Method for analyzing degradation of marine biological degradable plastics

Abstract

The invention provides an analysis method, which can obtain information of a degradation mechanism of marine biological degradable plastic. This analysis method is a method for analyzing the degradation of a marine biodegradable plastic, and is provided with: an extraction step in which a degradation intermediate, which adheres to the marine biodegradable plastic and / or a biofilm, is eluted into a solvent and extracted, and the biofilm is formed on the surface of the marine biodegradable plastic; and a detection step of detecting the degradation intermediate product by using the solution from which the degradation intermediate product has been extracted to perform mass spectrometry.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the degradation of marine biodegradable plastics. Background Art

[0002] Due to the problem of marine plastic waste, demand for marine biodegradable plastics, which are degraded by microorganisms in the ocean, has increased, and product development has progressed rapidly. Specifically, a biodegradable polyester composed of a polymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid has been proposed and commercially available (see Patent Document 1).

[0003] For such marine biodegradable plastics, the biodegradation mechanism, which is essential for developing products tailored to their intended uses, has yet to be elucidated. Conventional methods have evaluated the biodegradation of marine biodegradable plastics by quantifying the amount of gas consumed as degradation progresses (ISO 23977-2) and by comparing the weight of the plastic before and after biodegradation (ISO 22766).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2007-259708 Summary of the Invention

[0007] Technical problem to be solved by the invention

[0008] However, conventional methods are indirect and therefore cannot understand what degradation intermediates are generated during degradation and how they are distributed. Understanding such information on degradation intermediates is crucial for elucidating the degradation mechanism, and therefore the development of new analytical methods is required.

[0009] The object of the present invention is to provide an analytical method capable of obtaining information on the degradation mechanism of marine biodegradable plastics.

[0010] Solutions for solving the above technical problems

[0011] The analytical method of the first embodiment of the present invention is a method for analyzing the degradation of marine biodegradable plastics, comprising: an extraction step of extracting degradation intermediates attached to the marine biodegradable plastics and / or biofilms formed on the surface of the marine biodegradable plastics by dissolving them into a solvent; and a detection step of detecting the degradation intermediates by mass spectrometry using a solution containing the extracted degradation intermediates.

[0012] Effects of the Invention

[0013] According to the analysis method of the first embodiment, the presence or absence of degradation intermediates generated during the degradation of marine biodegradable plastics and their degree of polymerization can be ascertained, which can contribute to the elucidation of the degradation mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The experimental flow chart in Example 1 is shown. DETAILED DESCRIPTION

[0015] 1. First Implementation

[0016] The first embodiment of the present invention is an analysis method for analyzing the degradation of marine biodegradable plastics, specifically, the degradation intermediates thereof, and comprises a preparation step, an extraction step, and a detection step in sequence. Each step is described in detail below.

[0017] (Preparatory Steps)

[0018] In this step, a marine biodegradable plastic having a biofilm attached to its surface is prepared as an analysis sample.

[0019] Marine biodegradable plastics can be polymer compounds that degrade in seawater, for example, polyhydroxyalkanoates, etc. As monomers constituting polyhydroxyalkanoates, for example, 3-hydroxybutyric acid, 3-hydroxyisovaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxyhexadecanoic acid can be mentioned. In the first embodiment, as a representative example of an analysis sample of marine biodegradable plastics, a polymer compound having a structure represented by the following chemical formula (1), i.e., a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, can be mentioned. In the formula, x represents an integer greater than 1, and y represents an integer greater than 1.

[0020] [Chemistry 1]

[0021]

[0022] Marine biodegradable plastics with biofilms formed on their surfaces (hereinafter referred to as biofilm-attached plastics) are obtained by exposing them to seawater for a period of time or longer. Specifically, these include marine biodegradable plastics that have been floating or settled in the sea for a certain period of time, or marine biodegradable plastics that have been in contact with natural or artificial seawater for a certain period of time (i.e., marine biodegradable plastics used in biodegradation trials). Marine biodegradable plastics are typically degraded by microorganisms in seawater, forming a biofilm composed of these microorganisms on their surfaces. When this biofilm-attached plastic is removed from seawater, it becomes a structure consisting of the marine biodegradable plastic, the biofilm formed on its surface, and the interstitial water contained between them.

[0023] (Extraction Step)

[0024] In this step, a biofilm formed on the surface of at least one of (1) a marine biodegradable plastic in the process of degradation and (2) the marine biodegradable plastic is brought into contact with a solvent. Thus, degradation intermediates attached to the marine biodegradable plastic and / or the biofilm are extracted into the solvent.

[0025] Specifically, first, as a pre-treatment, in order to remove excess water such as interstitial water, the biofilm-attached plastic is subjected to a removal treatment such as centrifugal separation.

[0026] By centrifugation, the product is separated into a residue and a separated liquid. The residue contains a large amount of biofilm and marine biodegradable plastic, so the residue is used. In addition, a trace amount of biofilm also remains in the separated liquid. Therefore, in order to achieve more accurate quantification, the separated liquid can be filtered and the residue remaining on the surface of the filter paper can be added to the residue from the above centrifugation.

[0027] Next, the residue is subjected to solvent extraction. That is, the residue containing the marine biodegradable plastic and / or biofilm is brought into contact with the solvent, for example by immersing the residue in the solvent. In this case, ultrasonic extraction is preferably performed to reliably dissolve degradation intermediates adhering to the surface of the marine biodegradable plastic or biofilm into the solvent.

[0028] Examples of the solvent include organic solvents, of which water-soluble solvents such as methanol, ethanol, and 1-propanol are preferred because they have good affinity with the surface of the water-containing biofilm and can elute more degradation intermediates into the solvent.

[0029] After the solvent extraction, unnecessary substances in the solvent may be removed by filtration or the like, if necessary.

[0030] Thus, a solution containing the extracted degradation intermediates, ie, a biofilm extract, is obtained.

[0031] Furthermore, when liquid chromatography-mass spectrometry is performed in the detection step described later, in order to reliably perform component separation by liquid chromatography, in the extraction step, it is preferred that the solvent be transferred to the solvent used for liquid chromatography after solvent extraction. In the first embodiment, this transferred liquid (transfer solution) is also included in the "solution in which the degradation intermediates are extracted."

[0032] After the solution containing the degradation intermediates extracted by the above-mentioned solvent extraction is dried and solidified, the dried solid is redissolved in another solvent to perform resolubilization.

[0033] Examples of other solvents for dissolution include solvents used in mobile phases of liquid chromatography, which are appropriately determined according to the liquid chromatograph. Specifically, examples include acidic aqueous solutions such as formic acid, acetic acid, and phosphoric acid; and organic solvents such as methanol, ethanol, acetonitrile, and hexane.

[0034] The degradation intermediates extracted in this step are compounds eluted by degradation of the marine biodegradable plastic, and are monomers and / or oligomers constituting the marine biodegradable plastic, particularly both monomers and oligomers constituting the marine biodegradable plastic.

[0035] For example, when the marine biodegradable plastic is a polymer compound having a structure represented by chemical formula (1), the monomers are 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. The oligomer is an oligomer composed of at least one of these monomers, and is represented by the following chemical formula (2). In the formula, x represents, for example, an integer from 1 to 15, and y represents, for example, an integer from 1 to 15. The total degree of polymerization of the oligomer, that is, the total value of x and y, also depends on the type of mass spectrometer, but when a liquid chromatography tandem mass spectrometer is used as the mass spectrometer, it is, for example, 20 or less, preferably 12 or less, and for example, 1 or more.

[0036] [Chemistry 2]

[0037]

[0038] (Detection steps)

[0039] In this step, mass spectrometry is performed using the solution obtained in the extraction step, that is, the solution in which the degradation intermediates are extracted, thereby detecting the degradation intermediates of the marine biodegradable plastic.

[0040] Preferably, liquid chromatography-mass spectrometry is performed as the mass spectrometry method. That is, the solution from which the degradation intermediates have been extracted is subjected to component separation by liquid chromatography, and then subjected to mass spectrometry.

[0041] In liquid chromatography, any column capable of retaining the monomers and oligomers constituting the marine biodegradable plastic can be appropriately selected from known or existing columns. For example, a column filled with silica gel containing octadecyl groups can be used as the stationary phase. The mobile phase can be appropriately selected depending on the column and the marine biodegradable plastic, and for example, the aforementioned solvents can be used, with a mixed solvent of an acidic aqueous solution and an organic solvent being preferred.

[0042] In the first embodiment, a gradient elution method is preferably used. That is, the concentration of the organic solvent in the mobile phase is gradually increased from the beginning of the analysis. This allows efficient separation of multiple degradation intermediates with a wide molecular weight distribution, shortening the analysis completion time.

[0043] In mass spectrometry, examples of ionization methods include electron ionization (EI), electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI).

[0044] As the type of mass separation, i.e. the kind of mass spectrometer, for example, quadrupole mass spectrometer, magnetic field sector type mass spectrometer, time-of-flight mass spectrometer, ion trap type (including orbital trap type) mass spectrometer, ion cyclotron resonance type mass spectrometer etc. can be enumerated. In addition, it can also be the tandem mass spectrometer consisting of a plurality of mass spectrometers. That is, in the first embodiment, liquid chromatography tandem mass spectrometer (LC / MS / MS) can be adopted. As the kind of tandem mass spectrometer (MS / MS), for example, triple quadrupole (QQ) mass spectrometer, tandem time-of-flight type (TOF-TOF) mass spectrometer, quadrupole-time-of-flight type (Q-TOF) mass spectrometer, quadrupole-ion trap type (Q-IT) mass spectrometer, quadrupole-ion cyclotron resonance type (Q-ICR) mass spectrometer, ion trap-time-of-flight type (IT-TOF) mass spectrometer etc. can be enumerated. From the perspective of being able to perform precise mass measurement and easily distinguish and determine the chemical formulas of similar degradation intermediates, it is preferred to use an ion trap mass spectrometer, a time-of-flight mass spectrometer, or an ion cyclotron resonance mass spectrometer. From the perspective of easily performing accurate quantification of each degradation intermediate, it is preferred to use a quadrupole mass spectrometer, particularly a triple quadrupole mass spectrometer.

[0045] The liquid chromatography mass spectrometry apparatus used for the liquid chromatography mass spectrometry method can be a conventional or commercially available apparatus. The various setting conditions for the liquid chromatography and mass spectrometry method can be appropriately set according to the recommendations of the respective apparatuses with reference to the composition of monomers and oligomers in the degradation intermediates.

[0046] Thus, the degradation intermediate product is detected. That is, mass spectrum is obtained by each degradation intermediate product obtained by having carried out component separation by liquid chromatography. In mass spectrum, m / z and the peak intensity of the ion (precursor ion, fragment ion or product ion) of the monomer and / or oligomer constituting the degradation intermediate product are output. And, in these each mass spectrum, based on m / z and the peak intensity of various ions, each monomer and each oligomer constituting the degradation intermediate product can be determined.

[0047] In this step, the polymerization degree of each oligomer, i.e., the intermediate degradation products, can be further determined. Specifically, the m / z values ​​of these oligomers are calculated and correlated based on the structure and molecular weight of the intermediate degradation products, i.e., the oligomers, and the structure and molecular weight of the monomers that constitute the oligomers. In the mass spectrum obtained in the detection step, peaks derived from ions of the monomers and / or oligomers that constitute the intermediate degradation products are detected and compared with the corresponding m / z values ​​of these peaks to determine the polymerization degree of each oligomer.

[0048] The analytical method of the first embodiment enables analysis of degradation intermediates of marine biodegradable plastics. Previously, it was assumed that when marine biodegradable plastics degrade in seawater, their degradation products were immediately assimilated or inorganicized, or that they were not present at detectable concentrations due to dilution by seawater. In contrast, the present inventors focused on biofilms attached to marine biodegradable plastics. They hypothesized that within biofilms, where biodegradation is ongoing, degradation intermediates might be present at measurable concentrations. By measuring the biofilm surface using a mass spectrometer, they confirmed the presence of large amounts of degradation intermediates, thereby completing the invention of the first embodiment.

[0049] In particular, the analysis method of the first embodiment can determine the presence and degree of polymerization of degradation intermediates generated during the degradation of marine biodegradable plastics, contributing to the elucidation of degradation mechanisms. Furthermore, by performing this analysis method in parallel with conventional biodegradation evaluation methods, the relationship between biodegradation and degradation intermediates can be examined, potentially leading to the proposal of a new biodegradation evaluation method.

[0050] 2. Plan

[0051] It should be understood by those skilled in the art that the above exemplary embodiments are specific examples of the following schemes.

[0052] (Item 1) The analytical method involved in one scheme is a method for analyzing the degradation of marine biodegradable plastics, which may include: an extraction step of dissolving degradation intermediates attached to the marine biodegradable plastics and / or biofilms into a solvent for extraction, wherein the biofilms are formed on the surface of the marine biodegradable plastics; and a detection step of detecting the degradation intermediates by performing mass spectrometry using a solution from which the degradation intermediates have been extracted.

[0053] (Item 2) In the analysis method described in Item 1, in the extraction step, the biofilm may be brought into contact with the solvent after removing interstitial water present in the biofilm.

[0054] (Item 3) In the analysis method described in Item 1 or Item 2, the solvent may be an organic solvent.

[0055] (Item 4) In the analysis method described in any one of Items 1 to 3, the degradation intermediate product may be a monomer and / or oligomer constituting a marine biodegradable plastic.

[0056] (Item 5) In the analysis method of Item 4, the degradation intermediates may be monomers and oligomers constituting marine biodegradable plastics.

[0057] (Item 6) In the analysis method described in Item 4 or 5, the oligomer may be an oligomer having a total degree of polymerization of up to 20.

[0058] (Item 7) In the analysis method described in Item 6, the oligomer may be an oligomer having a total degree of polymerization of up to 12.

[0059] (Item 8) In the analysis method described in any one of Items 1 to 7, in the detection step, the solution from which the degradation intermediate product is extracted may be subjected to component separation by liquid chromatography and then to mass spectrometry.

[0060] (Item 9) In the analytical method described in Item 8, liquid chromatography can be performed by gradient elution.

[0061] Example

[0062] Next, the present invention will be described in detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited thereto.

[0063] <Example 1>

[0064] In the seawater collected from Osaka Bay, ammonium chloride was added in an amount of 19.1 mg / L and sodium hydrogen phosphate was added in an amount of 2.3 mg / L to prepare the test seawater. 300 mL was injected into each of 15 500 mL glass test bottles. As marine biodegradable plastic films, 15 pieces of Green Planet (registered trademark) (100 μm thick, 30 mg) represented by the following chemical formula were prepared, and one piece of film was immersed in one of the test bottles. The water temperature was maintained at 27°C, and the test seawater was stirred while being placed for a specified period (5 days, 8 days, 9 days, 10 days, and 13 days). During this period, the formation of a biofilm on the surface of the plastic film was visually confirmed.

[0065] [Chemistry 3]

[0066]

[0067] After each prescribed period, three biofilm-forming plastic films were removed and centrifuged at 1000g for 20 minutes to separate the residue and a separating liquid. The separating liquid was centrifuged at 2000g for 20 minutes using a PTFE filter (trade name "Ultrafree-CL," pore size 0.45 μm) to collect the residue attached to the filter. The two residues were combined and placed in 8 mL of 1-propanol and subjected to solvent extraction by irradiating with ultrasound for 10 minutes. Thus, the components attached to the surface of the marine biodegradable plastic film and the biofilm were dissolved and diffused into the solvent. Next, after removing the marine biodegradable plastic film and biofilm, the resulting solvent was filtered using a PTFE filter (trade name "GL Chromatograph 13P," pore size 0.45 μm) to collect the filtrate. The collected filtrate was dried and solidified at 30°C under a nitrogen stream. The dried solid was dissolved (transformed) in a 0.1% formic acid aqueous solution, and the solution was filtered using a PTFE filter (trade name "GL Chromatodisc 13A", pore size 0.2 μm) to obtain a filtrate, i.e., a biofilm extract (refer to Figure 1 ).

[0068] The obtained biofilm extract was analyzed using a liquid chromatograph / orbitrap mass spectrometer under the measurement conditions shown in Table 1. Specifically, the extract was separated into components by liquid chromatography, and the separated extract was analyzed using an orbitrap mass spectrometer.

[0069] [Table 1]

[0070]

[0071] The qualitative and relative quantitative results obtained are shown in Table 2 below. At this time, in the calculation of the relative quantification of each monomer and each oligomer, the peaks of each standard substance (concentration: 200 ng / mL each) relative to 3-hydroxybutyric acid and 3-hydroxyhexanoic acid were used as the benchmark. In addition, for the monoisotopic precision masses of 1 and 2 valences calculated based on the molecular formula of the degradation intermediate (oligomers with a total polymerization degree of 20 composed of 2 monomers of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid), the presence or absence of peak detection of the extracted ion chromatogram was confirmed under the condition of mass error ± 5 ppm. The numbers in Table 2 represent the amount of substance (ng) in the biofilm extract. In the compound column, the number after B represents the degree of polymerization of 3-hydroxybutyric acid, and the number after H represents the degree of polymerization of 3-hydroxyhexanoic acid. For example, B1 H2 represents an oligomer formed by the polymerization of 1 3-hydroxybutyric acid and 2 3-hydroxyhexanoic acids.

[0072] [Table 2]

[0073]

[0074] <Comparative Example 1>

[0075] In Example 1, the supernatant seawater from the test bottles containing the marine biodegradable plastic film and the test seawater was collected after a predetermined period (5 days, 8 days, 9 days, 10 days, and 13 days), filtered using a PTFE filter, and the filtrate was used as the extract of Comparative Example 1. The extract of Comparative Example 1 was analyzed using a liquid chromatography / orbitrap mass spectrometer under the same conditions as in Example 1, but no degradation intermediates of the marine biodegradable plastic were detected.

[0076] As clearly shown in Table 2, the monomeric and oligomeric components that make up the marine biodegradable plastic were detected in analyzable amounts in the biofilm extract. This confirms that intermediate degradation products, eluted from the degradation of the marine biodegradable plastic film, remain on the biofilm surface and on the surface of the marine biodegradable plastic and can be detected. This demonstrates that the analysis method of the first embodiment can provide information on the degradation components and their amounts in the marine biodegradable plastic.

Claims

1. A method for analyzing the degradation of marine biodegradable plastics, characterized in that: have: an extraction step of dissolving degradation intermediates attached to the marine biodegradable plastic and / or biofilm into a solvent for extraction, wherein the biofilm is formed on the surface of the marine biodegradable plastic; The detection step comprises performing mass spectrometry using a solution obtained by extracting the degradation intermediate product to detect the degradation intermediate product.

2. The analysis method according to claim 1, wherein In the extraction step, the biofilm is contacted with the solvent after removing interstitial water present in the biofilm.

3. The analysis method according to claim 1, wherein The solvent is an organic solvent.

4. The analysis method according to claim 1, wherein The degradation intermediates are monomers and / or oligomers constituting marine biodegradable plastics.

5. The analysis method according to claim 4, wherein The degradation intermediate products are monomers and oligomers constituting marine biodegradable plastics.

6. The analysis method according to claim 4 or 5, characterized in that The oligomer has a total polymerization degree of up to 20.

7. The analysis method according to claim 6, wherein The oligomer has a total polymerization degree of up to 12.

8. The analysis method according to claim 1, wherein In the detection step, the solution from which the degradation intermediate product is extracted is subjected to component separation by liquid chromatography and then subjected to mass spectrometry.

9. The analysis method according to claim 8, wherein Liquid chromatography was performed by gradient elution.

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