INTERNATIONAL ORGANISATION OF VINE AND WINE

GUIDELINES ON AUTOMATED COLORIMETRIC

ANALYSERS IN OENOLOGY

Resolution OIV-OENO 391-2010,

adopted at the OIV General Assembly the 25th June 2010 in Tbilisi

© OIV 2013
1
 Guidelines on automatic analysers in oenology

The first development of automatic analysis in oenology appeared in the beginning of the
1970’s. Up until that time, they were developed based on continuous flow analysers used
essentially in medical biology. The principle accessible parameters were volatile acidity,
reducing sugars, free and total sulfur dioxide. Enzymatic techniques were later acquired,
authorising particularly the dosage of glucose and fructose and malic and lactic acids.

In 1980 near infrared analysers enabled the rapid determination of alcoholic strength by
volume in wine and sugar found in musts.

The first sequential analysers, derived from the same medical analysis, appeared in the
beginning of the 1990’s. This allowed rapid access to numerous determinations based on
chemical reactions (total phenolic compounds, free and total sulphur dioxide, tartaric and
enzymatic acid (glucose and fructose, malic and lactic acid, citric acid…)
Later at the end of the 1990’s, infrared technology transformed (IRTF) completed by
processing automaton available to oenology laboratories.

All these automatic techniques are currently found worldwide. These techniques have
resulted in a significant lowering of oenological analysis costs which has lead to the
possibility of accessing, for a given cost, increasing parameters and thus increasing
information. This has enabled the continual and increasingly efficient analysis of the
steps of the life of a wine

Automatic analysis has quickly proven to be reliable and consistent. provided to setting
up adapted internal quality control techniques.

A fundamental characteristic of all automatic techniques is that these techniques are

strictly comparative. Automatic analysers must involve initial grading with scales with
known values. These gradings may be permanent, semi-permanent, daily or may
associated with each sample series. The quality of results obtained is directly dependant
on the set up grading scale. These scales may be for wine or musts presenting values
known for sought after parameters.They may also be synthetic solutions.

Automatic methods oftentimes remain internal methods for which each laboratory must
ensure the quality of results using different approaches including:
- The initial validation of the method
- Quality of grading and control scales used
- Very regular participation in interlaboratory comparison chains
- Regular comparison of results with results obtained by reference methods, etc…

The widespread use of automatic techniques in the wine world has made their
technological, commercial and control role genuine and fundamental for the economy and
for vitivinicultural exchanges. This situation justifies the publication of this guide which
serves not as a reference document but rather for information purposes. The objective of
this document is to:
- Describe the principle of implemented techniques, their application practice and
limits of use
- Provide details on methods usually applied taking into account that these methods
are oftentimes partially adapted by laboratories in accordance with the apparatus

Certified in conformity
Tbilisi, 25th June 2010
The General Director of the OIV
Secretary of the General Assembly

Federico CASTELLUCCI
© OIV 2010 2/40
used or matrixes implemented. The list proposed is in no case restrictive. Other
applications are possible. New developments are proposed on a regular basis.

3/40
 GUIDELINES FOR AUTOMATED COLORIMETRIC ANALYSERS
IN OENOLOGY

Warning; this guide is not a reference document and is only provided for its informative
value. Unlike the methods contained in the book of international wine and must analysis
methods of the OIV or in the book of international grape-based spirits’ analysis methods
of the OIV, the methods contained herein cannot be considered as reference methods.
Only the methods published in the book of international wine and must analysis methods
of the OIV or in the book of international grape-based spirits’ analysis methods of the
OIV are official and can be used for the settlement of any possible dispute that may
arise.

These analysers automate the various steps of a chemical or enzymatic analysis using colorimetric
detection. There are two main categories used in oenology:

• Continuous flow analysers;

• Sequential analysers.

1 CONTINUOUS FLOW ANALYSERS

The first work on the automation of chemical analysis by continuous flow in oenology was
completed by MORFAUX, SARRIS et al. between 1969 and 1972. It was however only from 1974
onward that this technique gained ground in oenology laboratories with the development of reliable
and repeatable methods.

1.1 Composition and principle of an automated continuous flow analyser chain

An automated continuous flow analysis chain is made up of separate modules that each carries out
a very specific task (figure 1).

DISTILLATION
ASSEMBLY

SAMPLE

AIR
DILUENT
AIR
REAGENT

RECORDER ANALYTICAL PERISTALTIC SAMPLE

CASSETTE PUMP DISTRIBUTOR
COLORIMETER

Assembly diagram of an automated continuous flow

analyser chain

1.2 The sample distributor

The distributor contains a plate which can generally hold 20 to 60 cups of samples of a volume
ranging from 1 to 3 ml.
A swivelling arm equipped with a needle takes the samples one after another. Between each
sample, the needle is immersed into a rinsing solution. The needle is connected to the remainder of
the analytical circuit by a PVC tube. The rinsing solution, a sample, more rinsing solution and a new
sample are successively sucked through the needle into the analytical circuit. Accordingly, the
analysis rate is regulated at the sample distributor level. If the needle samples for one minute then
rinses for one minute, the rate will be of 30 samples per hour. It is possible to regulate this rate as

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desired, as well as the ratio between rinse time and sampling time, which does not necessarily
have to be 1 as in the example above, but may also be 2, 1/2, 1/3, 1/4, etc.

1.3 A peristaltic pump

This is the heart of the automated analysis chain. All liquids moving through the analytical circuit
are regulated by the pump, which generally contains from 20 to 30 channels. Each one consists of
a tube, generally out of PVC, which is crushed against the housing by a series of rollers. The
advance of these rollers makes the liquid circulate inside the pump tube. The flow of each tube is
dependent on its internal diameter. Therefore, by using tubes of different diameters (0.005 to
0.110 cm) and although the rate at which the pump rollers advance is perfectly consistent, there is
a very wide range of flow rates.
This enables precise control of the rates at which the sample and various reagents enter the
analytical circuit.

1.4 An analytical cassette

The analytical cassette includes all the elements of the analytical circuit. At the cassette level, the
sample can be subjected to various treatments according to the analysis to be performed:
• dilution;
• dialysis;
• addition of reagent;
• mixing;
• distillation;
• transfer to thermostated water bath (variable temperature and duration).

Part of the circuit can be thermoregulated.

From the moment they are released from the pump, all liquid flows moving through the analytical
circuit are regularly segmented by air bubbles every 1 or 2 centimetres. Surface tension forces
enable these air bubbles to prevent the circulation of a compound solution from one section of the
liquid column – caught between two bubbles – towards the previous or following section.
Furthermore, the air bubble dries the wall of the glass or PVC tube in which it is moving.

This eliminates any risk of contamination from one sample to the other, and prevents a sample
from being dissolved by the rinsing solution that precedes and follows it.

This fundamental principle of separation of liquids by an air bubble is applied starting from the
sample distributor. Indeed, aspiration at the sampling needle level is constant; when the needle
goes from the rinsing bath to the sample cup, a small amount of air is taken, creating a bubble
that, in the sample tube, separates the rinsing solution from the sample.

A second generation of continuous flow equipment using the microflow technique, which avoids
segmentation, was proposed. However, due to clogging problems that are difficult to solve, this
type of equipment remains poorly widespread. The analytical principles remain the same as for
segmentation instruments.

1.5 A detector
The most common type of detection is colorimetry. The majority of automated chemical analyses
are based on a colometric method, particularly in the oenological field. However, other detectors
can be used:

• UV spectrophotometer;
• flame photometer;
• fluorescence meter;
• nephelometer;
• cell counter;
• refractometer;
• specific electrodes;

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 1.6 A recorder
The presence of the compound sought in the sample taken is indicated, at the detector level, by a
signal whose intensity is proportional to the concentration of the compound. This signal is recorded
as peaks (fig. 2).
Between each peak, the return to the baseline corresponds to the rinsing solution being taken.
The reliability of the continuous flow analysis method lies in reading the recorder using the
following principles:

1° Creating a state of equilibrium in the analytical circuit so that the return to the baseline
always occurs when the rinsing solution is being taken due to a given concentration in the sample
of the analysed compound shows a constant-height peak.

2° The determination method is a comparative method. All measurements are based on a

standard range whose concentrations are perfectly known.

Recording is affected when the circuit is not operating properly (drift of the baseline, variation in
peak shapes, variation in calibration peak heights, etc.), which limits analysis errors.
Response

Time in minutes

Fig. 2

Recording example obtained in the case of volatile acidity determination in wines at a 30-
sample per hour rate.
(The values shown are expressed in g/L of sulfuric acid)

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 1.7 A digital interface
This converts the analogical signal from the colorimeter into a digital value that can be directly
expressed in concentration of the compound measured. It generally includes a control system to
make sure that the determination process was not adversely affected.

2 SEQUENTIAL ANALYSERS

2.1 Principle and organisation of a sequence analyser

These analysers are generally multiparametric. They can therefore perform several determinations
on the same sample, and these determinations can be programmed for each sample. Each sample
to be analysed is taken, placed in a transparent measuring vessel where it receives the reagent(s).
A colorimetric measurement is performed directly on the vessel. Rates range from 50 to 1000
determinations per hour. This is mainly applied either in chemical analysis or in enzymatic analysis.
Necessary reagent volumes are low, which minimises the costs, particularly for enzymatic
determinations. A sequence analyser comprises several components.

2.1.1. Composition of a sequence analyser

2.1.1.1 A sampling plate

This can contain a variable number of cups in which the samples to be analysed are placed.

2.1.1.2. A reaction plate

This is composed of transparent vessels that receive the test specimen of the sample to be
analysed and the necessary reagent(s). They are maintained in a thermostated bath at constant
temperature and enable colorimetric reading. The vessels can be single-use or cleaned by a jet
spray before further use.

2.1.1.3. A reagent plate

This holds the reagent containers to perform desired determinations. They are placed in a cooled
enclosure, which allows the use of fragile reagents in the case of enzymatic methods.

2.1.1.4. Sampling arm(s)

Using an automatic syringe system, sampling arms take samples on the sampling plate as well as
reagents on the reagent plate and place them successively in the reaction vessel.

2.1.1.5. An optical reader

This enables colorimetric measurement of the reaction vessels, either at specific moments
(beginning and end of reaction for instance), or dynamically in order to produce a reaction curve
for each one of them. In this second case, the measurement of the initial speed of reaction can be
evaluated and used for calculating the concentration of the required compound.

2.1.1.6. An electronic/computerised module

This ensures system operation and calculations necessary to obtain the desired result.

2.1.2. Analytical scheme

The analytical scheme can be variable. The example below describes the most traditional cases. It
comprises the following steps:
Sampling
The wine sample, whose volume is given for each determination, is taken from the sample plate
and placed in a reaction vessel.

2.1.2.1. Addition of the first reagent

The first reagent (R1) is taken from the reagent plate, for a specific volume, and added in the
reaction vessel. The mixture is generally homogenised by an agitator system.

2.1.2.2. Time delay 1

The reaction mixture is left as is for a given amount of time (often 4 to 6 minutes).

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2.1.2.3. Absorbance measurement
The first absorbance measurement at the required wavelength is performed. It corresponds to level
zero of the implemented reaction and helps take into account any possible influence of the wine’s
colour.

2.1.2.4. Addition of the second reagent

The second reagent (R2) is added to the reaction vessel. This is the reagent that triggers the
desired colorimetric reaction.

2.1.2.5. Time delay 2

The reaction mixture is left as is for a given amount of time (often 4 to 6 minutes).

2.1.2.6. Absorbance measurement

The second absorbance measurement at the required wavelength is performed. It measures the
absorbance variation due to the reaction itself.
This basic scheme can vary. As a result, for most powerful instruments, the absorbance
measurement can be performed dynamically with a reading every 12 seconds, for instance. This
produces a reaction curve and measures the initial speed of reaction which, under specific
analytical conditions, can be proportional to the concentration of the required compound.

2.1.2.7. Calculation of concentrations

For oenological applications, this is always performed by calibration with wines of known
concentration in analyte or with synthetic solutions of this analyte. The number of calibration
standards can be variable, and higher if the reaction curve is not linear.
Only one reagent can be used with some materials, which eliminates the possibility of performing
certain determinations for which two distinct reagents are necessary. However, instruments
allowing the use of two reagents can easily use one only, if that is enough for the considered
method.

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 APPENDIX 1

EXAMPLES OF USUAL DOSAGE METHODS BY AUTOMATIC CONTINUOUS FLOW

ANALYSERS USED IN ENOLOGY

The methods described here are examples. Other methods may also be used.

1 Determination of content of volatile acidity in wines and musts

1.1 Principle
Volatile acidity is formed from fatty acids of the acetic series. Lactic, succinic, carbonic acids and
sulphur dioxide are excluded from volatile acidity. Acids of the acetic series are separated from
wine components by micro-distillation at 98°C under nitrogen current. Lactic acid is rectified during
distillation. Sulphur dioxide is oxidised into sulphuric acid by hydrogen peroxide and is eliminated
before the distillation step. Carbon dioxide does not interfere. The distillate of acids of the acetic
series is put in the presence of a redox reagent whose colouring intensity variations are
proportional to the volatile acidity content: potassium iodide or bromophenol blue.

1.2 Apparatus
Segmented flow chain.
1.3 Reagents and solutions
1.3.1 Hydrogen peroxide solution
• Hydrogen peroxide (CAS no[7722-84-1]) 30 to 35% (or 110 vol.) 0.5 ml
• demineralised water up to 200 ml.
Conservation: 1 day.

1.3.2 5% tartaric acid solution

• pure, crystallised tartaric acid (L, D or DL) 50 g
• demineralised water up to 1000 ml.
Conservation in a tinted bottle: 1 month at + 4°C.

1.3.3 Potassium iodide and iodate

• KI solution (CAS no[7681-11-0]) at 10% (p/v)
• KIO3 solution (CAS no[7758-05-6]) at 0.2% (p/v)
Conservation in a tinted bottle: 1 month.
The neutrality of KI and KIO3 solutions can be checked by mixing small quantities in a tube. The
mixture must remain colourless.

1.3.4 Bromophenol blue

• Coloured indicator
- bromophenol blue (CAS no[115-39-9]) 330 mg
- 0.1 M potassium phosphates (CAS no[7758-1-4 ]) 100 ml
- 0.1 M sodium tetrahydroborate (CAS no[16940-66-2]) 5 ml
Distilled water up to 1000 ml.
Stir and allow to rest for 3 days safe from light.
Before use, filter then adjust pH to 4.9 with the sodium tetrahydroborate solution.
1.4 Calibration solutions (any of the following)
1.4.1 Acetic acid solutions (CAS no[64-19-7]) from 0.2 to 1 g∙l-1 expressed in H2SO4 (from 4 to 20
mEq or 0.28 to 1.40 g∙l-1 in acetic acid)
Conservation: 1 day.

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1.4.2 Reference wines of known volatile acidity content
1.5 Procedure
The flow chart is provided in the appendix.
The samples do not undergo any preliminary treatment. The analysis rate can vary from 30 to 60
samples per hour depending on the assembly used.
1.6 Characteristics of the described method
Intralaboratory reproducibility: 0.05 g∙l-1 in H2SO4 (0.07 g∙l-1 of acetic acid)
Interlaboratory reproducibility: 0.10 g∙l-1 in H2SO4 (0.14 g∙l-1 of acetic acid)

1.7 Bibliography
DUBERNET M. Automatic determination of volatile acidity in wines. Connaiss. Vigne et Vin,
1976,10,3, 297-309.
PILONE G.J. Effect of lactic acid on volatile acid determination of wine. J of Oenol. 1967,18, 149-
156.
SARIS J, MORFAUX J.N., DERVIN L. Automatic determination of volatile acidity of wine. Ind. Alim.
Agric., 1970, 87, 115-121.

1.8 Appendices: Flow chart examples

1.8.1 Bromophenol blue

Pump Flow rates

Channel 1 160mb nitrogen

rotations rotations
95°C
5% tartaric acid
water
bath Sample
Channel
2
rotations

Sink

rotat.

Reactor Sink

rotations
Bromophenol blue
Colorimeter
15mm light path
Colorimeter recovery
Sink

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1.8.2 Potassium iodide

Assembly
diagram
Flow direction Flow rates (ml/min)
Nitrogen
(flow meter position 30
Distilling
Water
column or 30 L/min) Distilled water

Tartaric acid
Coolant

H2O2 (+acetic
acid)

Water
95°C water bath Sink
Bidistilled water

Sink Bubble trap

Sink

Sink
Spectrometer

Recorder
5V, max calib

2 Determination of content of L-malic and L-lactic acids in wines and musts

2.1. Principle
In the presence of nicotinamide adenine dinucleotide (NAD), the acid is oxidised in a reaction
catalysed by its specific enzyme:
• malate dehydrogenase (MDH) in the case of malic acid,
• lactate dehydrogenase (LDH) in the case of lactic acid.

It is an equilibrium reaction that is moved in the direction of NADH formation by a basic hydrazine
buffer and an excess of NAD+.
MDH
L-malate + NAD+ < = = > oxaloacetate hydrazone + NADH + H+
LDH
L-lactate + NAD+ < = = > pyruvate hydrazone + NADH + H+
The quantity of NADH produced is proportional to the acid concentration of the sample. Its
absorbance is measured at 340 nm.

2.2 Apparatus
Segmented continuous flow chain.

2.3 Reagents

2.3.1 Brij 35 (CAS no[9002-92-0]) at 30% (p/v)

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2.3.2 Buffer of a pH of about 9.5
For 500 ml:
• Pure glycine (CAS no[56-40-6]) 38 g
• Hydrazine sulphate (CAS no[10034-93-2]) 26 g
• EDTA (CAS no[60-00-1]) 1,0 g
• Sodium hydroxide (CAS no[1310-73-2]) 20 g
• Brij 35 30% 5 drops
• Bidistilled water up to 500 ml.
Filter if necessary. Maximum conservation: 2 months at +4°C.

2.3.3 NAD+, MDH and LHD

• β Nicotinamide adenine dinucleotide (reduced form) (NAD) (CAS no[53-84-9]) with purity
≥ 98%
• MDH suspension in ammonium sulphate (CAS no[9001-64-3])
• LDH suspension in ammonium sulphate (CAS no[9001-60-9])
Conservation:
• Unopened MDH or LDH bottle at -20°C until the expiry date.
• Opened bottle: +4°C, 6 months maximum or until the expiry date.
• NAD powder is stored dry at +4°C until the expiry date.

2.3.4 Enzymatic solutions for the analysis

• NAD solution with 100 mg to 10 ml of bidistilled water.
• MDH or LDH solution: 100 µl in 10 ml of bidistilled water. The quantities to be prepared
depend on the duration of the analysis.
Conservation: one week at +4°C.

2.3.4.1 Reference solutions of L-malic (CAS no[97-67-6]) or L-lactic (CAS no[79-33-4]) acid from
0.5 to 5 g/l

2.4 Procedure
The flow chart is provided in the appendix.
The samples do not undergo any preliminary treatment. In the case of samples with suspended
matter, centrifugation is essential.
The analytical rate is at least 30 samples per hour, with sampling time / rinse time = 1/1.
Note: For deeply coloured red wines, the absorbance due to colouring of the wine at 340 nm will be
subtracted from the absorbance measured, by running the samples using reagents without the
enzyme, but with the NAD+coenzyme.

2.5 Expression of the results

The results are expressed in grams per litre to one decimal place.

2.6 Characteristics of the described method

Interlaboratory reproducibility: 0.15 g∙l-1

2.7 Bibliography
Battle, J-L and Herdsman, J.C., Automation of enzymatic determination for oenological analysis,
Revue Française d’Œnologie (Cahier Scientifique), 1986, 26, (101): 38-43.
Battle, J-L, Joubert, R., Collon, Y. and Jouret, C, Continuous flow enzymatic determination of L-
malate and L-lactate in grape musts and wines, Ann. Fais. Exp. Chim., 1978, 71(766): 223-228.

Curvelo-Garcia, A. S., Reliability of segmented continuous flow methods applied to the analysis of
wines and musts, Feuillet Vert de l’OIV no941 (1993).

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2.8 Appendix: Flow chart example

exch./h
Sink ml/min
rotations
Sink
6 inch dialysis
Buffer

Buffer
MDH or LDH
rotations

10° delay

Sink

rotations

340 nm colo.

3. Determination of the content of tartaric acid in wines and musts

3.1 Principle
The Rebelein method is applied to dialysed wine samples to develop a red-orange colour at hot
temperature (37°C) with sodium metavanadate in alkaline medium. The absorbance is measured
at 520 nm. High malic acid and sugar contents may interfere.

3.2 Apparatus
Segmented continuous flow chain.

3.3 Reagents

3.3.1 Brij 35 (CAS no[9002-92-0]) at 30% p/v

3.3.2 Pure acetic acid (CH3COOH) (CAS no[64-19-7])

3.3.3 27% sodium acetate solution (NaCH3COO) (CAS no[127-09-3])

3.3.4 Ammonium vanadate (NH4 VO3) (CAS no[7803-55-6])

3.3.5 N solution of sodium hydroxide (NaOH) (CAS no[1310-73-2])

3.3.6 Acid solution of sodium acetate

• acetic acid 80 ml
• 27% sodium acetate 320 ml
• 30% brij 1 ml
• distilled water up to 1000 ml
Conservation: 1 week.

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3.3.7 Sodium metavanadate solution
• ammonium vanadate 4g
• NM NaOH solution 150 ml
• 27% sodium acetate 200 ml
• distilled water up to 500 ml
Conservation: 1 week.

3.3.8 Reference solutions of D+-tartaric acid (CAS no[147-71-7]) from 1 to 8 g/l

3.4 Procedure
The flow chart is provided in the appendix.
The samples do not undergo any preliminary treatment. In the case of musts or other samples with
suspended matter, centrifugation is essential.

The analytical rate is 30 samples per hour (sampling time / rinse time = 1/1).

3.5 Expression of the results

The results are expressed in grams per litre to one decimal place.

3.6 Characteristics of the described method

Interlaboratory reproducibility: 0.7 g∙l-1

3.7 Bibliography
Battle, J-L, Joubert, R., Colion, Y. and Jouret, C, Use of continuous flow for colorimetric
determination of tartaric acid in grape musts and wines using the Rebelein method, Ann. Fals. Exp.
Chim., 1978, 71(764), 155-158.
Curvelo-Garcia, A. S., Reliability of segmented continuous flow methods applied to the analysis of
wines and musts, Feuillet Vert de l’OIV no941 (1993).

Trossais J. and Asselin C. Influence of malic acid levels in musts on the determination of tartaric
acid by continuous flow colorimetry with metavanadate. Conn. Vigne Vin. 1985, 19, 4, 249-259.

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3.8 Appendix: Flow chart example

Sink

Flow rate
ml / min

Acid
Dialyser

Acid

Sink

15 min delay coil Recorder

4. Determination of content of citric acid in wines and musts

4.1 Principle

The automatic method for the fluorometric determination of citric acid is based on the
dicyclohexilcarbodimide (DDC) method. The sample reacts with the DDC in presence of
acetic acid in anhydrous environment, which gives rise to aconitine formation.
The transmission wavelength of aconitine is 490 nm and its excitation wavelength is 400
nm. The signal given out by the fluorometre is sent to an interface connected to the
system’s software.

4.2 Apparatus

Segmented flow chain

4.3 Reagents and solutions

4.3.1 Sodium chloride (NaCl)

Sodium chloride (NaCl) CAS N° [7647-14-5], 4,0 g

Acetic acid (CH3COOH) CAS N° [64-19-7], 180 cm3
Ethanol (CH3CH2OH) CAS N° [64-17-5], 20 cm3
Citric acid solution 10,0 g/dm3, 30 cm3

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Water type I (Standard ISO 3696) or identical up to 2000 cm3
Brij 35 (30%) N °CAS [9002-92-0], 6 cm3

Preparation:

In a graduated vial of 2000 cm3, dissolve sodium chloride in approx. 1600 cm 3 of water.
Delicately add the acid and leave to settle until ambient temperature is reached. Add
ethanol and critic acid solution and mix. Fill to the top with demineralised water, add Brij
and mix.
6 month conservation time

4.3.2 1-butanol (CH3(CH2)3OH) CAS N° [71-36-3]

4.3.3 Acetic acid solution at 1,7% (v/v)

Acetic acid (CH3COOH) CAS N° [64-19-7], 17 cm3
1-butanol (CH3(CH2)3OH) CAS N° [71-36-3] up to, 1000 cm3
Preparation :
In the bottle of 1-butanol (1 litre), carefully add the acid. Mix well.
12 month conservation time

4.3.4 dicyclohexilcarbodimide solution (DCC)

Dicyclohexylcarbodimide (C13H22N2) CAS N° [538-75-0], 41 g
1-butanol (CH3(CH2)3OH) CAS N° [71-36-3], up to, 1000 cm3
Preparation :
Weigh the dicyclohexilcarbodimide in a 1000 cm 3 Erlenmeyer flask and add the n-
butanol. Mix thoroughly.
12 month conservation time

4.3.5 Brij 35 CAS N° [9002-92-0]

4.3.6 Decontamination solution

1-butanol (CH3(CH2)3OH) CAS N° [71-36-3]

4.4 Preparing the sample

Let the gas out of the sample so as to eliminate as much CO 2 as possible, by strirring for
at least two minutes. If the sample is murky, it must be filtered.

4. 5 Calibration solutions

4.5.1 Citric acid solutions (C6H8O7) N° CAS [77-92-9], of 0,07 at 1,00 g/dm3 in citric acid

Conservation: stability is given by an internal control of the calibration process

4.5.2 Calibration with a solution of known content in citric acid

4.6 Procedure

The flow diagram is appended. The frequency of analyses is approximately 18 samples

per hour, depending on the assembly used.

4.7 Results

The results are given with a two-digit accuracy and expressed in g (citric acid)

4.8 Characteristics of the method described

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Reproducibility: 0.03 g (citric acid) L-1
Inter-laboratory reproducibility: : 0.03 g (citric acid) L-1
Uncertainty: uexp = 0.20 x concentration of the sample in g (citric acid) L-1

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4.9 Appendix: Flow chart example

Pompes Debits
Evier
ml/min

Fluorimètre

Émission 490 nm

L'extinction à 400 nm

2 mm d'épaisseur de cellule

Acide acétique

DCC
air
n-butanol

air

chlorure de sodium
échantillon
evier

evier
Sampler

o
* Membrane de dialyse, Ref SA5283

** Tubes à la bombe en aciflex

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 5. Determination of glucose and fructose content in wines and musts

5.1 Principle
The determination method helps determine glucose and fructose concentrations separately or the
sum of the two concentrations in wines or musts.

5.1.1 Glucose and fructose phosphorylation

This reaction is catalysed by hexokinase (HK):
HK
glucose + ATP < = = > glucose-6-phosphate + ADP (1)
HK
fructose + ATP < = > fructose-6-phosphate +ADP
ATP: adenosine triphosphate.
ADP: adenosine diphosphate.

5.1.2 Glucose-6-phosphate(G-6-P) oxidation

This is achieved by the action of nicotinamide adenine dinucleotide phosphate (NADP), in the
presence of its specific glucose-6-phosphate dehydrogenase (G6PDH) enzyme.
G6PDH
G-6-P + NADP+ < = = > gluconate-6-phosphate + NADPH + H+ (2)
The equilibrium is moved in the direction of NADH formation by carefully selecting the operating
conditions (pH 7.6 buffer and an excess of NADP). NADPH proportional to the glucose concentration
in the sample is determined by measuring its absorption at 340 nm.

5.1.3 Fructose-6-phosphate isomerisation

This reaction determines the sum of glucose + fructose.
The enzyme used is phosphoglucose-isomerase (PGI).
PGI
fructose-6-phosphate < = = > glucose-6-phosphate (3)
The formed glucose-6-phosphate reacts with NADP according to (2) to form NADPH, which is
determined at 340 nm.

5.2 Apparatus
Segmented continuous flow chain.

5.3 Reagents and solutions

Many suppliers on the market offer packages containing prepared reagents. An example is provided
below.

5.3.1Bottle 1:
Lyophilisate composed of:
• Triethanolamine buffer of pH = 7.6
• NADP (CAS no[53-59-8]) 64 mg
• ATP (CAS no[987-65-5]) 160 mg
• Magnesium sulphate MgSO4 (CAS no[7487-88-9])
• Stabilisers.

5.3.2 Bottle 2:
0.7 ml of enzymatic suspension composed of:
Hexokinase (CAS no[9001-51-8]) approximately 200 U
• Glucose-6-phosphate dehydrogenase (CAS no [9001-40-5]) 100 U solution in ammonium sulphate.

19/40
 5.3.3 Bottle 3:
0.7 ml of phosphoglucose-isomerase (CAS no[9001-41-6]) (approximately 490 U) in suspension in
ammonium sulphate.

5.3.4 Bottle 4:
7 ml of 0.5 g-1 D-glucose standard solution (CAS no[50-97-7]). Unopened package conservation at
-20°C until the expiry date.

5.3.5 NAD/ATP buffer solution

This is prepared from the enzyme package:
Dissolve the content of bottle 1 in 90 ml of bidistilled water.
Store the diluted buffer at -20°C for 3 months at the most.

5.3.6 Preparation of enzyme solutions for determination

Example of volumes to prepare for the proposed assembly:
Determinations Fructose and glucose + fructose

Glucose
Analysis duration NAD/ATP buffer HK/G6PDH PGI
1 hour 15 ml 100 µl 100 µl
4 hours 60 ml 500 µl 500 µl
8 hours 120 ml 1000 µl 1000 µl

The determination of fructose requires running the standards and samples twice; the duration of
analysis is doubled compared to the determination of glucose.
Conservation of enzyme solutions for determination: one week at +4°C.

5.3.7 Standard solutions

5.3.7.1Stock solution at 10.0 g/l

The use of glucose alone is enough for calibration: the isomerisation of fructose will be carried out
for samples only.
anhydrous D-glucose 2.50 g
• Distilled water up to 250 ml
• Allyl isothiocyanate (preservative) (CAS no[57-06-7]) 2 drops.

5.3.7.2 Daughter solutions of glucose:

• 3 g/l standard: 15 ml of stock solution in a 50 ml vial
• 2 g/l standard: 10 ml of stock solution in a 50 ml vial
• 1 g/l standard: 10 ml of stock solution in a 100 ml vial
These solutions are topped up to the filling mark with distilled water.
• 0,50 g/l standard: standard solution from the enzyme package
Conservation: at + 4°C for 1 month.

5.4 Procedure
The flow chart is provided in the appendix.
The analytical rate is 60 samples per hour, with sampling time / rinse time = 1/1.
The samples do not undergo any preliminary treatment. In the case of musts or samples with
suspended matter, preliminary filtration is essential.

5.5 Expression of the results

The concentration of glucose, fructose or glucose + fructose is reported in g∙l-1 to one decimal
place.

5.6 Bibliography
Battle J.L., Herdsman J.C. Rev. France Oenol., 1986, 101, 38-43.

20/40
5.7 Appendix: Flow chart example

0 to 3 g/l
Sample

Rinse
Flow direction exch./h
Sink

rotations
Sink
6 inch dialysis

Buffer

rotations
Sink

37°C water bath

(min delay: 10°) * Brij 35 at 30% p/v
Spectrometer
3 drops/litre
Recorder

6 Determination of content of free and total sulphur dioxide in wines and musts
following dialysis

6.1 Principle
After wine acidification, the free sulphur dioxide diffuses through a dialysis membrane. The
combined sulphur dioxide is released by alkaline hydrolysis. Two determinations are performed at
560 nm in the presence of pararosaniline (or basic fuschine). Segmentation is carried out with
nitrogen in order to avoid any risk of oxidation.
SO2 + rosaniline —> colourless complex + formaldehyde —> coloured complex.
The method is applicable for a range from LOQ to 200 mg∙l-1.

6.2 Apparatus
Segmented flow chain.

6.3 Reagents and solutions

6.3.1 Reagent specific to the determination of free sulphur dioxide

6.3.1.1 Solution with 10% of sulphuric acid (98% purity)

• H2SO4 (CAS no[57-06-7]) 20 ml
• distilled water up to 200 ml.

6.3.2 Reagents specific to the determination of total sulphur dioxide

6.3.2.1 Solution of 4 M sodium hydroxide

• NaOH (CAS no[1310-73-2]) 40 g
• distilled water up to 250 ml.

21/40
Conservation: 1 week.

6.3.2.2 Sulphuric acid solution (CAS no[57-06-7]) at 1/2 (v/v)

Conservation: 1 month.

6.3.3 Common reagents

6.3.3.1 R-type nitrogen (CAS no[7727-37-9])

6.3.3.2 Sulphuric acid solution (CAS no[57-06-7]) at 1% (v/v)
Conservation: 1 month.
6.3.3.3 Formaldehyde solution at 6% (v/v)
• formaldehyde 37% (minimum) (CAS no[50-00-0]) 1.2 ml
• distilled water up to 200 ml.
Conservation: 1 day in a bottle with stopper.
6.3.3.4 Rosaniline (coloured reagent).
• Stock solution: 2 g/l of basic fuschine (or rosaniline hydrochlorate) (CAS no[632-99-5])
Stir and allow to rest for 3 days and store safe from light.
Conservation: 3 months in a tinted bottle.

• Working solution
- stock solution 40 ml
- orthophosphoric acid (CAS no[7664-38-2]) 62 ml
- distilled water up to 500 ml.
Conservation: 2 weeks in a tinted bottle.
Note: This preparation is highly exothermic. Use with caution.

6.3.4 Synthetic standard solutions

6.3.4.1 Acid solution of a pH of about 3

• 1% H2SO4 (CAS no[57-06-7]) 5ml
• distilled water up to 1000 ml.
This solution will be used to top up the standard SO2 vials.
Conservation: 1 day.

6.3.4.2 Stock solution with 300 mg∙l-1 of SO2

• Pure sodium disulfite (CAS no[7681-57-4]) 227 mg
• pH 3 solution up to 500 ml.
Conservation: 1 day.

6.3.4.3 Standard daughter solutions

Preparation example of a standard solution at 120 mg∙l-1:
• stock solution with 300 mg∙l-1 50 ml
• pH 3 solution 40 ml
• water distilled up to 125 ml.

6.3.5 Wine standards

Reference wines or wines tested with the reference method.

6.4 Procedure
The flows charts are provided in the appendix. The samples do not undergo any preliminary
treatment. The analytical rate can reach up to 60 samples per hour with rinse / sampling times =
2/1.

Note:
At the end of the analysis, rinse the analytical circuit with water for at least 10 min.
Whenever necessary, wash the circuit with twenty-fold diluted liquid bleach for at least 30 min.
Rinse with water for at least 2 h.

22/40
6.5 Expression of the results
The sulphur dioxide values are expressed in mg per litre without decimal places.

6.6 Characteristics of the described method

Interlaboratory reproducibility for free sulphur dioxide: 7 mg∙l-1
Interlaboratory reproducibility for total sulphur dioxide: 27 mg∙l-1

6.7 Bibliography
DUBERNET Mr., 1997. Automation of chemical analysis in oenology. Revue Française d’Œnologie,
66, 45-61.
Scholten G, Woller R., Holbach B Automated wine analysis, 2nd communication. Die Wein. Wiss.,
1982, 38, 397-426.

23/40
6.8 Appendix: Flow chart examples

Determination of free SO2

Sample

Rinse
Flow direction
exch./h
Sink
Sink 3 inch dialysis rotations rotat.

Nitrogen
10% H2SO4
Nitrogen
1% H2SO4
Formaldehyde
Pararosaniline
rotat.

rotations
Sink

45°C water bath max calib.

Spectrometer
Recorder

Determination of total SO2

Sample

Rinse
Flow direction
exch./h
Sink
Sink 3 inch dialysis rotations rotat.

Nitrogen
4N sodium
½ H2SO4
rotations 1% H2SO4
Nitrogen
Formaldehyde
Pararosaniline

rotat.

Sink

40°C water bath

Spectrometer
Correction filter: Recorder
700 nm

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7 Determinations of content of free and total sulphur dioxide in wines and musts by
distillation

7.1 Principle
An alternative to the previous method is also largely used. It consists in replacing dialysis by
distillation in a micro-column. In the case of total sulphur dioxide, alkaline hydrolysis is
unnecessary, the total release of the sulphur dioxide is ensured by higher acidification and a higher
distillation temperature.

7.2 Reagents and solutions

7.2.1 Reagent specific to the determination of free sulphur dioxide

7.2.1.1 Solution with 1% of sulphuric acid (98% purity)

• H2SO4 (CAS no[57-06-7]) 2 ml
• distilled water up to 200 ml.

7.2.2 Reagents specific to the determination of total sulphur dioxide

7.2.2.1 Sulphuric acid solution (CAS no[57-06-7]) at 20% (v/v)

Conservation: 1 month.

7.2.3 Common reagents

7.2.3.1 R-type nitrogen (CAS no[7727-37-9])

7.2.3.2 Sulphuric acid solution (CAS no[57-06-7]) at 1% (v/v) with addition of 4 drops
per litre of Brij-35 wetting agent (CAS no[9002-92-0])
Conservation: 1 month.

7.2.3.3 Formaldehyde solution

• 40% formaldehyde (CAS no[50-00-0]) 10 ml
• distilled water up to 1000 ml.
Conservation: 1 day in a bottle with stopper.

7.2.3.4 Sodium hydroxide solution (NaOH) 0.2 M

• Sodium hydroxide (CAS no[1310-73-2]) 4g
• Brij 35 (CAS no[9002-92-0]) 2 drops
• distilled water up to 500 ml.

7.2.3.5 Rosaniline (coloured reagent).

• Stock solution: 2 g/l of basic fuschine (or rosaniline hydrochlorate) (CAS no[632-99-5])
Stir and allow to rest for 3 days and store safe from light.
Conservation: 3 months in a tinted bottle.
• Working solution
stock solution 70 ml
orthophosphoric acid H3 PO4 (CAS no[7664-38-2]) 70 ml
distilled water up to 500 ml.
Conservation: 2 weeks in a tinted bottle.
Note: This dilution is strongly exothermic. Use with caution.

7.2.4 Synthetic standard solutions

7.2.4.1 Acid solution of a pH of about 3

• H2SO4 1% (CAS no[57-06-7]) 5 ml
• distilled water up to 1000 ml.
This solution will be used to top up the SO2 standard vials.
Conservation: 1 day.

25/40
 7.2.4.2 Stock solution with 300 mg∙l-1 of SO2
• Pure sodium disulfite Na2S2O5(CAS no[7681-57-4]) 227 mg
• pH 3 solution up to 500 ml.
Conservation: 1 day.

7.2.4.3 Daughter standard solutions

Preparation example of a standard solution at 120 mg∙l-1:
• stock solution at 300 mg∙l-1 50 ml
• pH 3 solution 40 ml
• distilled water up to 125 ml.

7.2.5 Wine standards

Reference wines or wines tested with the reference method.

7.3 Expression of the results

The sulphur dioxide values are expressed in mg per litre without decimal places.

7.4 Characteristics of the described method

Interlaboratory reproducibility for free sulphur dioxide: 7 mg∙l-1
Interlaboratory reproducibility for total sulphur dioxide: 27 mg∙l-1

26/40
 7.5 Appendix: Flow chart examples

Determination of free sulphur dioxide

Rate: 90 exch. / hour

Distillation
column Sampling / rinse ratio :
5 1/1
rotation Nitrogen for
s practice Flow
Pump
rate: 40
Coolantml/min ml/mi
(cold n
0.1 Sample
water) 6
1.4 1% H2SO4
0
0.0
20 Nitrogen for
5
1.6
rotations bubbling M
0.2
0
0.0 NaOH
Nitrogen for bubbling
Sink 5
2.5
0

5 0.6
5 5 0
Colorimete rotation rotation rotation 1.2
1% H2SO4 + Brij
r s s s 0
0.3
560 nm Formaldehyde
48°C ± 5°C 2
0.3 Para-rosaniline
water bath 2
0.8
0
Colorimeter
recovery

Determination of total sulphur dioxide

Distillation
column Nitrogen for
practice Flow Rate: 90 exch. / hour
Coolant 105°C ±
rate: 40 Sampling / rinse ratio :
(cold 5°C water
ml/min 1/1
water) bath

Pump
ml/mi
n
0.1 Sample
6
1.4 10% H2SO4
0
0.0
20 Nitrogen for
5
rotation 1.4 bubbling
0.2
s 0
0.0 NaOH N
Nitrogen for
5
2.0 bubbling
Sink
0

0.6
3 x 20 5 0
Colorimete rotations rotation 1.2
1% H2SO4 + Brij
r s 0
0.3
560 nm Formaldehyde
2
0.3 Para-rosaniline
2
0.8
0
Colorimeter
APPENDIX 2
recovery
Examples of dosage methods usually used in enology with sequential analysers

The methods described hereunder are examples and other methods may be used.

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1 Determination of acetic acid in wines and musts

1.1 Principle
Enzymatic method.
In the presence of ATP, the acetic acid is converted into acetyl-phosphate in a reaction catalysed
by acetate kinase.
(1) Acetate + ATP —Acetate Kinase Acetyl-phosphate + ADP
The ADP formed by this reaction is converted back into ATP by reaction with phosphoenolpyruvate
in the presence of pyruvate kinase.
(2) ADP + Phosphoenolpyruvate —Pyruvate Kinase Pyruvate + ATP
The pyruvate is reduced to L-lactate by reduced nicotinamide-adenine-nucleotide (NADH) in the
presence of lactate-dehydrogenase.
(3) Pyruvate + NADH + H+ —Lactate dehydrogenase Lactate + NAD+ + H2O
The amount of NADH oxidised in reaction (3) is determined by the absorbance measurement at
340 nm, and is proportional to the acetic acid concentration of the wine.
A fourth reaction maintains the equilibrium of reaction 1 in the direction of acetylphosphate
formation by its elimination.

(4) Acetyl-phosphate + CoA —Phosphotransacetylase  Acetyl-CoA + Inorganic phosphate

The reaction medium is added with polyvinylpyrrolidone (PVP) to eliminate the interferences due to
the phenolic compounds of the wine.

1.2 Reagents

1.2.1 Preparation of the MOPS buffer

Add:
13 g of MOPS (sulphonic N-morpholino-3-propane acid) CAS [1132-61-2]
500 mg of magnesium chloride, 6H2O - CAS [7791-18-6]
1.5 g of potassium chloride (KCl) - CAS [7447-40-7]
250 ml of bidistilled water

Adjust the pH to 4.75 with a 1.5 m solution of potassium hydroxide (KOH) - CAS [1310-58-3].
Wait 5 minutes and readjust the pH to 7.45.
Top up to 300 ml with bidistilled water.
Buffer stability: 60 days at 4°C.

1.2.2 Preparation of the reagents

1.2.2.1 Reagent 1 (R1):

Add to 100 ml of MOPS buffer 400 mg of polyvinylpyrrolidone (PVP) - CAS
[9003-39-8].

1.2.2.2 Reagent 2 (R2)

Add to 100 ml of MOPS buffer:

300 to 350 mg of adenosine-5-triphosphate, disodium salt (ATP) - CAS [987-65-5]
50 mg of phospho-enolpyruvate of tricyclohexylammonium (PEP) - CAS [35556-70-8]
38 mg of β Nicotinamide adenine dinucleotide (reduced form) (NADH) - CAS [606-68-8] - Purity ≥
98%
25 mg of trilithium salt of coenzyme A - CAS [18439-24-2] – purity 2
Approximately 400 units of pyruvate kinase (PK) – CAS [9001-60-9] and lactate dehydrogenase
(LDH) - CAS [9001-60-9]
Approximately 600 units of phosphotransacetylase (PTA) – CAS [9029-91-8]
230 units of acetate kinase (AK) - CAS [9027-42-5]

Reagent stability: approximately 12 hours at + 4°C.

28/40
1.3 Preparation of the samples
The wine samples are diluted tenfold beforehand.

1.4 Analytical procedure

This can vary depending on the material used. The following description must be regarded as an
example.
The volume of the reaction vessels is 300 µl. The analytical cycle is 10 minutes, i.e. 50 rotations of
the reaction plate. A colorimetric reading is performed every 12 seconds, i.e. with every rotation of
the reaction plate, in order to plot the reaction curve.

10.4.1 Programming
The reading wavelength is 340 nm.
The volume of sample diluted tenfold, added to time zero is 20 µl.
The volume of reagent R1 added to time zero is 111 µl.
The volume of reagent R2 added after 5 minutes is 125 µl.
Total reaction volume is therefore of 256 µl.

1.4.2 Calibration
Calibration is performed at 4 points. A zero value is obtained by using a solution with 9‰ of
sodium chloride – CAS [7647-14-15]. The other three calibrants are solutions of increasing
concentrations of pure acetic acid – CAS [64-19-7] (0.25 g∙l-1; 0.50 g∙l-1; 1 g∙l-1) or standard wines
of known concentration in acetic acid.

1.4.3 Results
The figure below provides an illustration of the resulting reaction curve.
Absorbance at 340
nm

Number
of
rotations
Addition of the sample in the reaction
vessel
Addition of reagent no1 in the reaction
vessel
Addition of reagent no2 in the reaction
vessel
Colorimetric reading no1
Colorimetric reading no2

29/40
Under the implemented measurement conditions, the reaction curve observed after the addition of
reagent R2 is a straight line, in the selected working scale (0 to 1 g/l). It represents the initial
speed of reaction, proportional to the acetic acid concentration. The slope of this line can be
measured by a two-point reading at rotations no27 and 50. The value of acetic acid concentration
will be obtained by the formula:
C = K (L1-l2)
Where K is a reaction factor calculated by the instrument after each calibration according to the
following formula:
CEt − CB
K=
AEt − AB
where:
AB = blank absorbance
CB = blank concentration
AEt = standard absorbance
CEt = standard concentration

The results are given in g∙l-1 of acetic acid.

1.5 Characteristics of the described method

Intralaboratory reproducibility: 0.04 g∙l-1
Interlaboratory reproducibility: 0.10 g∙l-1

1.6 Bibliography

DONECHE B, and SANCHEZ P.J., 1985: Automation of continuous flow technique for the enzymatic
determination of acetic acid in wines, Connaissance de la vigne et du vin, 1985; no3, 161-169.

DUBERNET Mr., PENNEQUIN F and GRASSET F., 1997 Use of the Hitachi 717 sequence analyser
for acetic acid determination in wines, Feuillet vert OIV NO1001

McCLOSKEY Leo P., 1980: An improved enzymatic assay for acetate in juice and wine, Am. J
Enol. Vitic. Vol 31, NO2, 1980, pp 170-173.

2 Determination of total sulphur dioxide in wines and musts

2.1 Principle
Chemical method.
The sample to be determined is diluted in a phosphate buffer solution of pH 8. After stabilisation,
the reaction medium receives a buffered solution of DTNB acid (5,5'-Dithio-bis(2-nitrobenzoic acid)
(3,3'-6) or 3-Carboxy-4-nitrophenyl disulphide), known as "Ellman’s reagent". It is a specific
reagent allowing the modification and quantitative detection of disulphide bonds, the developed
formula of which is provided below:

O OH

O2N

S
S

NO2

O OH

The reaction develops a yellow colour measurable at a wavelength of 405 nm.

30/40
2.2 Measurement protocol

2.2.1 Equipment
Any sequential analysis equipment enabling the use of two reagents and allowing measurement at
405 nm can be used.

2.2.2 Reagents
Two reagents are implemented:
• Reagent 1

K2HPO4 (CAS [7758-11-4]) 17.4 g

Bidistilled water up to 1000 ml

The pH of the buffer solution prepared is adjusted by addition of pure H 3PO4 (CAS [7664-38-2]) to
obtain a value of 8 (maximum tolerable gap: ± 0.2 pH unit).
• Reagent 2

DNTB (CAS [69-78-3]) 760 mg

Reagent 1 900 ml
Pure ethanol 100 ml

2.2.3 Implementation
The following table summarises the various quantities of sample of wine and reagents to be added
in the reaction vessel over time:

Time in Sample in µl Reagent 1 in Reagent 2 in Bidistilled water

minutes µl µl in µl
0 8 80 170
5 80

The absorbance at 405 nm is read regularly over time – 10 minutes in total that may be cut given
the practically instantaneous speed of the reaction. The following graph shows the type of reaction
curve obtained:

Courbe réactionnelle
Reaction curve

8000

7000

6000

5000
Do (Valeurs relatives)

4000

3000

2000

1000

0
12

36

60

84

108

132

156

180

204

228

252

276

300

324

348

372

396

420

444

468

492

516

540

564

588

Temps
Timeeninsecondes
seconds

After the addition of reagent no1, stabilisation occurs rapidly. The addition of reagent no2
containing the DNTB is followed by an immediate coloured reaction whose plateau is reached within

31/40
a few seconds. Then, the plateau becomes stable. Reading is performed by measuring the
difference between absorbance OD1, read after the stabilisation of reagent no1, and the
absorbance OD2 read on the plate. This absorbance difference is proportional to the total SO 2
content in the tested sample.
Calibration is performed either with standard wines whose total SO 2 values are known and cover
the considered measurement range, or using potassium metabisulfite solutions stabilised in
sulphuric medium at 1% and of known concentration. Generally speaking, since the calibration
curve is not completely linear, 4 to 5 calibration points are necessary. A zero value is obtained by
using a 9‰ sodium chloride solution (CAS no[7647-14-5]).

2.3 Characteristics of the described method

Intralaboratory reproducibility: 12 mg∙l1
Interlaboratory reproducibility: 20 mg∙lL-1

2.4 Bibliography
ELLMAN G.L., 1959. For the modification and quantitative detection of sulfhydryl groups; Arch.
Biochem. Biophys. 82,70.
DUBERNET Mr., LEBOEUF J.P. and GRASSET F, 1998. Automated method for colorimetric
determination of total sulphur dioxide in wines. FV NO1068 OIV.
DUBERNET Mr., PENNEQUIN F and GRASSET F, 1995. Use of the Hitachi 717 sequence analyser for
sulphur dioxide determination in wines. FV NO997 OIV.

3 Determination of free sulphur dioxide in wines and musts

3.1 Principle
Chemical method.
The free sulphur dioxide is stabilised in acid medium. Its determination is ensured by the
development, in the presence of formaldehyde, of a pink colour by combination with Fuschine
bleached in phosphoric medium beforehand. Determination is corrected for a parasitic reaction due
to the phenolic compounds present in the wine.

3.2 Equipment
Any sequence analysis equipment enabling the use of two reagents and allowing measurement at
570 nm can be used.

3.3 Reagents
Two reagents are used:

3.3.1 Reagent 1

Basic stock solution of Fushine (Rosaniline Hydrochlorate)

Rosaniline hydrochlorate (CAS no[632-99-5]) 2g
Bidistilled water up to 1000 ml

Conservation: 6 months in a refrigerator

3.3.1.2 Working solution (R1)

Basic stock solution of Fuschine 3 ml
Phosphoric acid (CAS [7664-38-2]) at 10% 77 ml
Bidistilled water 20 ml

Conservation: 5 days at 4°C and safe from light.

3.3.2 Reagent 2
Formaldehyde (CAS no[50-00-0]) 5 ml
Bidistilled water up to 1000 ml

Conservation: 1 week at ambient temperature.

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3.4 Preparation of the samples
The wine samples are not diluted. It is recommended to carry out the analysis as quickly as
possible after sampling to avoid losses of free sulphur dioxide.

3.5 Analytical procedure

The reading wavelength is 570 nm.
The analytical sequence is as follows:
Rotation 1 (6 seconds): sampling of 20 µl of wine
Rotation 1 (12 seconds): addition of 250 µl of reagent R1
Rotation 24 (288 seconds): OD1 reading at 570 nm
Rotation 25 (300 seconds): addition of 106 µl of reagent R2
Rotation 28 (336 seconds): OD2 reading at 570 nm

3.6 Calibration
This is performed at two points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The standard value is given by a regularly titrated solution of potassium
metabisulfite (CAS no[16731-55-8]) stabilised in sulphuric medium at 1% and with a concentration
of approximately 50 mg∙l-1.

3.7 Reading of results

The following diagram shows the evolution of the absorbance at 570 nm.

OD at
570 nm
Reagent Reagent
no1 no2

OD 2

OD 1

Reading no1 Reading no2

Number
of
rotations

It has been observed that the mixture of the wine with the fuschine solution causes an initial
coloured reaction involving an increase in absorbance (reaction no1). The level of the latter is
proportional to the richness of the wine in phenolic compounds and constitutes a parasitic effect.
Reaction no2 occurs after the addition of reagent R2 and corresponds strictly to the combination of
free sulphur dioxide and fuschine. Only the latter must be taken into account. Reading is performed
by measuring the difference between basic absorbance OD1 and absorbance OD2. The difference in
absorbance is proportional to the free SO2 content in the tested sample.

3.8 Characteristics of the described method

Intralaboratory reproducibility: 6 mg∙l-1
Interlaboratory reproducibility: 12 mg∙l1

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3.9 Bibliography
DUBERNET Mr., PENNEQUIN F and GRASSET F, 1995. Use of the Hitachi 717 sequence analyser for
free sulphur dioxide determination in wines. FV NO998 OIV.

4 Determination of glucose and fructose in wines and musts

4.1 Principle
Enzymatic method.
The glucose and fructose are phosphoryled in the presence of adenosine triphosphate (ATP). The
reaction is catalysed by hexokinase (HK) and respectively produces glucose-6-phosphate (G6P)
and fructose-6-phosphate (F6P).
HK
1) Glucose + ATP ------> G6P + ADP
HK
2) Fructose + ATP ------> F6P + ADP

The glucose-6-phosphate is oxidised into gluconate-6-phosphate in the presence of nicotinamide-

adenosine-dinucleotide (NAD). The reaction is catalysed by glucose-6-phosphate dehydrogenase
(G6PDH).

G6PDH
3) G6P + NAD -----> Gluconate-6-Phosphate + NADH + H+

The fructose-6-phosphate is converted into glucose-6-phosphate in the presence of

phosphoglucose isomerase (PGI).

PGI
4) F6P -----> G6P

The increase in absorbance at the wavelength of 340 nm is proportional to the amount of D-

glucose and D-fructose present.

4.2 Equipment
Any sequence analysis equipment enabling the use of two reagents and allowing measurement at
340 nm can be used.

4.3 Reagents
The reagents used are commonly offered in an enzyme package by manufacturers, for easy
implementation by laboratories. An example is provided below.
• Reagent 1 (30 ml)
Buffer solution pH 7.5
NAD (CAS no[53-84-9]) 70 mg
ATP (CAS no[987-65-5]) 90 mg

• Reagent 2 (0.6 ml)

HK (CAS no[9001-51-8]) 160 U and G6PDH (CAS no[9001-40-5]) 200 U

• Reagent 3 (0.6 ml)

PGI (CAS no[9001-41-6]) 380 U

The single reagent used in the instrument is prepared by addition of the 3 reagents from the
package, to which is added 900 mg of PVP (Polyvinylpyrolidone) (CAS no[9003-39-8]). In the
above case, it helps perform approximately 600 tests. It may be stored for 1 month at +8°C.

4.4 Preparation of the samples

The wine samples are diluted 10 times. The must samples are diluted so as to obtain an initial
concentration lower than 1 g∙l-1.

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4.5 Analytical procedure
The reading wavelength is 340 nm.
The analytical sequence is as follows:
• Rotation 1 (6 seconds): sampling of 5 µl of wine
• Rotation 1 (12 seconds): addition of 50 µl of reagent and 200 µl of demineralised water
• Rotation 1: reading of absorbance OD1 at 340 nm
• Rotation 50 (600 seconds): reading of absorbance OD2 at 340 nm

4.6 Calibration
This is performed at 4 points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The standard values are given by solutions of glucose (CAS no[50-99-7])
and fructose (CAS no[57-48-7]) (50/50) covering the range from 0 to 10 g∙l-1 diluted 10 times at
the time of use. Calibration can also be performed with wines of known concentrations.

4.7 Reading of results

Reading is performed by measuring the difference between basic absorbance OD2 and absorbance
OD1. The difference in absorbance is proportional to the content of glucose and fructose in the
tested sample. The result is expressed in g of glucose + fructose per litre.

4.8 Characteristics of the described method

Intralaboratory reproducibility: 0.3 g∙l-1 from 0 to 5 g∙l-1 and 0.6 g∙l-1 over 5 g∙l-1
Interlaboratory reproducibility: 0.35 g∙l-1 from 0 to 2 g∙l1

5 Determination of L-malic acid in wines and musts

5.1 Principle
The L-malic acid, in the presence of nicotinamide-adenosine–dinucleotide (NAD), is oxidised into
oxaloacetate in a reaction catalysed by L-malate dehydrogenase (L-MDH). The reaction equilibrium
is in favour of malate. The reaction equilibrium is moved in the direction of oxaloacetate formation
by elimination of oxaloacetate which, in the presence of L-glutamate, is converted into L-aspartate.
This reaction is catalysed by glutamate-oxaloacetate-transaminase (GOT).

1) L-malate + NAD+ - > oxaloacetate + NADH + H+ (in the presence of L-malate

dehydrogenase)
2) Oxaloacetate + L-glutamate - > L-aspartate + 2-oxoglutarate (in the presence of
GOT)

NADH formation, measured by the increase in absorbance at the wavelength of 340 nm, is
proportional to the amount of L-malate present.

5.2 Equipment
Any sequence analysis equipment allowing measurement at 340 nm can be used.

5.3 Reagents
The reagents used are frequently offered in an enzyme package by manufacturers, for easy
implementation by laboratories. An example is provided below.
• Reagent 1 (30 ml)
Buffer solution pH 10 / glutamic acid (CAS no[56-86-0]) 440 mg

• Reagent 2 (6 ml)
NAD (CAS no[53-84-9]) 210 mg

• Reagent 3 (0.6 ml)

L-MDH (CAS no[9001-64-3]) 2400 U/GOT (CAS no[9000-97-9]) 160 U

The single reagent used in the instrument is prepared by addition of the 3 reagents from the
package, to which is added 300 mg of PVP (Polyvinylpyrolidone) (CAS no[9003-39-8]). It may be
stored for 1 month at 8°C.

5.4 Preparation of the samples

The must or wine samples are diluted 10 times.

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5.5 Analytical procedure
The reading wavelength is 340 nm.
The analytical sequence is as follows:
Rotation 1: (6 seconds): sampling of 5 µl of wine
Rotation 1: (12 seconds): addition of 20 µl of reagent and 200 µl of demineralised water
Rotation 1: reading of absorbance OD1 at 340 nm
Rotation 50: (600 seconds): reading of absorbance OD2 at 340 nm

5.6 Calibration
This is performed at 3 points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The standard values are given by L-malic acid solutions (CAS no[97-67-6])
covering the range from 0 to 3.5 g∙l-1 diluted 10 times at the time of use. Calibration can also be
performed with wines of known concentrations.

5.7 Reading of results

Reading is performed by measuring the difference between basic absorbance OD2 and absorbance
OD1. The difference in absorbance is proportional to the L-malic acid content in the tested sample.
The result is expressed in g of L-malic acid per litre.

5.8 Characteristics of the described method

Intralaboratory reproducibility: 0.4 g∙l-1.
Interlaboratory reproducibility: 0.6 g∙l-1

6 Determination of L-lactic acid in wines and musts

6.1 Principle
Enzymatic method.
The L-lactic acid, in the presence of nicotinamide-adenosine-dinucleotide (NAD), is oxidised into
pyruvate in a reaction catalysed by L-lactate dehydrogenase (L-LDH). The reaction equilibrium is in
favour of lactate. The elimination of pyruvate from the reaction medium moves the reaction
equilibrium in the direction of pyruvate formation. In the presence of L-glutamate, pyruvate is
converted into L-alanine. This reaction is catalysed by glutamate-pyruvate-transaminase (GPT).

1) L-lactate + NAD+ - > pyruvate + NADH + H+ (in the presence of L-lactate

dehydrogenase)
2) Pyruvate + L-glutamate - > L-Alanine + 2-oxoglutarate (in the presence of GPT)

NADH formation, measured by the increase in absorbance at the wavelength of 340 nm, is
proportional to the amount of lactate present.

6.2 Equipment
Any sequence analysis equipment allowing measurement at 340 nm can be used.

6.3 Reagents
The reagents used are frequently offered in an enzyme package by manufacturers, for easy
implementation by laboratories. An example is provided below.
• Reagent 1 (30 ml)
Buffer solution pH 10 / glutamic acid (CAS no[56-86-0]) 440 mg

• Reagent 2 (6 ml)
NAD (CAS no[53-84-9]) 210 mg

• Reagent 3 (0.6 ml)

GPT (CAS no[9000-86-6]) 1100 U

• Reagent 4 (0.6 ml)

L-LDH (CAS no[9001-60-9]) 3800 U

The single reagent used in the instrument is prepared by addition of the 4 reagents from the
package to which is added 300 mg of PVP (Polyvinylpyrolidone) (CAS no[9003-39-8]). It may be
stored for 1 month at 8°C.

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6.4 Preparation of the samples
The must or wine samples are diluted 10 times.

6.5 Analytical procedure

The reading wavelength is 340 nm.
The analytical sequence is as follows:
Rotation 1 (6 seconds): sampling of 5 µl of wine
Rotation 1 (12 seconds): addition of 20 µl of reagent and 200 µl of demineralised water
Rotation 1: reading of absorbance OD1 at 340 nm
Rotation 50 (600 seconds): reading of absorbance OD2 at 340 nm

6.6 Calibration
This is performed at 3 points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The standard values are given by L-lactic acid solutions (CAS no[79-33-4])
covering the range from 0 to 1.2 g∙l-1 diluted 10 times at the time of use. Calibration can also be
performed with wines of known concentrations.

6.7 Reading of results

Reading is performed by measuring the difference between basic absorbance OD2 and absorbance
OD1. The difference in absorbance is proportional to the L-lactic acid content in the tested sample.
The result is expressed in g of L-lactic acid per litre.

6.8 Characteristics of the described method

Intralaboratory reproducibility: 0.2 g∙l-1.
Interlaboratory reproducibility: 0.5 g∙l-1

7 Determination of total phenolic compounds in wines by Folin-Ciocalteu index

7.1 Principle
Chemical method.
The mixture of phenolic compounds present in the wine is oxidised by Folin-Ciocalteu reagent. The
latter is composed of a mixture of phosphotungstic acid and phosphomolybdic acid that is reduced,
during the oxidation of phenols, to a mixture of blue tungsten and molybdenum oxides. The blue
colour produced has a maximum absorption of about 750 nm. It is proportional to the amount of
phenolic compounds present in the wine. The proposed method is a direct automation of the
manual method.

7.2 Equipment
Any sequence analysis equipment enabling the use of 2 reagents and allowing measurement at
750 nm can be used.

7.3 Reagents
• Reagent 1
Folin Ciocalteu reagent

• Reagent 2
Sodium carbonate (CAS [497-19-8]) 20 g
Demineralised water up to 100 ml

7.4 Preparation of the samples

The must or wine samples are not diluted.

7.5 Analytical procedure

The reading wavelength is 750 nm.
The analytical sequence is as follows:
Rotation 1 (6 seconds): sampling of 2 µl of wine or must
Rotation 1 (12 seconds): addition of 50 µl of reagent 1 and 40 µl of demineralised water.
Rotation 25 (300 seconds): addition of 200 µl of reagent 2
Rotation 50 (600 seconds): reading absorbance C to 750 nm

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7.6 Calibration
A zero value is obtained by using a 9‰ sodium chloride solution (CAS no[7647-14-5]). A standard
value is given by a wine determined using the manual reference method.

7.7 Reading of results

The value of the Folin-Ciocalteu index is directly proportional to the measured absorbance OD.

7.8 Characteristics of the described method

Intralaboratory reproducibility: 4.5
Interlaboratory reproducibility: 8.5

8 Determination of -amino nitrogen in wines and musts

8.1 Principle
Chemical method.
The amount of α-amino nitrogen is determined by reaction with o-phthaldialdehyde. The intensity
of absorbance at 340 nm is compared with the intensities obtained for standards of known
isoleucine concentrations.

8.2 Equipment
Any sequence analysis equipment enabling the use of 2 reagents and allowing measurement at
340 nm can be used.

8.3 Reagents
• Reagent 1
NaOH (CAS no[1310-73-2]) 3.837 g
Boric acid (CAS no[10043-35-3]) 8.468 g
Demineralised water up to 1000 ml

• Reagent 2
o-phthaldialdehyde 99% (CAS no[643-79-8]) 0.671 g
N-acetyl–cysteine (CAS no[616-91-1]) 0.0816 g
Twofold ethanol (CAS no[64-17-5]) up to 100 ml

8.4 Preparation of the samples

The must or wine samples are not diluted.

8.5 Analytical procedure

The reading wavelength is 340 nm.
The analytical sequence is as follows:
Rotation 1 (6 seconds): sampling of 5 µl of wine
Rotation 1 (12 seconds): addition of 200 µl of reagent 1
Rotation 24 (294 seconds): reading of absorbance OD1 at 340 nm
Rotation 25 (300 seconds): addition of 200 µl of reagent 2
Rotation 50 (600 seconds): reading of absorbance OD2 at 340 nm

8.6 Calibration
This is performed at 6 points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The other 5 values are obtained with a range of L-isoleucine solutions.
• Stock solution of L-isoleucine
L-isoleucine (CAS no[73-32-5]) 0.936 g
Demineralised water up to 100 ml
Stir well until complete dissolution. This solution contains 1 g of α-amino nitrogen per litre. It
remains stable for 3 months.

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 • Calibration solutions
They are prepared according to the following table:

Quantity in ml of stock
solution for 100 ml of 1 5 10 15 20
demineralised water

Amino nitrogen concentration 10 50 100 150 200

in mg/L
Lifetime: 3 months

8.7 Reading of results

Reading is performed by measuring the difference between basic absorbance OD2 and absorbance
OD1. The difference in absorbance is proportional to the α-amino nitrogen content in the tested
sample. The result is expressed in α-amino nitrogen mg per litre.

9 Determination of ammonia nitrogenin wines and musts

9.1 Principle
Enzymatic method.
In the presence of reduced nicotinamide-adenosine-dinucleotide (NADH), in a reaction catalysed by
glutamate dehydrogenase (GLDH), NH4+ ions and 2-oxoglutarate form L-glutamate.

NH4+ + 2-oxoglutarate + NADH - > L-glutamate + NAD+ + H2O (in the presence of GLDH)

The amount of NADH oxidised in NAD+ is proportional to the amount of ammonia nitrogen present.

9.2 Equipment
Any sequence analysis equipment enabling the use of 2 reagents and allowing measurement at
340 nm can be used.

9.3 Reagents
The reagents used are frequently offered in an enzyme package by manufacturers, for easy
implementation by laboratories. An example is provided below.

• Reagent 1 (30 ml)

TRIS buffer, 2-oxoglutarate pH 8

• Reagent 2 (6 ml)
NADH (CAS no[606-68-8]) 14 mg

• Reagent 3 (0,6 ml)

GLDH (CAS no[9029-11-2]) 550 U

9.4 Preparation of the samples

The must or wine samples are diluted 10 times.

9.5 Analytical procedure

The reading wavelength is 340 nm.
The analytical sequence is as follows:
Rotation 1 (6 seconds): sampling of 5 µl of wine or must
Rotation 1 (12 seconds): addition of 100 µl of reagent and 100 µl of demineralised water
Rotation 24 (294 seconds): reading of absorbance OD1 at 340 nm
Rotation 25 (300 seconds): addition of 20 µl of reagent 2
Rotation 50 (600 seconds): reading of absorbance OD2 at 340 nm

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9.6 Calibration
This is performed at 6 points. A zero value is obtained by using a 9‰ sodium chloride solution
(CAS no[7647-14-5]). The other 5 values are obtained with a range of ammonium sulphate
solutions.
• Stock solution of ammonium sulphate
(NH4)2SO4(CAS no[7783-20-2]) 4.8563 g
Demineralised water up to 100 ml
Stir well until complete dissolution. This solution contains 12.5 g of ammonia nitrogen per litre. It
remains stable for 3 months.

• Calibration solutions
They are prepared according to the following table:

Quantity in ml of stock
solution diluted tenfold per 1 5 10 15 20
100 ml of demineralised
water

Ammonia nitrogen 12.5 62.5 125 187.5 250

concentration in mg∙l-1
Lifetime: 3 months

9.7 Reading of results

Reading is performed by measuring the difference between basic absorbance OD2 and absorbance
OD1. The difference in absorbance is proportional to the ammonia nitrogen content in the tested
sample. The result is expressed in ammonia nitrogen mg per litre.

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