Detection of vicinal diketones

Abstract

The present invention relates to the use of a compound of formula (I) as a condensation reagent for the photometric detection of vicinal diketones of formula (II)

Description

BACKGROUND OF THE INVENTIONThe quality assurance of the beer and the monitoring of the brewing process are at the focus of each brewing plant. The vicinal diketones (VDCs) play a decisive role here as false flavours as quality parameters in beer maturation and in defining the optimum storage time. An important representative among the VDCs is the diketone diacetyl, which already causes off-taste in the beer in relatively low concentrations (>0.1 mg / l). It arises during the fermentation process, but is successively degraded during the beer maturation.If the yeast cells during the brewing process can no longer sufficiently absorb the amino acid valine from the wort for the buildup of biomass, they themselves produce these from glucose. An intermediate product of this biosynthesis is the metabolic intermediate pyruvate, from which 2-acetolactate is formed by decarboxylation. Outside the yeast cells, 2-acetolactate non-enzymatically reacts to diacetyl by oxidative decarboxylated. Diacetyl causes an undesirable, colored off-note even in low concentrations in beer. Diacetyl can, however, be further reduced by the beer yeasts to 2,3-butanediol, which has a taste threshold value that is many times higher and is therefore no longer discernible in the beer. If the latter ceases to degrade or if release is forced by adverse conditions, diacetyl causes a distinct and unpleasant off-flavour. A similar problem is observed in wine. However, the taste limit is much higher due to the more intensive aroma of the wine (white wine 0.2 mg / l, red wine up to 3 mg / l). In addition, the mechanism of formation is coupled to the biological acid degradation, wherein the degradation of citric acid by bacteria first produces pyruvate and, from this, enzymatically diacetyl.The previous analysis of VDCs to monitor the brewing process is relatively expensive and complicated and comprises gas chromatography of cooled random samples by external laboratories. Furthermore, steam distillation has been used to enrich the VDCs, which is a time-consuming and cost-intensive process.J. M. Dechary et al., "Sptrophotometric Determination of Ethanediaf", Analytical Chemistry 26, 1954, pp. 449-452, relates to the photometric detection of ethanedial and 2-oxopropanal in animal samples. The reaction of 2,3-butanedione with 2,3-diaminophenazine is described.M. Mariaud et al., "New Quantitative Determination of 2,3-dioxobutane (diacetyl) in alcoholic procedures by an indirect spectrofluorometric procedure with use of 2,3-diaminonaphthalene as reagent of derivatization", Talanta, 41, 1994, 1, pp. 75-79, discloses photometric detection of diacetyl in alcoholic beverages with the aid of 2,3-diaminonaphthalene.A. Barros et al., "Determination of glyoxal, methylglyoxal, and diacetyl in selected beer and wine, by HPLC with UV spectrophotometric detection, after derivatization with o-phenylenediamine", J. Liq. Chrom. & Technol., 22, 1999, 13, pp 2061-2069 discloses photometric detection of diacetyl with the aid of 1,2-diaminobenzene.X. Song et al., "Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization", J. Chromatogr. A., 1361, 2014, pp. 162-168 describes the photometric detection of 4,5-dihydroxy-2,3-pentanedione with the aid of 2,3-diaminonaphthalene.The announcement "Development of a mobile detection system in handheld format for the quantitative determination of vicinal diketones from green beer" at the spring symposium of the American Chemical Society describes a rapid and simple on-site detection of diacetyl by binding and release of diacetyl by means of a crosslinked polyacroylhydrazide and subsequent photometric detection.It is therefore an object of the present invention to improve the analytics of VDCs. In particular, it is the object of the present invention to provide a fast and simple on-site detection.SUMMARY OF THE INVENTIONTo overcome these problems, the present invention proposes:A method for the detection of a vicinal diketone of formula (II) wherein R 1 and R 2 are methyl, the method comprising the steps of: (i) providing a sample containing the vicinal diketone; (ii) contacting the sample with a hydrazide functional polymer to covalently attach the vicinal diketone to the polymer; (iii) separating the sample and polymer; (iv) releasing the vicinal diketone from the polymer by changing the pH; and (v) photometric detection of the vicinal diketone, wherein the photometric detection comprises a step of condensing the vicinal diketone and a compound of formula (I) and detecting the condensation product of formula (III) wherein A and B are each N and C and D together with the carbon atom to which they are attached form a benzene ring, wherein the photometric detection takes place at a wavelength of between 500 and 700 nm, and wherein step (ii) takes between 5 seconds and 30 minutes and / or step (iv) takes between 5 seconds and 30 minutes.DESCRIPTION OF THE DRAWINGSFIG. 1 : Schematic longitudinal section of a capillary containing polyacryloylhydrazide (PAH). Such a capillary can serve as a connection and release unit.DETAILED DESCRIPTION OF THE INVENTIONPreferred embodiments of the method according to the invention are defined in the dependent claims. The invention will be described in detail below.The prefix "C x- C y" denotes the possible number of carbon atoms in the respective group.The term "C 1- C 6 alkyl" alone or as part of another group denotes a linear aliphatic carbon chain containing 1 to 6 carbon atoms or a branched aliphatic carbon chain containing 4 to 6 carbon atoms. Non-limiting exemplary (C 1- C 6) alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, and 2-butyl.As used herein, the singular forms such as "a" and "the" include both the singular and the plural, unless the context clearly indicates otherwise. For example, "a vicinal diketone" means a vicinal diketone or more than one vicinal diketone, i.e., "vicinal diketones.".The invention relates to a method for the detection of a vicinal diketone of formula (II) wherein R 1 and R 2 are methyl, the method comprising the steps of: (i) providing a sample containing the vicinal diketone; (ii) contacting the sample with a hydrazide functional group polymer to covalently bond the vicinal diketone to the polymer; (iii) separating the sample and polymer; (iv) releasing the vicinal diketone from the polymer by changing the pH; and (v) photometric detection of the vicinal diketone, wherein the photometric detection comprises a step of condensing the vicinal diketone and a compound of formula (I) and detecting the condensation product of formula (III) wherein A and B are each N and C and D together with the carbon atom to which they are attached form a benzene ring, wherein the photometric detection takes place at a wavelength of between 500 and 700 nm, and wherein step (ii) takes between 5 seconds and 30 minutes and / or step (iv) takes between 5 seconds and 30 minutes.The compound of formula (I) is 2,3-diaminophenazine.The detection of the condensation product takes place at a wavelength between 500 and 700 nm, preferably at a wavelength between 550 and 680 nm, particularly preferably at 623 nm. The condensation product of 2,3-diaminophenazine and diacetyl, 2,3-dimethylpyrazino[2,3-b]phenazine, has its absorption maximum at 623 nm and can be detected particularly well at this wavelength. It has surprisingly been found that the compound of the formula (I) is less sensitive to oxidation than established staining compounds. Furthermore, the condensation of the compound of the formula (I) with the vicinal diketone of the formula (II) takes place rapidly and reliably. Thus, the concentration of the vicinal diketone can be determined quickly and with excellent accuracy. Furthermore, the concentration can be detected accurately under different measurement conditions and over a further concentration range. A further advantage is that the condensation product is detectable at a wavelength of preferably between 550 and 680 nm. Thus, the measurement range is outside the intrinsic color of beer or wine.R 1 and R 2 of the vicinal diketone of formula (II) are methyl, i.e. the vicinal diketone is diacetyl. As described above, diacetyl can occur as a disadvantageous, undesirable by-product during an alcoholic fermentation process, for example during wine or beer production. In a preferred embodiment, the vicinal diketone is from a sample in wine or beer production. In other words, photometric detection of the vicinal diketone takes place to monitor the concentration of this compound.The concentration of the vicinal diketone can thus be determined (indirectly) via the concentration of the condensation product of the formula (III). The expression "photometric detection of vicinal diketone of the formula (II)" thus comprises indirect photometric detection via the condensation product of the formula (III) from vicinal diketone of the formula (II) and the compound of the formula (I). In other words, the use according to the invention relates to the use of the compound of the formula (I) as a condensation reagent with a vicinal diketone of the formula (II) in order to photometrically detect the condensation product.The concentration of the condensation product can be determined, for example, using a calibration straight line and such concentration determination is well known to those skilled in the art.The disclosure also relates to a method for enriching the vicinal diketone of formula (II), the method comprising the above steps (i) to (iv): (i) providing a sample containing the vicinal diketone; (ii) contacting the sample with a hydrazide functional polymer to covalently attach the vicinal diketone to the polymer; (iii) separating the sample and polymer; and (iv) releasing the vicinal diketone from the polymer by changing the pH.The process for the enrichment of the vicinal diketone is much simpler and more efficient than the established processes, such as steam distillation, and enables portable on-site analysis. In particular, the enrichment of the vicinal diketone by steps (i) to (iv) makes it possible to detect the vicinal diketone photometrically accurately, quickly, reproducibly and reliably.In one embodiment, the sample is from wine or beer production. The method is for the detection of the vicinal diketone during wine or beer production. By enriching the vicinal diketone by the polymer with functional hydrazide groups (also called "capture-release" polymer), it is possible to determine even very low concentrations of vicinal diketones. Furthermore, the beer matrix or wine matrix can be separated by the method, whereby the measurement accuracy is further improved.The chronological sequence of steps (i)-(iv) or (i)-(v) takes place in the sequence mentioned.The contacting of the sample with the hydrazide functional polymer in step (i) may be by mixing with polymer powder. The polymer can also be present as granules, matrix or porous structure. Alternatively, the sample may be contacted with the polymer by filtering over a filter system comprising, among other things, the polymer. Alternatively, a capillary or syringe comprising the polymer may be used to contact the sample with the polymer.During step (ii), the ketone groups of the vicinal diketone are attached to the hydrazide groups of the polymer. It is preferred that the pH of the sample for this purpose is not changed. Preferably, the pH of the sample is between 4 and 8.In other words, the method comprises the steps of: (i) providing a sample containing the vicinal diketone; (ii) contacting the sample with a hydrazide functional polymer to covalently attach the vicinal diketone to the polymer and provide a polymer having attached the vicinal diketone; (iii) separating the sample and polymer having attached the vicinal diketone; (iv) releasing the vicinal diketone from the polymer having attached the vicinal diketone by changing the pH.In step (iii), the remaining sample is separated from the polymer having the vicinal diketone covalently bonded. The step (iii), separating sample and polymer, can be realized by centrifugation, pumping or filtration.It is preferred that after step (iii), the separation of sample and polymer, the polymer is washed, for example, with water in order to avoid non-specific absorption. Thus, it is preferred that the process comprises the step of: (iiia) washing the polymer.In step (iv), the vicinal diketone is released. The release of the vicinal diketone from the polymer by changing the pH, especially with an aqueous acidic solution, is well known to those skilled in the art. It is preferred that the pH be changed by contacting it with an acidic solution to effect acid catalyzed hydrolysis. The pH of the solution is preferably between -1 and 4.The release takes place preferably for 0.5-60 minutes, more preferably for 1-30 minutes. The term "release" is to be understood as meaning that the diketone is released completely or approximately completely. Also, the term "contacting to covalently bond the vicinal diketone to the polymer" is understood to mean that the vicinal diketone in the sample is completely or approximately completely bonded. Approximately complete denotes at least 90%, preferably at least 95%, particularly preferably at least 98%. In a preferred embodiment, the contacting takes place for 10 seconds to 30 minutes. Step (ii) lasts between 5 seconds and 10 minutes and / or step (iv) lasts between 5 seconds and 10 minutes.It is preferred that steps (i) to (iv) not only separate the vicinal diketone of the formula (II) from the remaining constituents of the sample but also enrich the vicinal diketone of the formula (II). For example, the vicinal diketone of the formula (II) is liberated in step (iV) with 0.1 to 10 ml, preferably with 0.5 to 5 ml, of an acidic aqueous solution. In one embodiment, the concentration of the vicinal diketone after step (iv) is at least 5 times, preferably at least 10 times, more preferably at least 20 times higher than the concentration of the vicinal diketone in the sample.It is preferable that the hydrazide functional polymer is a polyacryloylhydrazide (also called polyacryloylhydrazide). The polyacryloylhydrazide is preferably crosslinked. The polyacroylhydrazide is obtainable from a polyacrylate and hydrazine.It is preferred that the hydrazide functional polymer is a homopolymer. In a further embodiment, the polymer may be a copolymer composed of at least two (chemically) different repeat units.Polyacrylates, and also suitable monomers and comonomers for their preparation, are described, for example, in "Penzel, E.; Ballard, N.; Asua, J. M. Polyacrylates. Ullmann's Encyclopedia of Industrial Chemistry; Wiley-VCH: Weinheim, Germany, 2018". Non-limiting examples of monomers suitable for forming the hydrazide functional polymer in the present invention are: methacrylate (MA). Ethyl acrylate (EA), hydroxyethyl acrylate, n-butyl acrylate, isobutyl acrylate, n-propyl acrylate, hydroxyethyl methacrylate (HEMA), hydroxyethoxyethyl methacrylate (HEEMA), hydroxydiethoxyethyl methacrylate (HDEEMA), methoxyethyl methacrylate (MEMA), methoxyethoxyethyl methacrylate (MEEMA), methoxydiethoxyethyl methacrylate (MDEEMA), ethylene glycol dimethacrylate (EGDMA), acrylic acid (AA), acrylamide, and N-(2-hydroxypropyl) methacrylamide (HPMA). After polymerization, hydrazine is added to form the hydrazide functional polymer.The hydrazide functional polymer is preferably cross-linked. Preferably, the hydrazide group is used as a group to crosslink the polymer. The hydrazide groups can be crosslinked, for example, with a dicarboxylic ester such as diethylmalonate, a dicarboxylic acid or a dicarboxylic anhydride as a crosslinking agent.The typical proportion of hydrazide groups which are cross-linked is 1-30% based on the amount of all hydrazide groups in the polymer. In one embodiment, the polymer is a polyacryloylhydrazide of the formula (IV): In one embodiment, n and I are independently between 95 and 80 mol %, and m is between 5 and 20 mol %.The number average molecular weight of the polyacryloylhydrazide is preferably from 6000 to 150000 g / mol.It has surprisingly been found that the polymer with hydrazide functional groups has a high selectivity and reactivity towards diketones. The binding and release thus take place quickly and selectively. Furthermore, the polymer is stable even under acidic conditions and no appreciable degradation of the polymer or hydrazide functional groups occurs. This allows reuse of the polymer. Due to the high number of hydrazide groups, even small amounts of the polymer are sufficient for complete attachment of the vicinal diketones from the sample.Furthermore, the method can be adapted to different conditions, such as different diketone concentration. However, due to the high number of hydrazide groups in the polymer, this is usually not necessary and the hydrazide functional polymer can be used for enrichment of both high and low amounts of vicinal diketone. In one embodiment, the concentration of the vicinal diketone of formula (II) in the sample is between 0.005 and 10 mg / L, preferably between 0.01 and 5 mg / L.The polymer may be located in a capillary, a filter (system), a syringe or a pipette tip. The dimension of the capillary can have, for example, a length of 20 to 200 mm, preferably 50 to 150 mm, particularly preferably 90 mm. The volume of the capillary is preferably 20 to 2000 μl, more preferably 50 to 1000 μl, particularly preferably 250 μl. Furthermore, a pumping unit may be used to contact a sample with the polymer. A suitable pump unit is, for example, a peristaltic pump with a sufficient number of channels to process a large number of samples in parallel. As described above, the hydrazide functional polymer may be in a filter system. The filter system can serve as a plug-on filter system for a syringe. The filter system comprises a membrane filter which serves to separate the sample and the polymer. For this purpose, a screwable syringe filter with a diameter of 25 mm can be used, for example. A polycarbonate membrane with a pore size of 10 μm and a diameter of 25 mm can be used as filter insert, for example. A 20 ml disposable syringe with a Luer lock connection can be used as the syringe, for example, so that the connection between syringe and filter cannot be released even at higher pressure.In a particularly preferred embodiment, the method of the invention comprises the steps of: (i) providing a sample containing the vicinal diketone of formula (I)); (ii) contacting the sample with a polymer which is polyacryloylhydrazide to covalently bond the vicinal diketone to the polymer; (iii) separating the sample and polymer; (iiia) optionally washing the polymer; (iv) releasing the vicinal diketone from the polymer by contacting the polymer with an acidic aqueous solution having a pH between -1 and 4; and (v) photometrically detecting the vicinal diketone.The present disclosure further relates to a detection system for determining the concentration of vicinal diketones of the formula (II): the detection system comprising: (i) a bonding and release unit which is designed such that it can bond the vicinal diketones and, after changing the pH value, can release the vicinal diketones.The detection unit can furthermore comprise: (ii) a detection unit which is designed in such a way that the concentration of the cardiovascular diketones can be determined, preferably photometrically. The attachment and release unit and the detection unit can be spatially separate from one another.The present disclosure further relates to an enrichment system for enrichment of vicinal diketones of the formula (II), wherein the enrichment system comprises a binding and release unit which is designed such that it can bind the vicinal diketones and release the vicinal diketones after changing the pH. Preferably, the attachment and release moiety comprises a polymer having hydrazide functional groups. The attachment and release unit may comprise a capillary, a filter system or a pipette. In addition to enrichment, the attachment and release unit also achieves a separation of the vicinal diketone from the further constituents in a sample and it can thus be separated from the beer matrix from a beer sample, for example.ExamplesThe following exemplary embodiment is intended to illustrate and explain the method according to the invention further without restricting the subject matter according to the invention.Example 1: Preparation of a cross-linked polyacryloylhydrazideThe monomer methyl acrylate (15 ml, dest.) was dissolved in DMF c Monomer= 5 mol / l). AIBN (0.5 mol %) was added and nitrogen was passed through the mixture for 30 min to remove oxygen. The mixture was stirred and heated to 75°C. After 60 min, the mixture was cooled and the polymer precipitated in methanol. It was dissolved three times in acetone and precipitated in methanol. After freeze drying, for example, a yield of 9.5 g (66%) polymethyl acrylate was obtained.A solution of polymethylacrylate (9 g, 104 mmol) was dissolved in THF (270 mL) of THF. Hydrazine hydrate (35 g, 689 mmol) and TBAB (9 g, 27 mmol) were added. The mixture was stirred at 60°C for 12 hours. After cooling, the aqueous phase was transferred to methanol to precipitate the product. The obtained white polymer was washed several times with methanol to remove low molecular weight impurities and dried under vacuum at room temperature. For example, a yield of 8.2 g (91%) of polyacryloylhydrazide (PAH) was obtained.To 85 mL of an aqueous solution of polyacryloylhydrazide (93 mmol) was added diethylmalonate (32 mmol, 0.34 eq.) and the mixture was stirred until a homogeneous solution formed. The solution was then maintained at 70°C for 3 hours. The formed hydrogel was cooled to room temperature, purified by dialysis, and then freeze-dried. The resulting product was ground to a fine white polymer powder.Example 2: Method for the detection of vicinal diketoneThe procedure of the "Capture&Release" process for determining the diketone concentration in, for example, a beer sample proceeds as follows:The sample volume of 10 ml is brought into contact with the functional polymer PAH for 15-120 min. This can be done in a capillary by pumping, free in solution or in a syringe with a syringe filter placed on top. After the diketones have been bound, the polymer is separated from the sample and washed with water to avoid non-specific absorption. Depending on the method, this is achieved by pumping with the peristaltic pump, centrifugation or by actuation of the syringe. For release, a small volume of acid is placed in contact with the polymer. For this purpose, for example, 1 ml of concentrated hydrochloric acid having a pH of -1 is used. After a contact time of 1-30 min, the acid with the released diketones is separated from the polymer likewise by pumping with the peristaltic pump, centrifugation or by actuation of the syringe. The polymer can be reused after washing several times with water. The release solution is stained with 2,3-diaminophenazine for photometric determination.Example 3: Staining and photometric detectionTo the release solution from the "Capture&Release" process is added a solution of 2,3-diaminophenazine (DAP) in ethanol (e.g., to 1 ml release solution 50 μl DAP in ethanol, 1 mg / ml).The stained release solution is measured in a polystyrene disposable cuvette in a commercial photometer in the wavelength range from 500 to 800 nm. The measurement is measured at the time t=0 min as a baseline (baseline correction). The diketone concentration is determined by measuring the absorption at 623 nm after a reaction time of 15-120 min and by comparing the obtained absorption with a calibration straight line prepared beforehand.The stained release solution is placed between the LED and photodiode in a polystyrene disposable cuvette in the portable photometer and the photocurrent is measured. The measurement is measured at the time t=0 min as a baseline (baseline correction). The diketone concentration is determined by measuring the photocurrent after a reaction time of 15-120 min and by comparing the absorption obtained with a calibration straight line prepared beforehand.

Claims

A method for the detection of a vicinal diketone of formula (II) wherein R 1 and R 2 are methyl, the method comprising the steps of: (i) providing a sample containing the vicinal diketone; (ii) contacting the sample with a hydrazide functional group polymer to covalently attach the vicinal diketone to the polymer; (iii) separating the sample and polymer; (iv) releasing the vicinal diketone from the polymer by changing the pH; and (v) photometric detection of the vicinal diketone, wherein the photometric detection comprises a step of condensing the vicinal diketone and a compound of formula (I) and detecting the condensation product of formula (III) wherein A and B are each N and C and D together with the carbon atom to which they are attached form a benzene ring, wherein the photometric detection takes place at a wavelength of between 500 and 700 nm, and wherein step (ii) takes between 5 seconds and 30 minutes and / or step (iv) takes between 5 seconds and 30 minutes.The method of claim 1, for the detection of a vicinal diketone in wine or beer, wherein the sample is from wine or beer production.The method according to claim 1 or 2, wherein the hydrazide functional polymer is a polyacryloylhydrazide, preferably a cross-linked polyacryloylhydrazide.The method according to any one of claims 1 to 3, wherein the polymer is contacted with the sample as a powder, granules, matrix or porous structure.The method of any one of claims 1 to 4, wherein the polymer is located in a capillary, a filter, a syringe or a pipette tip.