WO2025262196A1

WO2025262196A1 - Optical method for the determination of total or volatile acidity

Info

Publication number: WO2025262196A1
Application number: PCT/EP2025/067220
Authority: WO
Inventors: Antonio Luis MEDINA CASTILLO; Melany Gisell LÓPEZ AVEIGA; María Dolores FERNÁNDEZ RAMOS; Vanessa MARTOS NÚÑEZ; Luis Fermín CAPITÁN VALLVEY; Antonio GONZÁLEZ CASADO
Original assignee: Universidad de Granada
Current assignee: Universidad de Granada
Priority date: 2024-06-20
Filing date: 2025-06-18
Publication date: 2025-12-26
Anticipated expiration: 2026-12-20
Also published as: EP4667914A1

Abstract

The present invention refers to an optical method for the determination of the total or volatile acidity of a sample, which comprises: - a) carrying out a calibration of an optically pH-sensitive material, which is a material comprising a solid support functionalized with an optically pH-sensitive probe through covalent interactions - b) contacting a sample with the optically pH-sensitive material, - c) measuring a change in an optical property of the optically pH-sensitive material produced by a reaction in heterogenous phase between the free hydronium ions (H₃O⁺) of the sample and the optically pH-sensitive material, during a constant reaction time, - d) detecting a signal corresponding to the change in the optical property.

Description

OPTICAL METHOD FOR THE DETERMINATION OF TOTAL OR VOLATILE ACIDITY

FIELD OF THE INVENTION

The present invention is encompassed in the field of analytical chemistry, more specifically in the area of devices for measuring total or volatile acidy in a liquid sample, for example, in the food industry.

BACKGROUND OF THE INVENTION

The acidity of food and beverages plays a fundamental role in the food industry and its control and quantification is essential. The main short-chain soluble organic acids present in beverages (vinegar, milk, wine, juice, etc.) are lactic, tartaric, citric, malic, succinic, and acetic (these acids are produced naturally during fermentation processes). In addition to being natural components of many foods and beverages, lactic, tartaric, citric, malic, succinic, and acetic acids are also widely used in the food industry as antioxidants, flavor enhancers, or additives to control pH. in Article 120 octies, first paragraph, Regulation (EC) No 1234/2007 of Council (DO L 299 de 16.11.2007) two parameters (total or titratable acidity: TA and volatile acidity: VA) are established to regulate and control the acid content in wine and other beverages. TA is the concentration of all the protons available in a sample (both those that are free in solution as HaO⁺ and those that are part of undissociated acids) expressed as g/L of the majority acid present in the sample. On the other hand, VA refers to the concentration of all the protons coming only from the volatile acids present in the sample (a volatile acid is one that has a high vapor pressure at room temperature and, therefore, can be perceived as a smell), and is expressed as g/L of the major volatile acid of the sample. The European Union establishes two official methods, based on classical titration with sodium hydroxide, for the analysis of TA (method: AS-313-01-ACITOT) and VA (method: AS-313-02-ACIVOL for VA.

As examples, wherein the control of these parameters is very important and mandatory, we can highlight the wine and vinegar. The main acids present in vinegar are tartaric, citric, malic, succinic, and acetic (these acids are produced naturally during fermentation processes), with acetic acid being the majority. Therefore, the TA of vinegar is the concentration of all available protons in a vinegar sample (both those that are free in solution as HaO⁺ and those that are part of undissociated acids: tartaric, citric, malic, succinic, acetic, etc.) expressed in g/L of acetic acid (majority acid). On the other hand, considering that acetic acid is the main volatile acid present in the vinegar, and that its concentration is extremely high with respect to the rest of the volatile acids, the VA of vinegar is expressed as the concentration of acetic acid. Just like in vinegar, TA of a wine is the result of the contribution of non-volatile acids, such as malic and tartaric, plus volatile acids. However, in wine, the majority acid component is tartaric acid, so, TA is expressed as g/L of tartaric acid (majority acid). A measure of VA is used routinely as an indicator of wine spoilage. Although generally VA in wine is interpreted as acetic acid content (in g/L), it also includes all those steam-distillable acids, and thus, significant contributions to VA (by steam distribution) of wine may be made by carbon dioxide (as carbonic acid); sulfur dioxide (as sulfurous acid).

Although fast, simple and inexpensive analytical techniques have made great strides in recent years, surprisingly no such methodology exists for determining TA and VA in real samples, with classical titration therefore being the only method capable of accurately determining TA and VA economically and in a relatively short time. Titration consists of direct titration with sodium hydroxide in the presence of phenolphthalein. However, this method has some drawbacks, such as the visual identification of the endpoint can lead to quantification errors, is non-eco-friendly (generating corrosive and toxic sodium hydroxide solutions containing disposable pH colorimetric dyes), does not allow the possibility of quick and easy analysis integrated into the production area, and is timeconsuming: samples must be collected, transported and stored, and the analysis must also be performed in a laboratory by a specialized technician. Nowadays, enzymatic methods have been developed as an alternative to titration to specifically analyze the concentration of acetic acid in food samples (volatile acidity). However, these methods require very expensive and unstable reagents (enzymes), long analysis times, present many interferences, and have a low reproducibility. Alternatively, conventional instrumental methods such as capillary electrophoresis, ion exclusion chromatography with conductimetric detection, and liquid and gas chromatography coupled with mass spectrometry can also be used to determine TA and VA. However, these techniques have many drawbacks, including tedious sampling methods, sample transport and storage, tedious and time-consuming sample and analysis treatments, large quantities of not environmentally friendly solvents, they use very expensive methods that require expensive equipment and specialized technicians, and do not offer the possibility of quick and easy analysis integrated into the production system. Therefore, the development of simple and compact sensors capable of quantifying TA and VA in real samples would provide the food industry with a valuable tool for process and quality control. Gas sensor devices are a promising alternative for real-time monitoring of volatile organic acids in food samples. Most gas sensors are based on a metal oxide semiconductor (MOS), whose electrochemical response is quite fast and reproducible. However, these gas sensors are commonly very sensitive to humidity and, other volatile compounds such as alcohols, aldehydes, ketones, etc, which greatly affect the precision and accuracy of the measurements.

Gas sensors measure the concentration of compounds in the atmosphere (in the gas phase, not in solution) and the humidity present in the atmosphere (gas phase) negatively interferes in the measurements of this type of sensors. With the sensor of the present invention humidity does not affect at all, since the measurement is directly carried out in solution.

Optical sensors have some advantages over electrochemical sensors, such as the absence of electrical interferences, and measurement at different wavelengths with a wide variety of possibilities: transmittance, reflectance, luminescence (with its multiple modes: intensity or polarization, decay time, energy transfer, and quenching efficiency), infrared and Raman spectroscopy, interferometry, and surface plasmon resonance. Other advantages include the possibility to work in humid/aggressive/corrosive/radioactive environments where electrochemical sensors are not operational, cost-effectiveness, the possibility of measuring remotely and at a distance, they can be read without physical contact with the sample, easy miniaturization, etc [1 , 2], Therefore, portable fiber-optic sensors are, an attractive solution for analysed many parameters in the food industry [3], Many optical sensors have been developed to sense gaseous acetic acid and other similar volatile compounds in the atmosphere (gas phase) [3, 4], However, to the best of our knowledge, only three optical sensors have been developed for the quantification of the concentration of short-chain soluble organic acids in solutions, but none of them meet the requirements for application in real samples (e.g. vinegar, wine, etc.). Kurauchi et al [5] reported a fiber-optic sensor having a chitosan/poly(vinyl alcohol) cladding for the detection of the concentration of organic acids in an aqueous solution. However, this fiber-optic sensor presents a strong interference from ethanol, and the authors cannot use it to measure the acetic acid concentration or TA of real samples. On the other hand, Jesus C. et al [6] have developed a sensing probe based on a fiber Bragg grating (FBG) Fabry-Perot cavity, coated with a thin film of sol-gel-PVP (polyvinylpyrrolidone) composite material. The polymeric thin film renders the interferometric output sensitive to the presence of carboxylic acid species. This optical sensor has a linear and reversible response and short response times with adequate reproducibility and repeatability. However, as the authors state, their results show that the sensor responds non-specifically to both short-chain carboxylic species and other types of molecules of similar size such as alcohols, aldehydes, etc., which does not allow its use for the analysis of real samples.

The method with the sensor of the present invention measures total concentration of acid. Unlike other methods, the method based on the use of the sensor of the invention is not affected by the presence of other types of molecules present in the real samples, such as aldehyde alcohols, ketones.

Many optical probes with different pKa values are available for measuring pH in acidic or basic media. This allows pH to be measured optically, both in acidic and basic media: the optical properties of these probes (luminescent, absorbance, color, etc.) increase or decrease as the pH of the medium varies, and this allows calibration to be carried out based on measurements of the intensity of the optical property vs. pH. Optical pH sensors based on the immobilization (using different techniques) of these optically pH- sensitive probes, into fiber optic devices, are already on the market (https://www.pyroscience.com/en/products/laboratory-solutions). However, these devices can only measure pH changes in certain ranges, with sensitivities not exceeding 0.1 pH units (at most), so they are not useful for measuring the concentration of acid (TA and VA) in samples with similar pH (e.g. different vinegar or wine samples where pH variations between samples do not exceed of 0.01 pH units). To perform an optical quantification of the TA and VA in samples like vinegar and wine (acid samples) through pH changes, an optical pH-sensitive probe capable of detecting changes in the order of 0.01 pH units would be required. To the best of our knowledge, there are no fluorescent pH probes with such a high sensitivity. To quantitatively analyze the above statement, let's use vinegar as an example. The total acidity of commercial vinegar ranges between 5% and 8% (w/v), which can be translated into a minimum (0.83M) and maximum (1.33M) concentration of acetic acid, respectively. Furthermore, considering the acidity constant of acetic acid (Ka=1.75 x 10'⁵), the minimum concentration (0.83M) is translated into [HaO⁺] = 0.0038M^pH=2.41 , and for the maximum concentration (1.33M), we have [HaO⁺] = 0.0048M— >pH=2.31. Therefore, to quantify the total acidity of vinegar by measuring pH changes with an optical pH-sensitive probe, it should be able to detect changes of 0.01 pH units in the pH interval [2-3], which far exceeds by far the sensitivity limits of available pH-sensitive probes (as mentioned above, the maximum sensitivity of these probes is 0.1 pH units). For this reason, optical sensors based on optically pH- sensitive probes have not been developed for the quantification of total and volatile acidity in real samples. E. Asijati et al [7] have developed an optical sensor based on polyaniline (PANI) for the quantification of acetic in vinegar samples. However, this sensor cannot be used for measurements in solution, and therefore requires a tedious setup to expose the sensor to sample vapours. Furthermore, this sensor has a dramatically low detection limit (20 g/l) which makes it impossible to use it for measurements in samples with lower acidity (wine, juice, must, etc.).

Analysis using solid supports, so-called test sticks, has recently increased in importance in wine analysis, and have been used for determination of total acid concentration in wine [8], The main advantages of these chemical methods include, simple handling and straightforward disposal owing to the small amount of reagent. In this method all or most of the reagents necessary for the analysis reaction are physically adsorbed on simple swellable solid supports, to which the sample is applied. Then the analysis reaction proceeds after contact of the reaction zone with the sample. The colour formed is a measure of the concentration of analyte to be determined that is evaluated visually i.e. semi-quantitatively or quantitatively, using reflectometry or absorbance [8], However, current test strips for total acidity measurements have several drawbacks, such as:

• Physical adsorption of pH indicators on a solid swellable support has the disadvantage of leaching indicators during the analytical reaction, which, among other things, can lead to irreproducible measurements.

• Leaching of the pH indicator also prevents current tests from being reversed, washed, and reused [8],

• For current test sticks to work, the samples must be buffered [8], This can introduce great irreproducibility in measurements, as different types of samples (e.g., wine) change considerably in their chemical composition (alcohol content, sugar concentration, phenolic profile, etc.), and thus the buffer effect can be drastically influenced by the non-acidic chemical composition of the sample.

• The problem of leaching also prevents the test from being in contact with the liquid sample for a long time. Therefore, the sensitivity of current test strips is very low, with a detection limit of 1 g/l [8], and they lack the sensitivity needed to measure the volatile acidity of distilled wine samples (the volatile acidity of wine ranges from 0.2 to 0.8 g/l).

• The pH indicators used in the current test sticks are colorimetric pH-probes [8], Therefore, these tests can only be evaluated by absorption spectroscopic techniques (colorimetry, reflectometry, absorbance) which are much less sensitive than emission (fluorescence) techniques.

Although conventional dry-chemical methods (test sticks) are currently used to determine the total acid concentration [8], it has not yet been possible to achieve an improved dry-chemical method that overcomes all the drawbacks mentioned above. The present invention therefore has the object of providing a method that overcomes the aforementioned drawbacks for test strips in quantifying the total acid concentration.

One way to overcome the two drawbacks mentioned above of current colorimetric test strips (pH probe filtration and low sensitivity) would be to use more sensitive optical pH probes (e.g., fluorescent probes) than conventional optical pH probes used in test strips (colorimetric pH-probes) [8], and covalently immobilizing them to the solid support to prevent their leaching, thereby manufacturing improved test strips based on a pH- sensitive optical material.

The present invention solves the problems of the conventional colorimetric test sticks mentioned above, by developing an optical technology that allows, to determine in a simple, direct, fast, clean, and cheap way, the total acid concentration in real samples (wine, wine distillate, vinegar, juices, water and others).

According to the present invention, an optical method for the measurement of total and/or volatile acidity in real samples using a conventional optically pH-sensitive probe is provided.

DESCRIPTION OF THE INVENTION

It is well known that the rate of an elementary chemical reaction is proportional to the concentration of reactants in solution. Therefore, based on the protonation rate (in solution) of an optical pH-sensitive probe, a calibration method has been developed for the determination of total and volatile acidity in real samples (wine, wine distillate, vinegar, water, etc.). Once the calibration is made, there is no need to repeat it.

For the development of the method of the invention, it is necessary to establish, experimentally, the relationship between the total concentration of acid in solution and the % of protonation of an optical pH-sensitive material (which is equivalent to the to the change in the optical properties of said material, for a constant reaction time. In this way, at a constant reaction time, the higher the concentration of reagent (acid), the greater the protonation rate of the optically pH-sensitive probe, and, therefore, the greater the change in the optical property (thus obtaining the calibrated [acid] curve vs. optical intensity; t reaction= constant). However, the development of this calibration method using the protonation reaction of optically pH-sensitive probes presents a problem: it is well known, that, no matter how small the concentration of acid (protons) in solution is, the protonation reaction of an optical pH-probe (homogeneous phase reaction) is extremely fast (milli- or microseconds). Therefore, it is impossible, at an experimental level, to establish a useful relationship between the total concentration of acid in solution and the change in the optical properties (fluorescence, absorbance, etc.) of the optical pH-sensitive probe, at a constant reaction time, since the experimental measurement times should be on the order of milli- or microseconds (these time ranges are impossible to manage in the development of simple and inexpensive analysis techniques).

To solve the previous problem and establish a useful reaction time at an experimental level (in the order of seconds), the present invention is based on the foundations of chemical reactions in a heterogeneous phase. In theory, due to the diffusion processes (in this case the protons, have to first diffuse from the liquid phase to the solid phase, so that the reaction can subsequently occur), the protonation reactions of optical pH- sensitive probes in the heterogeneous phase are very much slower than reactions in solution (homogeneous phase).

Based on these principles, we consider the protonation reaction of an optical pH- sensitive probe covalently immobilized (to avoid the leaching problems that occur in conventional test sticks) on a solid support, such as a porous membrane, a heterogeneous reaction in the solid phase. This reaction should be much slower than the protonation reaction in the homogeneous phase (the optical pH-sensitive probe alone dissolved in the medium). In the heterogeneous phase, the protonation reaction depends on the following experimental variables: i) Acid concentration in the solution (proportional to the concentration of free hydronium ions: HaO⁺), ii) Reaction time, iii) Physicochemical properties of the support used for the immobilization (type of material, polarity, porosity, thickness, etc.), iv) Temperature. Therefore, by fixing the variables ii), iii), and iv), the slowing down of the protonation reaction of the optical pH-sensitive probe covalent immobilized on the solid support allows experimentally establish a useful relationship (in terms of reaction time) between the total concentration of acid and the change in the optical properties (fluorescence, absorbance, reflectance, etc.) of the immobilized pH- sensitive probe.

The present invention provides an optically pH-sensitive material, such that by setting a constant reaction time, it is possible to experimentally establish a relationship (calibration curve) between the total concentration of acid in a solution (concentration of protons) and the intensity of an optical property of the optically pH-sensitive material.

A first object of the invention is an optical method for the measurement of the acidity, of a liquid sample, which comprises:

- a) carrying out a calibration of an optically pH-sensitive material, which is a material comprising a solid support functionalized with an optically pH-sensitive probe through covalent interactions

- b) contacting a sample with the optically pH-sensitive material,

- c) measuring a change in an optical property of the optically pH-sensitive material produced by a reaction in heterogenous phase between the free hydronium ions (HaO⁺) of the sample and the optically pH-sensitive material, during a constant reaction time,

- d) detecting a signal corresponding to the change in the optical property.

Step a) is carried out by measuring the change of the optical properties of intensity of the optically pH-sensitive material vs. acid concentration and obtaining a calibration curve, wherein the acidity is selected among one of the following:

- total acidity

- volatile acidity and

- total acidity and volatile acidity measured simultaneously. Total acidity (TA) is the total concentration of acids in a sample. Therefore, total acidity includes all non-volatile acids plus volatile acids. Whereas volatile acidity (VA) refers only to the total concentration of volatile acids present in the sample. Non-volatile acids are those that cannot be separated from the liquid sample by distillation (e.g. malic, succinic, tartaric, etc.), whereas volatile acids are those that can be separated by distillation (acetic acid formic acid, etc.).

“Reaction in heterogenous phase” means reactions in which the reactants are components of two or more phases (solid and gas, solid and liquid, two immiscible liquids) or in which one or more reactants undergo chemical change at an interface, e.g., on the surface of a solid catalyst.

The solid support used for the fabrication of the optically pH-sensitive material, can be any of the materials commonly used in the fabrication of optical sensors for test. The most widespread is filter paper, but it is also possible to use other materials:

The solid support can be selected among porous or non-porous organic, inorganic or hybrid (organic-inorganic), based materials.

The solid support of the optically pH-sensitive material can be an organic, inorganic or hybrid (organic-inorganic) material.

The solid support of the optically pH-sensitive material can be zero-dimensional (0D: 0D means the group of materials that have all three dimensions less than 100 nm), onedimensional (1 D: 1 D means the group of materials that have two dimensions less than 100 nm and one dimension higher than 100 nm), two-dimensional (2D: 2D means the group of materials that have one dimension less than 100 nm and two dimensions higher than 100 nm) or three-dimensional (3D: means the group of materials that have all three dimensions higher than 100 nm). Therefore, the solid support of the optically pH- sensitive material can be shaped as membranes, nanoparticles, microparticles, fibers and others.

The optically pH-sensitive material has the same dimension number as the solid support, meaning that if, for example, the solid support is a two-dimensional material, the optically pH-sensitive material resulting from the said support, will be a two-dimensional material as well. The optically pH-sensitive material of the invention can respond by means of a change in any of its optical properties to small changes in the acidity of the medium (on the order of hundredths of a pH unit), in the order of seconds.

The optical property of the optically pH-sensitive material can be selected among transmittance, reflectance, luminescence (with its many modes: intensity or polarization, decay time, energy transfer, and quenching efficiency), infrared and Raman spectroscopies, interferometry and surface plasmon resonance.

The optically pH-sensitive materials according to the present invention are obtained in two stages: i) activation of the surface of solid support with reactive functional groups, ii) covalent reaction between the reactive groups of the support and some of the chemical groups of optical pH-probe.

The solid support of the optically pH-sensitive material of the invention can be a 0D, 1 D, 2D, 3D material that by itself does not have the natural ability to change its optical properties depending on changes in the pH of the medium, covalently functionalized with an optical pH-probe.

According to a preferred embodiment, the solid support is a cellulose membrane, which is functionalized with the pH-probe Nile blue

The optically pH-sensitive materials are water insoluble solids, wherein the protonation reaction is a heterogeneous one.

In the optically pH-sensitive materials functionalization with the optical pH-probe is performed by covalent bonding to prevent subsequent leaching of the pH-probe.

The solid support of the optically pH-sensitive organic-based materials can be manufactured using one or more of the following polymers or copolymers: : celluloses, nylon, polyacrylonitriles, polyimides, methacrylates, acrylates, polystyrenes, polyacrylamides, polycarbonates, polyethylenes, polyacetylenes, polypropylenes, tetrafluoroethylenes, polytetrafluoroethylenes, polyurethanes, polysulfones, polythiophenes, polyanilines, polypyrroles, polybenzimidazoles, polyketones, chitosan, alginate, DNA, proteins, and any natural or synthetic organic polymer that possesses native luminescence.

The solid support of the optically pH-sensitive inorganic-based materials can be manufactured with one or more of the following raw materials, such as: glass, aluminium oxide, silicon dioxide, zirconium oxide, zeolites, carbon, cordierite, silicon carbide, silicon nitride, mullite, bentonite, zeolite, aluminium oxide, tin oxide, silicon carbide, iron oxide, zinc oxide, silicon, zirconium, copper, gold, silver, titanium, iron, and any natural or synthetic inorganic material that possesses native luminescence.

The solid support of the optically pH-sensitive hybrid-based materials can be manufactured using a combination of the organic and inorganic materials mentioned above.

According to particular embodiments, optically pH-sensitive probes for the manufacture of optically pH-sensitive materials are selected from:

- (i) organic probes, such as Nile Blue, Nile Red, Rose Bengal, Brilliant Red, Brilliant Blue, Brilliant Yellow, phthalocyanines derivatives, porphyrins derivatives, fluoresceines derivatives, rhodamines derivatives, cumarines derivatives, eosines derivatives, cyanines derivatives, acridines derivatives, dansyl-based fluorescent molecules, ruthenium or platinum or palladium or copper or silver or iron, or zinc organometallic complexes,

- (ii) biological probes, such as: protein-based optical probes, DNA-based optical probes, and cells-based optical probes.

- (iii) inorganic probes, such as: inorganic particles, for example, metallic particles such as Au, Ag, metal oxide particles, metal colloids, carbon nanotubes, graphene, carbon dots, and quantum dots,

-(iv) hybrid probes (organic/inorganic/biological), can be manufactured using a combination of the organic, inorganic and biological probes mentioned above.

The sample can be any type of liquid sample, such as vinegar, wine, distilled wine, alcoholic beverages (Brandy, cohac, rum), juice, milk, and water. The optical property can be selected among transmittance, reflectance, luminescence (with its many modes: intensity or polarization, decay time, energy transfer, and quenching efficiency), infrared and Raman spectroscopies, interferometry and surface plasmon resonance.

The method of the invention measures total acidity (TA), or volatile acidity (VA) or both simultaneously total and volatile acidity in a liquid sample.

The total acidity (TA) and/or the volatile acidity (VA) are measured in the same way: by contact of the optically pH-sensitive material with the liquid sample, regardless of the type of sample. For example, in wine the total acidity (TA) can be measured according to a particular embodiment, by introducing a membrane -as the optically pH-sensitive material- directly into the wine, then its fluorescence is measured, and the result is extrapolated by means of a calibration curve previously prepared with tartaric acid (tartaric acid, because in wine it is the major acid). In distilled wine samples only volatile acids are measured, so first the wine must be distilled (this is common to all methods for volatile acidity), a specific amount of wine is distilled - such as 50 ml - and 40 ml of distillate are collected (the distillate is now the sample). After this distillation step, the total acid concentration is measured exactly the same way as in the case of wine samples. The calibration step previous to the measuring of the change in the optical property is carried out with the majority acid presented in the sample, for example, acetic acid for distilled wine samples, and tartaric acid in the case of wine samples. , According to this particular embodiment, for determine the total concentration of volatile acids of wine (VA) a optically pH-sensitive material is introduced in the distilled wine sample . Then its fluorescence is measured, and the result is extrapolated by means of a calibration curve previously prepared with acetic acid.

An optical portable device for carrying out the method previously defined, can comprise:

- an optical recognition element based on an optically pH-sensitive material

- an optical signal transducer element capable of measuring an optical property of the optically pH-sensitive material, capable of transforming the optical signal into an electrical signal and capable of amplifying the signal,

- optionally, a multi-sample distillation system for volatile acidity measurements, for which samples must be distilled,

- a calibration software. The optical signal transducer element can be for example, an spectrometer, a photodiode array detector, a single photodiode detector, a photomultiplier tube, a colorimeter device.

The method of this invention presents significant advances over conventional test sticks, such as [8]: 1) Prevents leaching of the pH probes, 2) No need to buffer samples, 3) It is robust, reversible and reusable, 4) It is highly sensitive: it allows accurate measurement below 0.2 g/l of acid (the sensitivity of conventional test sticks is 1 g/l) [8], 5) The concentration of acetic acid in distilled wine samples ranges between 0.2 and 0.8 g/L. Thus, the method according to the invention, and the optically pH-sensitive material according to the invention offer, for the first time, the possibility of determining volatile acidity in distilled wine samples, 6) The optically pH-sensitive material of this invention has a dual optical response (colorimetric and fluorescent) which, unlike conventional test sticks, can also be evaluated by fluorescence measurements,

The present invention presents significant advances over titration and enzymatic methods such as 1) It is a simple method in which the measurements are performed by introducing the optical recognition element directly into the sample, 2) It is fast, the sensor response is obtained in 15 s, 3) It is environmentally friendly as it does not require additional reagents: titration methods generate corrosive and toxic sodium hydroxide solutions containing disposable pH dyes, and enzymatic methods require expensive and extremely unstable reagents (enzymes) , 4) It is cost-effective, the optical recognition element (Paper-FM) developed in this invention is reversible, reusable and, extremely stable (a membrane of 0.9 mm diameter can be used to carry out at least 50 consecutive measurements), 5) It is portable and could be easily implemented in a miniaturized optoelectronic device, 6) Total or volatile acidity measurements are not affected by the presence of acidic gases in the samples such as carbon dioxide (carbonic acid); or sulphur dioxide (sulphurous acid).

References:

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Jorge, J.M. Baptista, Measurement of acetic acid using a fiber Bragg grating interferometer, Meas. Sci. Technol, 20 (2009) 125201 , https://iopscience.iop.org/article/10.1088/0957-0233/20/12/125201

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Figure 1 : shows an example of the preparation of an optically pH-sensitive material, namely the synthesis of Paper-FM: Activation of the hydroxyl groups of the paper with vinyl sulfone groups (1), covalent immobilization of NB by Michael reaction between the vinyl sulfone groups of activated membrane and the amine groups of NB (2).

Figure 2: Measurement set-up showing the homemade interface formed by three plastic pieces (indicated as 1 , 2, and 3 in the figure) manufactured by 3D printing for the implementation of Paper-FM (indicated as 4 in the figure) in a portable optical device.

Figure 3: Optical characterization of Paper-FM: Fitting the data to equation 1 , where experimental data are denoted with the symbol (o) and the theoretical plot is indicated with a grey dashed line (A), fluorescence emission spectra at different pH values of Paper-FM (B).

Figure 4: Protonation rate of Paper-FM versus the concentration of acetic acid (AA) expressed as % (w/v).

Figure 5: Schematic of the measurement procedure (A), Sensor response: AIF=Ref.lFmax-IF_x vs. AA (%) w/v of the standard solution for a constant reaction time of 15 s (B), and linear range (C).

Figure 6: Effect of temperature on the calibration curve.

Figure 7: Short-term repeatability of Paper-FM: 10 measures for four levels of AA (%) w/v: 0.10%, 0.13%, 0.15%, and 0.18%).

Figure 8: Shewhart control chart for checking the long-term stability of Paper-FM.

Figure 9: Comparison between TA calculated using the proposed fiber-optic sensor and those, obtained by titration.

Figure 10: Sensor response: AIF=Ref.lF_max-IF_x vs. tartaric acid concentration (g L'¹) of the standard solutions for a constant reaction time of 15 s, and linear ranges.

EXAMPLES

EXAMPLE 1

An example of the fabrication of an optically pH-sensitive material (Paper-FM) and its implementation into a portable optical device for measuring total concentration of acid in real samples: wine (total acidity; TA), and, distilled wine (volatile acidity) was developed. For this purpose, we relied on the kinetic principles of heterogeneous solid-phase reactions (slow reactions due to diffusion processes) to carry out the calibration of (Paper-FM) for the quantification of TA. In this example of the calibration step of the method of the invention, a linear relationship between the concentration of acid and the protonation rate of Paper-FM is established. Paper-FM was synthesized by the covalent immobilization of the optically pH-sensitive probe Nile Blue on a porous paper membrane. In addition, a 3D-printed interface was designed to implement Paper-FM into an optical portable device.

2. PREPARATION

2.1. Chemicals and Materials

Divinyl sulfone (DVS), ethylene diamine (TEA), Nile blue chloride (NB), methanol (MeOH), glacial acetic acid (AA), sodium carbonate (SC), citric acid (CA), potassium dihydrogen phosphate (PDP), tris(hydroxymethyl)aminomethane (TRIS), potassium chloride (KCI), sodium tetraborate decahydrate (STBDH), sodium hydroxide (NaOH), and hydrochloric acid (HCI) were purchased from Sigma-Aldrich, cellulose filter paper (ref. 13058: 73 gm-2 and 170 pm thickness) from FILTER-LAB® (Barcelona, Spain), and decolorizing-carbon (Cod. 434507) from Grupo Montedison CARLO ERBA (Divisione Chimica/Milano/ltalia). Throughout the experiment, all aqueous solutions were prepared using reverse osmosis-type quality water (Milli-RO 12 plus Milli-Q station from Millipore, conductivity 18.2 pQ cm).

2.2. Synthesis of Paper-FM (fluorescence membrane)

Paper-FM was prepared by functionalizing porous filter paper (FILTER- LAB 1305) with the pH optically sensitive probe Nile Blue (NB). Functionalization was carried out in two steps [7, 8] (see Fig.1 ): 1) Activation of the hydroxyl groups of the paper with vinyl sulfone groups, 2) Covalent immobilization of NB by Michael reaction between the vinyl sulfone groups of the activated membrane and the amine groups of NB.

2.3. Equipment and measurement protocol

The fiber optic setup used in this work to conduct the fluorescence measurements is based on a 0.5 mm diameter optical fiber (from Ocean Insight) connected to a portable high-resolution spectrometer (measurement range: 100 nm to 1000 nm) equipped with seven excitation LED sources with central wavelengths of: 310 nm (power: 11.5 pW), 365 nm(power: 645.9 pW), 405 nm (Power: 991.6 pW), 457 nm (Power: 1071.9 pW), 523 nm (Power: 331.9 pW), 590 nm (Power: 86.8 pW), and 660 nm (Power: 568.1 pW) from Spyroistech company (Calle Tajonar 22, Edificio Jeronimo de Ayanz. 31006 Pamplona, Navarra, Spain). To reproducibly fit a circular piece 0.9 mm diameter of Paper-FM at a distance of 1 mm the optical fiber, an interface based on three assembled components was designed and fabricated (by 3D printing) (see Fig. 2).

The excitation and emission wavelengths used for Paper-FM were Aexc = 523 nm and Aem = 630 nm, respectively.

Measurement protocol:

I) Preparation of standard solutions of acetic acid. Four stock solutions of acetic acid in purified water (AA (%) w/v: 4%, 5%, 6%, and 7% w/v) were prepared, and subsequently, the standard solutions were prepared by diluting 40 times (1/39, stock solution/distilled water, v/v) the stock solutions,

II) Preparation of universal buffer pH=11 , 0.05 M. Tris (3.02 g), citric acid (5.25g), sodium tetraborate decahydrate (9.53g), potassium dihydrogen phosphate (3.54g), and potassium chloride (1.86 g) were dissolved in 500 mL of water, and then the pH was adjusted to 11 using a solution of NaOH.

III) Procedure for obtaining the calibration curve. Paper-FM (8x2.53cm) was cut into circles of 0.9 mm diameter. The reference state of maximum fluorescence emission (Ref.lFmax) of Paper-FM was established by introducing the membrane in universal buffer pH 11 , 0.05 M for 1 min. Therefore, the calibration curve was carried out as follows: a 0.9 mm diameter circle of Paper-FM was immersed in universal buffer pH 11 , 0.05 M for 1 min, and then the Ref.lFmax value was measured using the set-up shown in Fig.2. Next, the membrane was introduced into the corresponding standard solution of acetic acid for 15 s, and its fluorescence (IF_X; x= AA (%) w/v of the standard solution) was also measured with the set-up of Fig.2. The measurement cycle: universal buffer pH 11 (1 min) <- AA standard solution (15 s) was repeated with all the acetic acid standard solutions. Subsequently, the calibration curve was obtained by representing the sensor response (AIF= Ref.lFmax-IFx) versus AA (%) w/v of the standard solution (x). Three replicates were measured for each concentration.

IV) Measurements of total acidity (TA) in vinegar samples. Seven different types of vinegar (apple cider vinegar, white wine vinegar, rice vinegar, cider vinegar, red wine vinegar, Sherry vinegar, and Modena vinegar) and three brands of each type were selected to evaluate the performance of the sensor. The TA measurements of vinegar samples were performed following the same procedure used for the calibration curve: Firstly, Ref.lF_max was obtained by introducing a 0.9 mm diameter circle of Paper-FM into universal buffer pH 11 , 0.05 M for 1 min. The membrane was then introduced into the diluted vinegar sample (1/39, vinegar/distilled water, v/v) for 15 s, and its fluorescence (I FS_X; Sx = Sample x) was measured with the setup shown in Fig.2. The AT, expressed as AA (%) w/v, was obtained by extrapolating the sensor response (AIF= Ref.lF_max-IFS_x) on the calibration curve. Three replicates of each sample were measured to evaluate the error (stZ- n): where s is the standard deviation, t is the student-t at 95% probability and n-1 freedom degrees, and n is the number of replicas. Each membrane of 0.9 mm in diameter was used for 24 measurement cycles: 4 calibration points x 3 replicates per point and, 4 vinegar samples x 3 replicates per sample. The TA of the non-coloured samples (white wine, apple, rice, and cider vinegar) was measured by introducing the membrane directly into the sample for 15 s. However, in the case of coloured samples (red wine, Sherry, and Modena vinegar), colour significantly interferes with the TA measurement. The interference due to colour was eliminated by decolorizing the samples with activated carbon by the following protocol: The vinegar samples were diluted (1/39, vinegar/purified water, v/v), subsequently, 0.4 g of activated carbon was added to 20 ml of the diluted vinegar, and the sample was shaken for 10 min. Finally, the sample was filtered to remove the carbon. In the case of Sherry and Modena samples, the same protocol was carried out, but for the total decolorization process to occur in 10 min, it was necessary to increase the amount of carbon by 1 .5 g for each 20 ml of diluted vinegar (1/39, vinegar/purified water, v/v).

EXAMPLE 2

To validate the calibration method of an optically pH-sensitive material of the invention for the quantification of acid concentration in wine (total acidity; TA), and distilled wine (volatile acidity; VA), first, we focussed on the design of an optically pH-sensitive material type (II) (Paper-FM) with the above-mentioned characteristics according to example 1 (pKa > 9). For this, the optically pH-sensitive probe Nile Blue (organic probe) was covalently immobilized on a porous paper membrane using the following two-step protocol (see Fig.1 : 1) Activation of the hydroxyl groups of paper with vinyl sulfone groups. A piece of the paper (8x2.3 cm) was introduced into 40 mL of a solution of divinyl sulfone (0.33M) in sodium carbonate buffer (333 mM) at pH=12.00 for 1h. Subsequently, the membranes were washed three times with purified water for 15 min and dried at 50°C in a vacuum oven, 2) Covalent immobilization of NB. Vinyl sulfone groups can easily react with the amine group by a Michael-type reaction. Thus, to functionalize the membranes with the pH-probe NB, a piece of paper (8x2.3cm) previously activated with vinyl sulfone groups was introduced into 40 mL of a solution of 100 mg L'¹ of NB in methanol. Then 2 mL of triethylamine was added, and the immobilization reaction was carried out at 50°C under shaking (roller) for 24 h. Subsequently, the membrane was washed with methanol under sonication until the presence of NB was not detected in the supernatant (the presence of NB in the supernatant was analyzed by measuring the fluorescent emission at 630 nm). Finally, the membrane was dried at 50 °C in a vacuum oven. Then, the characterization of Paper FM was carried out by introducing the membrane (0.9 mm diameter circle) into universal buffer solutions at different pH values: 7, 8, 9, 9.5, 9.8, 10, 10.5, 11 , and 12. Subsequently, the fluorescence spectra were measured using the setup shown in Fig. 2, and the data were fitted using the following equation [9-11]: where I is the fluorescence at each pH value, Io is the fluorescence intensity of the basic form, and B is the fluorescence intensity of the acid form. Fig.3 shows the results of the optical characterization of Paper-FM.

As deduced from the results reported in Fig. 3, Paper-FM has an apparent pKa of 9.60 ± 0.08, which is ideal for developing a calibration method. Subsequently, the relationship between luminescence (fluorescence in this case) quenching of the membrane and acetic acid concentration for a fixed reaction time of 15 s was studied according with point 3 of the measurement protocol described above. For this purpose, stock solutions with different AA (%) w/v were prepared (2%, 4%, 6%, 8%, and 10%). On the other hand, Paper-FM was cut into circles of 0.9 mm diameter, and the fluorescent intensity at pH=11 was selected as the maximum reference fluorescence value (Ref.lF_max). To set the Ref.lFmax value, the membrane was introduced in 10 mL of universal buffer pH 11 , 0.05 M for 1 min, and then its fluorescence was measured using the setup shown in Fig. 2. Then, the membrane was introduced into the acetic acid stock solutions for 15 s, and its fluorescence (IF_X) was also measured with the set-up shown in Fig. 2. To determines the optimal concentration range of acetic acid, stock solutions were progressively diluted, and the sensor began to respond when the stock solutions were diluted 40 times (1/39, acetic acid stock solution /distilled water, v/v). Fig. 4 shows the sensor response (AIF= Ref.lFmax-IFx) versus the concentration of AA (%) w/v after dilution: 0.05%, 0.10%, 0.15%, 0.20%, and 0.25%.

The experimental results reported in Fig. 4 agree well with the proposed hypothesis: by selecting a constant reaction time within the interval [10s-40s] a relationship (calibration curve) between the concentration of acetic acid in solution and the luminescence quenching of Paper-FM can be established. The calibration curve was represented according with the point 3 of the measurement protocol described above stock solutions of acetic acid with the following concentrations (AA (%) w/v): 1 %, 2%, 3%, 4%, %5, 6%, 7%, 8%, and 9%, were prepared, and subsequently, they were diluted 40 times with purified water (1/39, AA stock solution/purified water, v/v) to obtain the following standard solutions: 0.03%, 0.05%, 0.08%, 0.10%, 0.13%, 0.15%, 0.18%, 0.20%, and 0.23%. Then, the sensor response (AIF= Ref.lF_max-IF_x) was measured for all standard solutions, and the calibration curve was obtained by representing AIF= Ref.lF_max-IF_x versus AA (%) w/v. Fig. 5 shows a schematic of the measurement procedure and the calibration curve. in Fig. 5A, the response of the sensor versus the concentration of AA corresponds to a type of sigmoidal curve in which two linear ranges are observed, one at low concentrations (% w/v) of AA [0.03%-0.08%] and, the other at higher concentrations [0.1 %-0.18%].

The experimental data presented in Figs. 4 and 5 validated the calibration method based on the relationship between acid concentration in solution and quenching of the luminescence of a conventional pH-sensitive fluorescent membrane with a basic pKa. Either of the two linear ranges in Fig. 5 could be used for the calculation of TA (expressed as AA (%) w/v), we have chosen the higher linear range (0.1 %-0.18%) (see Fig. 5C) simply because of its greater amplitude, and because it is closer to the TA of the vinegar, thus requiring less dilution of the samples. The linear range (0.1 %-0.18%) has a good correlation coefficient (R²= 0.996) with detection and quantification limits of 0.0024% and 0.0083%, respectively. The detection and quantification limits were determined using the IIIPAC method (LOD = 3sb/m; LOQ=10 sb /m). The standard deviation of the blank (Sb) was obtained by measuring (10 measurements) the response of the sensor for a pH=11 , 0.05 M universal buffer solution.

Considering that the proposed calibration method is based on the protonation reaction of immobilized pH-probe, the rate constants and diffusion coefficients of this reaction can be affected by temperature. Therefore, to check the influence of temperature, the calibration was carried out at different temperatures (15°C, 20°C, 25°C, 30°C, and 35°C). As shown in Fig. 6 between 15°C and 25°C calibration is not affected by temperature, however, as expected, above 25°C the rate of the protonation reaction increases considerably, and although good linearity is still maintained, the slope of the calibration begins to decrease (decrease in sensitivity). Even so, the sensitivity of the calibration at 35°C (black line of fig. 6) is still quite good, with a detection limit (0.004 AA% w/v) and quantification limit (0.013% AA w/v) well below the TA values in the vinegar samples (TA in vinegar is between 5% AA w/v and 8% AA w/v).

3.2. Stability of Paper-FM

To analyse the stability and reusability of the Paper-FM, short-term precision (repeatability) and long-term precision (reproducibility) were studied. For short-term precision, the sensor response (AIF= Ref.lF_max-IF_x) was measured for four concentration levels: AA (%) w/v: 0.10%, 0.13%, 0.15%, and 0.18% (10 measurements were performed for each concentration), and all the measurements (50) were performed consecutively in less than 2 h using a single membrane of 0.9 mm diameter. As can be seen in Fig. 7, Paper-FM is extremely stable: each membrane of 0.9 mm diameter can be used to perform at least 50 measurements.

(Fig. 7) The long-term stability was studied by measuring the sensor response for a single concentration level (0.14 AA (%) w/v) for 18 days (after each measurement, the membrane was dried at room temperature (RT) and stored in the absence of light). All measurements were performed using the same 0.9 mm diameter membrane. The results were interpreted using a Shewhart diagram (Long-term stability was defined as the signal that remains within the control lines on the Shewhart chart). As shown in Fig. 8, after 18 days, the response of the membrane remained within the established control limits, showing excellent long-term stability.

The results reported in Figs. 7 and 8 show extraordinary stability and reusability of Paper- FM: a single 0.9 mm diameter membrane can be used to perform more than 50 consecutive measurements, and can also be stored and reused for at least 18 days (although after 18 days the limits set in the Shewhart control chart are exceeded, the membrane is still responsive, and thus it could be recalibrated and used further). Therefore, with commercial use in mind, Paper-FM is extremely efficient and cost- effective.

3.3. Real-world application and validation

To evaluate the efficiency in the analysis of real samples, the total acidity (TA) of 21 samples of vinegar and the total and volatile acidity of 7 samples of wine of different origins were analysed by both the method proposed in this invention and the reference methods (titration method was used for TA of vinegar samples and for wines samples titration method and enzymatic method were used for total and volatile acidity respectively). Considering that the majority acid in wine is tartaric acid, to calculate the total acidity in the wine samples, a calibration using tartaric acid (see Fig. 9) was previously established (the calibration was carried out following the same protocol as that described above for acetic acid), and the total acidity was expressed as g L'¹ of tartaric acid. The total acidity of the samples was measured by introducing the Paper- FM directly into the sample (vinegar and wine) for 15 s, and then measuring its fluorescence. In the case of coloured samples (red wine, Sherry, and Modena vinegar), colour significantly interferes with the TA measurement. The interference due to colour was eliminated by decolorizing the samples with activated carbon using the protocol described above.

For the measurements of volatile acidity of wine samples, in a first step volatile acids were extracted by distillation: 50 mL of wine were taken and distilled until 40 mL of distillate was collected. Then, volatile acidity was measured by introducing Paper-FM directly into the distillate for 15 s, and measuring its fluorescence with the setup of Fig, 2. Each membrane of 0.9 mm in diameter was used for 24 measurements (four points for the calibration curve and three vinegar samples: three replicates for each measure). Table 1 summarizes the results of TA (expressed as % w/v of acetic acid) of vinegar samples given by the manufacturer, obtained with the optical technology of this invention, and those obtained by the official reference method (titration: AS-313-01- ACITOT), and Table 2 summarizes the results of total acidity (TA: expressed as g L'¹ of tartaric acid) and volatile acidity (VA: TA: expressed as g L'¹ of acetic acid) of wine samples obtained with the technology of this invention, and those obtained by the official reference methods (titration and enzymatic methods) Table 1 : Results of TA of vinegar samples by manufacturer, the optical technology of this invention and the official reference method (titration).

Table 2: Results of TA and VA of wine samples by the optical technology of this invention, and the official reference method (titration). VA was calculated using the official reference methods: i) Titration methods: OIV-AS-313-01-ACITOT for total acidity and AS-313-02-ACIVOL (Vol.) for volatile acidity, ii) Enzymatic method: RESOLUCION OIV-OENO 621-2019 (Enz) for volatile acidity. The results reported in Figs. 5, 6, 7, 8, 9, and Tables 1 and 2 demonstrate, that the proposed optical method allows simple, fast, cost-effective, and environmentally friendly quantification of acid concentration in wine (total acidity; TA), and distilled wine (volatile acidity; VA)

EXAMPLE 3

Sensor Validation

The optical technology developed was tested by analyzing 21 vinegar samples and 7 wine samples of different origins according to example 2 above, and the results were successfully validated with the official reference method.

In the case of the analysis of TA of vinegar samples, the results using the optical technology of the invention, were validated by the official reference method (OIV-AS- 313-01-ACITOT), which consists of direct titration with sodium hydroxide. The titration was carried out using a METTLER TOLEDO® Compact G20S according to the following protocol: Initially, the solution of NaOH was standardized with potassium hydrogen phthalate (KHP). For titration, the sample (2 mL of vinegar) was introduced into the sample cup, distilled water was added until the electrode was covered, and then titration was performed. The operation was repeated three times for each sample, and the volumes of NaOH spent were recorded and used for the calculation of total acidity, expressed as AA (%) w/v.

The validation of the results obtained for TA and VA in the wine samples with the optical technology developed in this invention, were validated using the official reference methods by the laboratory Dolmar (https://dolmarlaboratorio.com/) in La Rioja (laboratory authorized to carry out official analyses in the wine sector R.(EU)2018/273), and accredited by ENAC) using the official methods, titration methods: OIV-AS-313-01- ACITOT for total acidity and AS-313-02-ACIVOL for volatile acidity, and enzymatic method: RESOLUCION OIV-OENO 621-2019 (Enz) for volatile acidity.

The results show that the optical technology presented in the invention allows a direct, simple, and fast (15 s response time) quantification of TA and VA in beverages (vinegar and wine). It is environmentally friendly, because, unlike titration methods, it does not generate toxic waste, it is reversible, and is extremely stable (a 0.9 mm diameter circle of Paper-FM allows at least 50 consecutive measurements).

Claims

1. An optical method for the measurement of the acidity, of a liquid sample, which comprises:

- b) contacting a sample with the optically pH-sensitive material,

- d) detecting a signal corresponding to the change in the optical property.

- total acidity

- volatile acidity and

- total acidity and volatile acidity measured simultaneously.

2. The method according to claim 1 , wherein the solid support of the optically pH- sensitive material is a solid support selected among:

- porous or non-porous organic, inorganic or hybrid organic-inorganic, based materials.

3. The method according to claim 1 or 2, wherein the solid support of the optically pH- sensitive material is selected among:

- zero-dimensional materials,

- one-dimensional materials,

- two-dimensional materials and

- three-dimensional materials.

4. The method according to any of claims 1 to 3, wherein the optically pH-sensitive material is able to detect changes in the acidity of a medium on the order of hundredths of a pH unit, with a maximum optical response for a reaction time in the order of seconds.

5. The method according to claim 1 or 4, wherein the solid support of the optically pH- sensitive material comprises one or more of the following organic polymers or copolymers: celluloses, nylon, polyacrylonitriles, polyimides, methacrylates, acrylates, polystyrenes, polyacrylamides, polycarbonates, polyethylenes, polyacetylenes, polypropylenes, tetrafluoroethylenes, polytetrafluoroethylenes, polyurethanes, polysulfones, polythiophenes, polyanilines, polypyrroles, polybenzimidazoles, polyketones, chitosan, alginate, DNA, proteins.

6. The method according to claim 5, wherein the solid support of the optically pH- sensitive material of type comprises one or more of the following raw inorganic materials: glass, aluminium oxide, silicon dioxide, zirconium oxide, zeolites, carbon, cordierite, silicon carbide, silicon nitride, mullite, bentonite, zeolite, tin oxide, silicon carbide, iron oxide, zinc oxide, silicon, zirconium, copper, titanium, gold, silver, iron.

7. The method according to any of claims 1 to 6, wherein the optically pH-sensitive probe is selected from: i) organic probes, preferably selected from Nile Blue, Nile Red, Rose Bengal, Brilliant Red, Brilliant Blue, Brilliant Yellow, phthalocyanines derivatives, porphyrins derivatives, fluoresceines derivatives, rhodamines derivatives, cumarines derivatives, eosines derivatives, cyanines derivatives, acridines derivatives, dansyl-based fluorescent molecules, ruthenium and platinum organometallic complexes, ii) biological probes, preferably selected from protein-based optical probes, DNA-based optical probes and cells-based optical probes. iii) inorganic probes, preferably selected from carbon dots, quantum dots, metallic particles, carbon nanotubes, graphene, metal oxide particles and metal colloids, and iv) hybrid optically pH-sensitive probes, preferably selected from:

- organometallic complexes of ruthenium, or platinum, or copper or silver or iron, or zinc,

8. The method according to any of the preceding claims 1 to 7, wherein the sample is a liquid sample, selected from vinegar, wine, distilled wine, alcoholic beverages, juice, milk, and water.

9. Method according to any of the preceding claims 1 to 8 wherein the optical property is selected from transmittance, reflectance, luminescence infrared and Raman spectroscopies, interferometry and surface plasmon resonance.

10. Method according to any of claims 1 to 9, wherein the optically pH-sensitive material determine acid concentrations levels lower than 0.2 g/l, in reaction times between 15 and 120 s, in a reversible and reusable manner.