICPROPERTIES
1. WINES AND MUSTS1.1.Definitions
The chromatic properties of a wine are defined as its luminosity and
its chromaticity.
Theluminosity is represented by the transmittance and it varies
inversely with the colour intensity of the wine.
Thechromaticity 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 prop-
erties 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 ofthemethods
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 proper-
ties are expressed by convention as follows:
Thecolour intensity is givenby the sum of the absorben ces at wave-
lengths af 420, 520 and 620 nm for radiation traversing a 1 cm
optical path inthe sample.
Thetintis 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 inpairs, 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. Preparationof 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, inthe glass cell should be so chosenthat 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, ina 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
beA
445,A495,A550,A625.
1.3.3. Calculations
Together with Table I, use these values of the absorbences for the
optical path, bcm to obtain the corresponding transmittances ( T%).
Let these be T445,T495,T550andT625.
— Calculate the tristimulus values X, YandZexpressed as decimal
fractions from the following expressions:1990R2676 — EN — 18.03.2003 — 007.001 — 161
▼B
X= 0,42T625+ 0,35T550+ 0,21T445
Y= 0,20T625+ 0,63T550+ 0,17T495
Z= 0,24T495+ 0,94T445
— Calculate the chromaticity coordinates xandyfrom:
x¼X
XþYþZy¼Y
XþYþZ
1.3.4. Expression of results
1.3.4.1. The relative luminosity is given by the value of Yexpressed 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 chroma-
ticity 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
andyo= 0,3163, representing daylight of average brightness.
— Dominant wavelength
Plot the point C with coordinates x,yonthe 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 corre-
sponding 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:
100/C2distance from C to O
distance from O to S
If the point C is inside the triangle AOB, the purity is given as a
percentage by the ratio:
100 ×distance from C to O(1)
distance from O to P
where P is the point where the straight line OC cuts the line of
purples (line AB).
Purity is also givendirectly by chromaticity diagrams from the
known values of xandy(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 inwhich the measuremen ts were made.
1.4.Usualmethod
1.4.1. Apparatus
1.4.1.1. Spectrophotometer enabling measurements to be made between 300
and 700 nm.
1.4.1.2. Glass cells inpairs, with optical paths, b, equal to 0,1, 0,2, 0,5 and
1 cm.1990R2676 — EN — 18.03.2003 — 007.001 — 162
(1) This distance must be given in a direction going from O to C.
▼B
1.4.2. Preliminary 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.
1.4.3. Procedure
The optical path, b, inthe glass cell should be chosenso that the
measured absorbence A lies between 0,3 and 0,7.
The spectrophotometric measurements should be made using
distilled water, ina cell with the same optical path, b, as reference
liquid to establish the zero of the absorbence scale at wavelengths
420, 520 and 620 nm.
1.4.4. Expression of results
1.4.4.1. Calculations
Calculate the absorbences at the three wavelengths for an optical
path of 1 cm by dividing the measured absorbances by bincm. Let
these beA420,A520andA620.
The colour intensity Iis by convention given by the following
expression:
I=A420+A520+A620
It is quoted to three decimal places.
The tintNis by convention given by the following expression:
N¼A420
A520
It is quoted to three decimal places.1990R2676 — EN — 18.03.2003 — 007.001 — 163
▼B
TABLE 1
Transformationofabsorbencestotransmittances(T%)
Method of useFind 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 between2 an d 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 inter-
polation.
0123456789
0 1000
23977229552293321912218912087120851198321981319
1 79418776187591774117724167081669216676156611564615
2 63114617146031458914575135621354913537125251251312
3 50111490114791146811457104479436942710417104079
4 3989389938093718363835583478339833173248
5 3167309730272957288628272756269626362576
6 2516245524062345229522452195214520952045
7 1994195519041864182417841744170416641624
8 1583155415131484144414131383135313231293
9 1263123312031172115311221103107210531022
Example:
Absorbence 0,47 1,47 2,47 3,47
T% 33,9 3,4 0,3 0
Transmittances T% are to be expressed to the nearest 0,1 %.1990R2676 — EN — 18.03.2003 — 007.001 — 164
▼B
FIGURE 1
Chromaticitydiagramincludingallcoloursinthespectrum1990R2676 — EN — 18.03.2003 — 007.001 — 165
▼B
FIGURE 2
Chromaticity diagram for unadulterated red (bright red) wines
andred-brown(brickred)wines1990R2676 — EN — 18.03.2003 — 007.001 — 166
▼B
FIGURE 3
Chromaticity diagram for unadulterated red (bright red) wines
andred-brown(brickred)wines1990R2676 — EN — 18.03.2003 — 007.001 — 167
▼B
FIGURE 4
Chromaticity diagram for unadulterated red (bright red) wines
andpurplewines1990R2676 — EN — 18.03.2003 — 007.001 — 168
▼B
FIGURE 5
Chromaticity diagram for unadulterated red (bright red) wines
andpurplewines1990R2676 — EN — 18.03.2003 — 007.001 — 169
▼B
FIGURE 6
Chromaticity diagram for red-brown (brick red) wines and
purplewines1990R2676 — EN — 18.03.2003 — 007.001 — 170
▼B
2. RECTIFIED CONCENTRATED MUSTS
2.1.Principle ofthemethod
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) (25º 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) (25º
Brix) prepared as described inthe chapter ‘pH’, section4.1.2. 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 Aat the same wavelength of the solution
containing 25 % sugar (25º Brix) prepared as in 2.3.1 and placed in
a cell with anoptical path of 1 cm.
2.4.Expressionofresults
The absorbence at 425 nm of the rectified concentrated must in a
solutionwith 25 % sugar (25º Brix) is quoted to two decimal places.1990R2676 — EN — 18.03.2003 — 007.001 — 171
▼B
41.FOLIN-CIOCALTEUINDEX
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 oxidationof the phen ols, is reduced to a
mixture of the blue oxides of tungsten (W8O23) and molybdenum
(Mo8O23).
The blue colorationproduced has a maximum absorptioninthe
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
(Na2WO4·2 H2O) and 25 g of sodium molybdate (Na2MoO4·2 H2O)
in700 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.Redwine
Introduce the following into a 100 ml volumetric flask (4.1) strictly
inthe order given :
1 ml of the wine, previously diluted 1:5,
50 ml of distilled water,
5 ml of Folin-Ciocalteu reagent (3.1),
20 ml of sodium carbonate solution (3.2).
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.Whitewine
Carry out the same procedure with 1 ml of undiluted wine.1990R2676 — EN — 18.03.2003 — 007.001 — 172
▼B
5.3.Rectifiedconcentrated must
5.3.1. Preparation of sample
Use the solution with a sugar concentration of 25 % (m/m) (25º
Brix) prepared as described inthe chapter ‘pH’, section4.1.2.
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 RESULTS6.1.Methodofcalculation
The result is expressed in the form of an index obtained by multi-
plying 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).1990R2676 — EN — 18.03.2003 — 007.001 — 173
▼B
42.SPECIAL METHODS OF ANALYSIS FORR ECTIFIED CONCEN-
TRATEDGRAPEMUST
(a) TOTAL CATIONS1. 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 approxi-
mately 300 mm, fitted with a draintap.
2.2. pH meter with a scale graduated at least in0,1 pH un its.
2.3. Electrodes:
— glass electrode, kept indistilled water,
— calomel/saturated potassium chloride reference electrode, kept in
a saturated solutionof potassium chloride,
— or a combined electrode, kept in distilled water.
3. REAGENTS
3.1. Strongly acid cation exchange resin in H+form pre-swollenby
soaking in water overnight.
3.2. Sodium hydroxide solution, 0,1 M.
3.3. Paper pH indicator.
4. PROCEDURE4.1.Preparationofsample
Use the solution obtained by diluting the rectified concentrated must
to 40 % (m/v) as described inthe chapter ‘Total acidity’, section
5.1.2.
4.2.Preparationoftheionexchangecolumn
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.Ionexchange
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 solutionun til the pH is 7 at
20 ºC. The alkaline solution should be added slowly and the solution
continuously shaken. Let nml be the volume of 0,1 M sodium
hydroxide solutionused.
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 milliequiva-
lents per kilogram (see ‘Total acidity’, section 6.1.2): a
— Total cations in milliequivalents per kilogram of total sugars:
2;5n/C0a
P/C21001990R2676 — EN — 18.03.2003 — 007.001 — 174
▼B
Where
P= percentage concentration (m/m) of total sugars.
(b) CONDUCTIVITY1. 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 ºC.
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 ºC (20 ± 2 ºC).
3. REAGENTS
3.1. Demineralized water with specific conductivity below 2 µS cm−1at
20 ºC.
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 ºC, in demineralized water
(3.1). Make up to one litre with demineralized water (3.1). This solu-
tionhas a con ductivity of 1 000 µS cm−1at 20 ºC. It should not be
kept for more thanthree mon ths.
4. PROCEDURE4.1.Preparationofthesampletobeanalysed
Use the solution with a total sugar concentration of 25 % (m/m) (25º
Brix) as described inthe chapter ‘pH’, section4.1.2.
4.2.Determinationofconductivity
Bring the sample to be analysed to 20 ºC by immersion in a water-
bath. Maintain the temperature to within ± 0,1 ºC.
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 inmicrosiemen s per cm (µS cm−1)a t2 0º Ct o
the nearest whole number for the 25 % (m/m) (25º 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 ºC, add the correction; if the temperature is
above 20 ºC, subtract the correction.
TABLE I
Corrections to be made to the conductivity for temperatures different from 20 ºC (µS cm−1)
Conduc-
tivityTemperature (ºC)
20,2
19,820,4
19,620,6
19,420,8
19,221,0
19,021,2
18,821,4
18,621,6
18,421,8
18,222,0 (1)
18,0 (2)
0 000000000 0
5 0 001111122 2
1 0 0 011223334 41 5 0 112334556 71990R2676 — EN — 18.03.2003 — 007.001 — 175
▼B
Conduc-
tivityTemperature (ºC)
20,2
19,820,4
19,620,6
19,420,8
19,221,0
19,021,2
18,821,4
18,621,6
18,421,8
18,222,0 (1)
18,0 (2)
2 0 0 123345678 9
2 5 0 12346789 1 01 13 0 0 1345789 1 1 1 21 3
350 1 3 5 6 8 9 11 12 14 15
400 2 3 5 7 9 11 12 14 16 184 5 0 2 3 6 81 01 21 41 61 8 2 05 0 0 2 4 7 91 11 31 51 82 0 2 2550 2 5 7 10 12 14 17 19 22 24600 3 5 8 11 13 16 18 21 24 26
(1) Subtract the correction.
(2) Add the correction.
(c) HYDROXYMETHYLFURFURAL (HMF)
1. PRINCIPLE OF THE METHODS
1.1.Colorimetric method
Aldehydes derived from furan, the main one being hydroxymethyl-
furfural, react with barbituric acid and paratoluidine to give a red
compound which is determined by colorimetry at 550 nm.
1.2.High-performanceliquidchromatography(HPLC)
Separationthrough a columnby reversed-phase chromatography an d
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, indistilled water an d
heat slightly over a waterbath at 100 ºC. Make up to 100 ml with
distilled water. The solutionkeeps for about a week.
2.2.2. Paratoluidine solution, 10 % (m/v).
Place 10 g of paratoluidine, C6H4(CH3)NH2, ina 100 ml volumetric
flask; add 50 ml of isopropanol, CH3CH(OH)CH3,a nd1 0m lo f
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 inthe chapter ‘Total acidity’, section
5.1.2. Carry out the determination on 2 ml of this solution.1990R2676 — EN — 18.03.2003 — 007.001 — 176
▼B
2.3.2. Colorimetric determination
Into each of two 25 ml flasks aandbfitted with ground glass stop-
pers place 2 ml of the sample prepared as in2.3.1. Place ineach
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 flaska. 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 bfor a wavelength of
550 nm. Follow the variation in the absorbence of the contents of
flaska;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 aand
band treat them as described in 2.3.2.
The graph representing the variation of absorbence with the hydro-
xymethylfurfural concentration in mg/l is a straight line passing
through the origin.
2.4.Expressionofresults
The hydroxymethylfurfural concentration in rectified concentrated
musts is expressed inmilligrams per kilogram of total sugars.
2.4.1. Method of calculation
The hydroxymethylfurfural concentration Cmg/l inthe sample to be
analysed is that concentration on the calibration curve corresponding
to the absorbence Ameasured onthe sample.
The hydroxymethylfurfural concentration in milligrams per kilogram
of total sugars is givenby:
250/C2C
P
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:
— a loop injector, 5 or 10 µl,
— spectrophotometer detector for making measurements at 280 nm,— column of octadecyl-bonded silica (e.g. Bondapak C18 - Corasil,
Waters Ass.),
— a recorder, possibly anin tegrator.
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, CH
3OH, 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 hydroxymethylfur-
fural, C6H3O6, accurately weighed, and make up to the mark with1990R2676 — EN — 18.03.2003 — 007.001 — 177
▼B
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 ina brownglass bottle ina refrigerator, this solutionwill
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 inthe 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
sevenmin utes.
3.4.EXPRESSIONOFRESULTS
The hydroxymethylfurfural concentration in rectified concentrated
musts is expressed inmilligrams per kilogram of total sugars.
3.4.1. Method of calculation
Let the hydroxymethylfurfural concentration in the 40 % (m/v) solu-
tion of the rectified concentrated must be Cmg/l.
The hydroxymethylfurfural concentration in milligrams per kilogram
of total sugars is givenby:
250/C2C
P
where
P= percentage (m/m) concentration of total sugars in the rectified
concentrated must.
(d) HEAVY METALS
1. PRINCIPLE OF THE METHODS
I.Rapidmethodforevaluationofheavymetals
Heavy metals are revealed in the suitably diluted rectified concen-
trated must by the colorationproduced by the formationof
sulphides. They are assessed by comparisonwith a stan dard lead
solution corresponding to the maximum admissible concentration.
II.Determination of lead content by atomic absorption spectropho-
tometry
The chelate given by lead with ammonium pyrrolidinedithiocarba-
mate 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.1990R2676 — EN — 18.03.2003 — 007.001 — 178
▼B
2.1.4. pH 3,5 buffer solution.
Dissolve 25 g of ammonium acetate CH3COONH4, in25 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 solutionC2H5SN, 4 % (m/v).
2.1.6. Glycerol solution, C3H8O3, 85 % (m/v), (nD20 ºC= 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 solutionan d 20 ml of
glycerol (2.1.6). Heat ona waterbath at 100 ºC for 20 secon ds.
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 solutionwith water to two parts in1000 (v/
v) inorder to obtaina 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 concen-
trated 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 ABSORP-
TION SPECTROPHOTOMETRY
3.1.Apparatus
3.1.1. Atomic absorptionspectrophotometer equipped with anair-acetylen e
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.1990R2676 — EN — 18.03.2003 — 007.001 — 179
▼B
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 solu-
tion containing 0,010 g/l of lead (3.2.4). Treat these in the same way
as the solutionto be examin ed.
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 solu-
tions.
3.4.Expressionofresults
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 concentra-
tion corresponding to the solution to be examined.
Extrapolate the straight line joining the points until it cuts the nega-
tive part of the concentration axis. The distance of the point of
intersection from the origin gives the lead concentration in the solu-
tionto be examin ed.
(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 recti-
fied concentrated musts.
1. PRINCIPLE OF THE METHOD
Simple distillationof the liquid. Oxidationof the ethan ol inthe
distillate by potassium dichromate. Titrationof the excess dichro-
mate with aniron(II) solution .
2. APPARATUS
2.1. Use the distillationapparatus described inthe chapter ‘Alcoholic
strength by volume’, section 3.2.
3. REAGENTS
3.1.Potassiumdichromatesolution.
Dissolve 33,600 g of potassium dichromate, K
2Cr2O7, insufficien t
quantity of water to make one litre of solution at 20 ºC.
One millilitre of this solution oxidizes 7,8924 mg of alcohol.
3.2.Iron(II)ammoniumsulphatesolution.
Dissolve 135 g of iron(II) ammon ium sulphate, FeSO4· (NH4)2SO4·6H2O, 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 solutionmore or less correspon ds to half its volume of
dichromate solutionwhenjust prepared. Subsequen tly, it oxidizes
slowly.
3.3.Potassiumpermanganatesolution.
Dissolve 1,088 g of potassium permanganate, KMnO4, ina sufficien t
quantity of water to make one litre of solution.1990R2676 — EN — 18.03.2003 — 007.001 — 180
▼B
3.4.Dilutesulphuricacid,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.Ferrousorthophenanthrolinereagent.
Dissolve 0,695 g of ferrous sulphate, FeSO4·7H2O, in100 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. PROCEDURE4.1.Distillation
Place 100 g of rectified concentrated must and 100 ml of water in
the distillationflask. Collect the distillate ina 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 titrationof the iron(II) ammon ium sulphate solution
(3.2) with respect to the potassium dichromate solutionby placin g
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 perman-
ganate solution (3.3). Subtract a tenth of the volume of this solution
used from the volume of the iron(II) solutionadded. Let the differ-
ence benml.
Proceed inthe same way with the ‘con trol’ 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.Methodofcalculation
n′ml of ferrous solutionreduces 20 ml of dichromate solutionwhich
oxidizes 157,85 mg of pure ethanol.
One millilitre of iron (II) solution has the same reducing power as
157 ;85
n0mg of ethanol :
n−n′ml of iron(II) solutionhave the same reducin g power as
157 ;85n/C0n0ðÞ
n0mg ethanol
Ethanol concentration in g/kg of rectified concentrated must is given
by:1990R2676 — EN — 18.03.2003 — 007.001 — 181
▼B
7;892/C2n0/C0n ðÞ
n
Ethanol concentration in g/kg of total sugars is given by:
789 ;2/C2n0/C0n ðÞ
n0P
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. infused silica, coated with OV 1, film thick-
ness of 0,15 µm, length 25 m and internal diameter of 0,3 mm)
Operating conditions:
— carrier gas: hydrogenor helium,
— carrier gas flow rate: about 2 ml/minute,— injector and detector temperature: 300 ºC,
— programming of temperature: 1 minute at 160 ºC, 4 ºC per
minute to 260 ºC, constant temperature of 260 ºC 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 Teflonstopper.
3.7. Oven.
4. METHOD OF OPERATION
An accurately weighed sample of about 5 g of rectified concentrated
must is placed ina 50 ml flask. 1 µl of stan dard solutionof 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 ºC 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:1990R2676 — EN — 18.03.2003 — 007.001 — 182
▼B
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 solutionis weighed an d 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 over-
leaf), 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 ingrams per kilogram of
must.1990R2676 — EN — 18.03.2003 — 007.001 — 183
▼B
Chromatogram in gaseous phase of meso-inositol, scyllo-inositol and sucrose1990R2676 — EN — 18.03.2003 — 007.001 — 184
▼M4
43.DETERMINATION OF THE ISOTOPIC RATIO18O/16O OF THE
WATERCONTENTINWINES
I. DESCRIPTION OF THE METHOD
1.Methodobjective
The objective of the present method is to measure the isotopic ratio
18O/16O of waters of different origins. The isotopic ratio18O/16O can
be expressed indeviation δ‰ inratio to the value of isotopic ratio
of the international reference V.SMOW:
δiè0½/C138 ¼Ri
RSMOW/C01/C20/C21
/C21000
2.Principle
The isotopic ratio18O/16O is determined by mass spectrometry of
isotopic ratios (MSIR) from ionic currents m/z 46 (12C16O18O) and
m/z 44 (12C16O2) produced by carbondioxide obtain ed after an
exchange with the water in wine according to the reaction:
C16O2+H218O↔C16O18O+H216O
The carbondioxide inthe gaseous phase is used for an alysis.
3.Reagents
— Carbondioxide for an alysis
— SMOW (Standard Mean Ocean Water)
— GISP (Greenland Ice Sheet Precipitation)
— SLAP (Standard Light Arctic Precipitation)
— Reference water specific to the laboratory carefully standardized
in relation to the reference sample of the International Agency of
Atomic Energy in Vienna (IAEA).
4.Laboratoryequipment
— mass spectrometer of isotopic ratios with an internal repeatability
of 0,05 ‰
— triple collector for simultaneous recording of ions m/z 44, 45 and
46 or, by default, a double collector for measuring ions m/z 44
and 46
— thermostated system (± 0,5 ºC) to carry out the equilibration
betweenCO2and the water content in wine
— vacuum pump able to reach an internal pressure of 0,13 Pa
— phials for samples having 15 ml volume and a capillary annex
tube with anin terior diameter of about 0,015 mm
— Eppendorf pipette with plastic throw-away cone.
5.Experimentaldeterminations
5.1. Manual method
Operational mode of the equilibration method
Introduction of the sample
— Take the Eppendorf pipette at the fixed volume of 1,5 ml, adapt
a cone and pump the liquid to be analysed in order to insert it in
a balloon flask. Then, place silicon grease around the neck of the
balloonflask an d attach the balloonflask to the valve while veri-
fying that it is tightly shut,
— Repeat the operationfor each balloonflask onthe work ramp
while introducing the laboratory's reference water into one of
the balloons.
Degasing of the ramp
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.1990R2676 — EN — 18.03.2003 — 007.001 — 185
▼M4
Equilibrationof the water an d the CO2
Cool the work ramps to − 70 ºC (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
CO2introduction system. Insert the gaseous CO2into the work
ramp and, after having isolated it from the rest of the system, intro-
duce the ramp ina thermostated bath at 25 ºC (± 0,5 ºC) 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.
Transfer of the CO2exchanged in the measuring cells
A sample holder which supports as many measuring cells as balloon
flasks containing exchanged CO2is 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
Inorder 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 ºC. The sample slides containing the frozen
products are adapted to the equilibration system, and after having
created a vacuum inthe system, carbondioxide is in troduced at a
pressure of 800 hPa.
The balance is reached at a temperature of 22 ± 0,5 ºC after a
minimum period of five hours and with moderate agitation. Since
the equilibrationdurationdepen ds onthe phial's geometry, the
optimum durationshould be determin ed 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.Calculationandexpressionoftheresults
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:
δ0sample ¼I46=I44 ðÞ sample
I46=I44 ðÞ reference/C01/C20/C21
/C21000
The18O content of the sample compared to the reference V.SMOW
onthe V.SMOW/SLAP scale, is givenby the relation :
δ018O¼δ0sample /C0δ0SMOW
δ0SMOW /C0δ0SLAP/C20/C21
/C255;5
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
— the repeatability (r) is equal to 0,24 ‰
— the reproductibility (R) is equal to 0,50 ‰.1990R2676 — EN — 18.03.2003 — 007.001 — 186
▼M7
44.DETERMINATION OF ETHYL CARBAMATE IN WINE: SELEC-
TIVE DETECTION METHOD USING GAS CHROMATOGRAPHY/
MASSSPECTROMETRY
(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.Apparatusandchromatographicconditions(example)
(a) Gas chromatogram/mass spectrometer (GC/MS) and if necessary a
sample filter and data treatment system or equivalent
Capillary columnof fused silica 30 m (1) × 0,25 mm internal diameter,
0,25 µm of Carbowax 20M
Operation: injector 180 ºC, helium gas vector at 1 ml/minute at 25 ºC,
injection by splitless method
Temperature programme: 40 ºC for 0,75 minutes, rising thereafter by
10 ºC/minute up to 60 ºC, then by 3 ºC/minute up to 150 ºC(2), rising
to 220 ºC and maintaining that temperature for 4,25 minutes. Specific
retention time for ethyl carbamate is 23 to 27 minutes, that for propyl
carbamate is 27 to 31 minutes.
Gas chromatogram/spectrometer (GC/MS) interface: transfer line 220 ºC.
Mass spectrometer parameters manually tuned with perfluorotributyla-
mine and optimised for a lower mass sensitivity, SIM acquisition mode,
solvent delay and acquisition start time 22 minutes, dwell time/ion
100 ms.
(b) Vacuum rotary evaporator or concentration system similar to Kuderna
Danish.
(NB:the rate of recovery of ethyl carbamate from the test sample, C(g)
must be between 90 and 110 % during the process.)
(c) Flask — pear-shaped, 300 ml, single ground neck
(d) Concentration tube — 4 ml, graduated, with a teflon-coated joint and a
cork
C.Reagents
(a) Acetone — quality LC
(NB:check each batch before use in GC/MS for absence of response for
m/z 62, 74 and 89 ions.)
(b) Dichloromethane
(NB:analyse each batch before use in GC/MS after 200-fold concentra-
tion, to check for absence of response for m/z 62, 74 and 89 ions.)
(c) Ethanol — anhydrous
(d) Ethyl carbamate (EC) standard solutions
1. Stock solution— 1,00 mg/ml. Weigh 100 mg EC (purity ≥99 %) in
a 100 ml volumetric flask and dilute with acetone.
2. Standard working solution — 10,0 µg/ml. Transfer 1 ml of the stock
EC solution to a 100 ml volumetric flask and dilute with acetone up
to the mark.1990R2676 — EN — 18.03.2003 — 007.001 — 187
(1) For certainparticularly rich win es, a 50 m capillary columnmay be desirable.
(2) For certain particularly rich wines, a temperature programme of 2 ºC/minute may be
desirable.
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(e) Propyl carbamate (PC), standard solutions
1. Stock solution— 1,00 mg/ml. Weigh 100 mg PC (reagen t grade) ina
100 ml volumetric flask and dilute with acetone up to the mark.
2. Standard working solution — 10,0 µg/ml. Transfer 1 ml of the stock
PC solution to a 100 ml volumetric flask and dilute with acetone up
to the mark.
3. Internal standard solution PC — 400 ng/ml. Transfer 4 ml of the
standard PC working solution to a 100 ml volumetric flask and dilute
with water up to the mark.
(f) Standard calibrated solutions EC-PC
Dilute EC standard working solution (d)(2) and PC standard working
solution (e)(2), with dichloromethane to obtain:
1. (100 ng EC and 400 ng PC)/ml;
2. (200 ng EC and 400 ng PC)/ml;
3. (400 ng EC and 400 ng PC)/ml;
4. (800 ng EC and 400 ng PC)/ml;
5. (1 600 ng EC and 400 ng PC)/ml.
(g) Test sample — 100 ng EC/ml in 40 % of ethanol
Transfer 1 ml of EC standard working solution (d)(2) to a 100 ml volu-
metric flask and dilute with 40 % of ethanol up to the mark.
(h) Solid phase extractioncolumn— disposable material, pre-packed with
diatomaceous earth, capacity 50 ml
NB:Before analysis, check each batch of extraction columns for the
recovery of EC and PC and the absence of response for ions of 62,74
and 89 m/z. Prepare 100 ng EC/ml of test sample (g). Analyse 5,00 ml
of the test sample as described inD(a), E an d F. The recovery of 90 to
110 ng of EC/ml is satisfactory. Absorbents whose particle diameter is
irregular canlead to a slow flow which affects the recovery of EC an d
PC. If 90 to 110 % of the test sample value is not obtained after several
trials, change the column or use a corrected calibration recovery curve to
quantify EC. To obtain the corrected calibration curve, prepare standard
solutions as described in (f) by using 40 % ethanol instead of dichloro-
methane.
Analyse 1 ml of the standard calibration solution as described in D, E
and F.
Establish a new calibration curve by using the EC/PC ratio of the
extracted standards.
D.Preparationofthetestsample
Place the following volumes of test material in two separate 100 ml beakers:
(a) wines containing over 14 % vol of alcohol: 5,00 ± 0,01 ml;
(b) wines containing maximum 14 % vol of alcohol: 20,00 ± 0,01 ml.
To each beaker, add l ml of internal standard PC solution C(e)(3) and water,
to obtaina total volume of 40 ml (or 40 g).
E.Extraction
Extractionshould be carried out un der anextractor 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 dichlor-
omethane. Collect the eluate in a 300 ml conical flask.
Evaporate the eluate from 2 to 3 ml ina water bath rotary evaporator at
30 ºC. (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.1990R2676 — EN — 18.03.2003 — 007.001 — 188
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F.GC/MSanalysis
(a) Calibrationcurve
Inject 1 µl of each standard calibration solution C(f) into the GC/MS.
Plot the graph of EC-PC area ratio for the m/z 62 ionrespon se onthe
vertical axis and the quantity of EC in ng/ml on the horizontal axis
(100, 200, 400, 800, 1600 ng/ml).
(b) EC quantification
Inject 1 µl of concentrated extract prepared under E into the GC/MS
system and calculate the EC-PC area ratio for the m/z 62 ion. Establish
the concentration of EC (ng/ml) in the extract by using the internal stan-
dard calibration curve. Calculate the EC concentration in the test sample
(ng/ml) by dividing the quantity of EC (ng) in the extract by the test
sample volume (ml).
(c) Confirmation of EC identity
Determine whether the responses for the m/z 62, 74 and 89 ions appear
during the period of EC retention. These responses are features of the
mainfragmen ts (M - C2H2)+and (M - CH3)+and molecular ion (M)+
respectively. The presence of EC is confirmed if the relative ratios ofthese ions are within 20 % of the ratios for an EC standard. The extract
may need to be further concentrated in order to obtain a sufficient
response for the m/z 89 ion.
G.Collaborativeanalysis
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 (RSD
R) tends to decrease as the concentration of
ethyl carbamate increases.
Performance of the method for the determination of ethyl carbamate EC in alcoholic
beveragesbyGC/MS
SampleAverage
EC found
(ng/ml)Recovery
of added
EC
(%)SrSRRSDr(%)RSDR(%)
Wines > 14 % vol 40 1,59 4,77 4,01 12,02
80 89 3,32 7,00 4,14 8,74
162 90 8,20 11,11 5,05 6,84
Wines≤14 % vol 11 0,43 2,03 3,94 18,47
25 93 1,67 2,67 6,73 10,73
48 93 1,97 4,25 4,10 8,861990R2676 — EN — 18.03.2003 — 007.001 — 189
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45.DETERMINATION BY ISOTOPE MASS SPECTROMETRY OF
THE13C/12C RATIO IN WINE ETHANOL OR ETHANOL
OBTAINED BY THE FERMENTATION OF MUSTS, CONCEN-
TRATED MUSTS OR RECTIFIED CONCENTRATED MUSTS
1. FIELD OF APPLICATION
The method enables measurement of the13C/12C isotope ratio inwin e
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 reproduci-
bility 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 carbon13 (13C) to carbon12 (12C) isotopes for a
givensample.
δ13C: carbon 13 content (13C) expressed inparts 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
inSouth Carolin a (USA). Its13C/12C isotope ratio or RPDBis 0,0112372. PDB reserves have beenexhausted for along 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 rela-
tionto 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 C
3metabolism (Calvin
cycle) and the C4metabolism (Hatch and Slack). These two photosynth-
esis mechanisms present a different type of isotope fractionation.
Products of C4plants, such as sugars and alcohol derived from fermenta-
tion, have higher levels of carbon 13 than similar products of C3plants.
Most plants, including vines and sugar beets, belong to the C3group.
Sugar cane and maize belong to the C4group. Measuring the carbon 13
content enables the detection and evaluation of sugars of C4origin(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 C3and C4plants 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 (13C16O2and12C17O16O) and 46
(12C16O18O), resulting from the different possible combinations of isotopes
18O,17O,16O,13Ca nd12C, are determined from the ionic currents
measured by three different collectors of a mass isotopic spectrometer.
The contributions of isotopomers13C17O16Oa nd12C17O2may be disre-
garded given their low levels. The ionic current for m/z = 45 is
corrected for the contribution of12C17O16O, which is calculated according
to the current intensity measured for m/z = 46, while taking the relative
abundance of18Oa nd17O 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.1990R2676 — EN — 18.03.2003 — 007.001 — 190
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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 insealed metal capsules or the in jectionof liquid samples
through a septum using a syringe.
Depending on the type of instrument used, the following reference mate-
rials, reagents, and consumables may be used:
— reference materials
— available from the IAEA:
Name Materiel δ13C relative to V-PDB (9)
— IAEA-CH-6 Sucrose - 10,4 ‰
— IAEA-CH-7 Polyethylene - 31,8 ‰
— NBS22 Oil - 29,7 ‰
— USGS24 Graphite - 16,1 ‰
— available from the IRMM in Geel (B) (Institute for Reference Mate-
rials and Measurements):
Name Material δ13C relative to V-PDB (9)
— CRM/BCR 656 Wine alcohol - 26,93 ‰
— CRM/BCR 657 Glucose - 10,75 ‰
— CRM/BCR 660 Hydroalcoholic solution
(ASV 12 %)- 26,72 ‰
— a standard working sample with a known13C/12C ratio calibrated
against international reference materials,
— the following is an indicative list of consumables for continuous-flow
systems:
— helium for analysis (CAS 07440-59-7),
— oxygenfor an alysis (CAS 07782-44-7),
— carbon dioxide for analysis, used as a secondary reference gas for
the carbon 13 content (CAS 00124-38-9),
— oxidation reagent for the furnace of the combustion system, for
example copper ( ΙΙ)oxide for elemental analysis (CAS 1317-38-
0),
— a desiccant to eliminate water produced in combustion, for
example anhydrone for elemental analysis (magnesium perchlo-
rate) (CAS 10034-81-8) (This is not necessary for apparatuses
equipped with a water elimination system using cryogenic traps
or selectively permeable capillaries).
6. APPARATUS AND EQUIPMENT
6.1.Isotoperatiomassspectrometer (IRMS)
Isotope ratio mass spectrometer (IRMS) capable of determining the rela-
tive13C content of naturally occurring CO2gas with an internal accuracy
of 0,05 ‰ or better expressed as a relative value (point 9). Internal accu-
racy 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 spec-
trometer 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
carbondioxide from the other combustionproducts before measuremen t
inthe mass spectrometer.
6.2.Combustionapparatus
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 contin-
uous-flow system integrated with the mass spectrometry apparatus (point1990R2676 — EN — 18.03.2003 — 007.001 — 191
▼M8
6.2.1) or a separate combustionsystem (poin t 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:
— calibrated microsyringe or micropipette with appropriate tips,
— balance with µg accuracy or better,
— tweezers for encapsulation,— tincapsules for liquid samples,
— tincapsules for solid samples.
Note:
inorder to reduce the risk of evaporationof ethan ol samples, anabsor-
bent 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 preparationsystem:
— syringe for liquids,
— flasks equipped with airtight closing systems and inert septa.
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 carbondioxide resultin g from the combustionof the
samples to be analysed and the reference sample are collected in bulbs
which are thenplaced inthe dual in let of the spectrometer for isotopic
analysis. Several combustion apparatuses described in the literature may
be used:
— closed combustion system filled with circulating oxygen,
— elemental analyser with helium and oxygen flow,
— sealed glass bulb filled with copper ( ΙΙ)oxide as anoxidationagen t.
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 inpoin t 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 composi-
tionof 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
evaporationmay be used for the preparationof carbondioxide for
isotopic analysis.
Experimental procedure based on the use of an elemental analyser:
(a) placing the samples in capsules:
— use capsules, tweezers and a preparation tray, all of which must
be clean,
— take anappropriate-sized capsule usin g the tweezers,
— introduce an appropriate amount of liquid into the capsule using a
micropipette,1990R2676 — EN — 18.03.2003 — 007.001 — 192
▼M8
—Note:
3,84 mg of absolute ethanol or 4,17 mg of distillate with an
alcohol strength of 92 % m/m are necessary to obtain 2 mg ofcarbon. The appropriate quantity of distillate must be calculated
on that basis, according to the quantity of carbon necessary given
the sensitivity of the mass spectrometry apparatus,
— close the capsule with the tweezers,— each capsule must be completely sealed. If not, it must be
discarded and a new capsule prepared,
— two capsules must be prepared for every sample,— place the capsules inthe appropriate place onthe tray of the auto-
matic sampler of the elemental analyser. Every capsule must be
carefully identified by a serial number,
— systematically place capsules containing working references at the
beginning and the end of the sample series,
— regularly insert control samples in the sample series;
(b) checking and adjusting the elemental analysis and mass spectrometry
apparatus:
— adjust the temperature of the elemental analyser furnaces and the
helium and oxygen flows for optimal combustion of the sample,
— check the elemental analysis and mass spectrometry system for
leaks (for example by checking the ionic current where m/z = 28
for N
2),
— adjust the mass spectrometer to measure the ionic currents where
m/z = 44, 45 and 46,
— check the system using known control samples before starting to
measure the samples;
(c) carrying out a series of measurements
The samples placed onthe automatic sampler of the elemen tal
analyser (or of the chromatograph) are introduced in turn. The carbon
dioxide from each sample combustionis eluted towards the mass
spectrometer which measures the ionic currents. The interfaced
computer records the ionic currents and calculates the δvalue for
each sample (point 9).
9. CALCULATION
The purpose of the method is to measure the13C/12C isotope ratio
ofethanol extracted from wine or from products derived from grapes
following fermentation. The13C/12C isotope ratiocanbe expressed by its
deviation from a working reference. The isotopic deviation of carbon 13
(δ13C) is thencalculated ona delta scale per thousan d ( 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 δ13C values are expressed inrela-
tion to the working reference as follows:
δ13Csam/ref‰ = 1 000 × (Rsam-Rref)/Rref
where Rsamand Rrefare respectively the13C/12C isotope ratios of the
sample and of the carbon dioxide used as the reference gas.
The δ13C values are expressed inrelationto V-PDB as follows:
δ13Csam/V-PDB‰= δ13Csam/ref+δ13Cref/V-PDB+(δ13Csam/ref×δ13Cref/V-PDB)/1 000,
where δ13Cref/V-PDBis 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 δ13C 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 varia-
tion, 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.1990R2676 — EN — 18.03.2003 — 007.001 — 193
▼M8
10. QUALITY ASSURANCE AND CONTROL
Check that the13C value for the working reference does not differ by
more than0,5 ‰ from the admissible value. If n ot, 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 than0,3 ‰. The fin al result for a given
sample is the average value for the two capsules. If the deviationis
greater than0,3 ‰, the measuremen t 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 distil-
lation systems do not produce significant variation in the determination of
theδ13C 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.Jointstudyondistillates
Year of interlaboratory tests: 1996
Number of laboratories: 20Number of samples: Six samples indouble-blin d comparisonAnalyte: δ
13C of ethanol
Sample codeAlcohol of vinous
originBeet alcohol Sugar cane alcohol
A&G 8 0% 1 0% 1 0%
B & C 90 % 10 % 0 %D & F 0 % 100 % 0 %
E & I 90 % 0 % 10 %
H & K 100 % 0 % 0 %J & L 0 % 0 % 100 %
Samples A/G B/C D/F E/I H/K J/L
Number of labora-
tories retained after
eliminating aberrant
results19 18 17 19 19 19
Number of results
accepted38 36 34 38 38 38
Average value
(δ13C) ‰- 25,32 - 26,75 - 27,79 - 25,26 - 26,63 - 12,54
Sr20,0064 0,0077 0,0031 0,0127 0,0069 0,00411990R2676 — EN — 18.03.2003 — 007.001 — 194
▼M8
Samples A/G B/C D/F E/I H/K J/L
Repeatability standard
deviation(Sr) ‰0,08 0,09 0,06 0,11 0,08 0,06
Limit of repeatability
r (2,8 × Sr)‰0,22 0,25 0,16 0,32 0,23 0,18
SR20,0389 0,0309 0,0382 0,0459 0,0316 0,0584
Reproducibility stan-
dard deviation(SR)‰0,20 0,18 0,20 0,21 0,18 0,24
Limit of reproduci-
bility R (2,8 × SR)0,55 0,49 0,55 0,60 0,50 0,68
11.2.Interlaboratorystudyontwowinesandonealcohol
Year of interlaboratory tests: 1996
Number of laboratories: 14 for distillationof win e of which seven
also measured δ13C of wine ethanol,
Eight for measuring δ13C 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: δ13C of ethanol
Samples Red wine White wine Alcohol
Number of labora-
tories778
Number of results
accepted778
Average value ( δ13C) ‰- 26,20 - 26,20 - 25,08
Reproducibility
variance SR20,0525 0,0740 0,0962
Reproducibility stan-
dard deviation(SR)‰0,23 0,27 0,31
Limit of reproduci-
bility R (2,8 × SR)‰0,64 0,76 0,87
Different distillation systems were used by the participating laboratories.
The isotopic determinations ( δ13C) carried out ina sin gle 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 variationinresults (S2= 0,0059) is comparable to the repeat-
ability variances Sr2in the joint study on distillates (point 11.1).
11.3.Results of the exercises carried out to monitor proficiency in
performingisotopictests
Since December 1994, international proficiency tests for the determina-
tion 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 determination1990R2676 — EN — 18.03.2003 — 007.001 — 195
▼M8
ofδ13C for wine and distillate ethanol are summarised in the following
table:
DateWines Distillates
NSRS2
RRNSRS2
RR
December 1994 6 0,210 0,044 0,59 6 0,151 0,023 0,42
June 1995 8 0,133 0,018 0,37 8 0,147 0,021 0,41
December 1995 7 0,075 0,006 0,21 8 0,115 0,013 0,32
March 1996 9 0,249 0,062 0,70 11 0,278 0,077 0,78
June 1996 8 0,127 0,016 0,36 8 0,189 0,036 0,53
September 1996 10 0,147 0,022 0,41 11 0,224 0,050 0,63
December 1996 10 0,330 0,109 0,92 9 0,057 0,003 0,16
March 1997 10 0,069 0,005 0,19 8 0,059 0,003 0,16
June 1997 11 0,280 0,079 0,78 11 0,175 0,031 0,49
September 1997 12 0,237 0,056 0,66 11 0,203 0,041 0,57
December 1997 11 0,127 0,016 0,36 12 0,156 0,024 0,44
March 1998 12 0,285 0,081 0,80 13 0,245 0,060 0,69
June 1998 12 0,182 0,033 0,51 12 0,263 0,069 0,74
September 1998 11 0,264 0,070 0,74 12 0,327 0,107 0,91Weighted average 0,215 0,046 0,60 0,209 0,044 0,59
N: number of participating laboratories.
11.4.Limitsofrepeatabilityandreproducibility
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:
limit of repeatability r: 0,24
limit of reproducibility R: 0,6.1990R2676 — EN — 18.03.2003 — 007.001 — 196