JP7804498B2 - Mass spectrometry method for polyisoprene - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies. 1. Japan Society for Analytical Chemistry, Polymer Analysis Research Council, 26th Polymer Analysis Symposium, Abstracts, October 21, 2021, http://www.pack.jp/storage/1b48cc9b30d492c9aaf6ee49b7a847f9.pdf 2. Japan Society for Analytical Chemistry, Polymer Analysis Research Council, 26th Polymer Analysis Symposium (held online), October 29, 2021

This disclosure relates to a mass spectrometry method for polyisoprene that uses a laser desorption/ionization time-of-flight mass spectrometer to measure the molecular weight of polyisoprene contained in a sample.

Natural rubber, whose main component is polyisoprene, has excellent properties such as high tensile strength and low heat generation due to vibration, and is therefore used in a variety of rubber products such as tires, vibration-damping rubber, and belts. Consumption of natural rubber is predicted to increase due to the growing demand for rubber products. Because natural rubber is a natural resource extracted from the Para rubber tree, there is a growing need for synthetic rubber (synthetic polyisoprene) with properties equivalent to natural rubber from the perspectives of nature conservation and sustainable use.

Generally, measuring molecular weight and molecular weight distribution is important for understanding the physical properties of polymeric materials such as synthetic rubber. For example, mass spectrometry (MS) is a method for directly measuring the molecular weight of polymeric materials. Mass spectrometry ionizes atoms or molecules, moves them in a high vacuum using an electric or magnetic field, and then separates and detects them based on the differences in the mass of the ions. Methods for ionizing substances include electron ionization (EI), chemical ionization (CI), electrospray ionization (ESI), and laser desorption ionization (LDI). Methods for separating the generated ions include quadrupole (Q), magnetic ionization (BE), and time-of-flight (TOF). LDI-TOFMS, which combines LDI and TOF, is widely used for measuring polymeric materials. Matrix-assisted laser desorption ionization (MALDI), which ionizes polymeric materials mixed with an organic matrix agent, is particularly useful for measuring the mass of components that are difficult to ionize.

For example, Patent Document 1 describes a method for analyzing polymer compounds in which a sample containing the polymer compound is dispersed in an organic matrix reagent such as dithranol and mass spectral data is obtained using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS). Patent Document 2 describes a method in which a sample such as a protein is co-crystallized with an organic matrix molecule such as sinapinic acid in the presence of porous microparticles such as silica gel to prepare a sample, and then matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is performed using the sample.

JP 2017-138273 A Japanese Patent Application Laid-Open No. 2006-189391

For example, if the sample to be measured is polyisoprene alone, it can be measured using MALDI-TOFMS. However, during the synthesis of polyisoprene, catalysts and other additives are added and various reactions occur, resulting in the resulting product containing many impurities in addition to the target polyisoprene. When such a product is measured using MALDI-TOFMS, the impurities interfere with the measurement, making it impossible to accurately measure the polyisoprene itself. Therefore, the inventors first developed the product using thin-layer chromatography (TLC) to separate polyisoprene from the impurities. They then attempted to directly ionize the polyisoprene separated on the TLC plate and perform mass spectrometry. However, the inventors' investigations revealed that polyisoprene cannot be ionized on the TLC plate, either with the sample alone or using the organic matrix agents commonly used in MALDI-TOFMS.

The aforementioned Patent Documents 1 and 2 do not describe the use of TLC on samples containing impurities. For example, in the analytical method described in Patent Document 1, the desired components are extracted using a solvent before a sample containing a polymer compound is measured using MALDI-TOFMS. In the analytical method described in Patent Document 2, the effects of impurities are suppressed by adding porous microparticles to the sample. Furthermore, Patent Documents 1 and 2 do not consider combining TLC with MALDI-TOFMS or ionizing the substance to be measured on the TLC plate.

The present disclosure has been made in light of these circumstances, and aims to provide a mass spectrometry method for polyisoprene that combines thin layer chromatography and laser desorption/ionization time-of-flight mass spectrometry to measure the molecular weight of polyisoprene contained in a sample.

To solve the above-mentioned problems, the disclosed method for mass spectrometry of polyisoprene comprises a separation step in which polyisoprene contained in a sample is separated by thin layer chromatography; a measurement plate preparation step in which an ionizing agent solution containing a transition metal compound is applied to the polyisoprene separated on a thin layer chromatography plate to prepare a measurement plate; and a measurement step in which the measurement plate is measured using a laser desorption ionization time-of-flight mass spectrometer to obtain a mass spectrum.

According to the disclosed method for mass spectrometry of polyisoprene, the polyisoprene to be measured is first separated from a synthetic polyisoprene sample containing contaminants using thin-layer chromatography (TLC). This eliminates the influence of the contaminants, and the molecular weight of the polyisoprene can be accurately measured using a laser desorption/ionization time-of-flight mass spectrometer (LDI-TOFMS). Polyisoprene does not readily absorb laser light. Therefore, polyisoprene cannot be ionized simply by irradiating it with laser light. After extensive research, the inventors discovered that polyisoprene can be ionized using an ionizing agent solution containing a transition metal compound. The ionization mechanism of polyisoprene is believed to be as follows: When an ionizing agent solution containing a transition metal compound is irradiated with laser light, the ionizing agent solution is heated, generating transition metal nanoparticles. The generated transition metal nanoparticles are desorbed, and cations are added to the polyisoprene, causing the polyisoprene to also be desorbed and ionized.

The polyisoprene mass spectrometry method disclosed herein uses an ionizing agent solution containing a transition metal compound to ionize polyisoprene, but does not exclude the use of conventionally used organic matrix agents. However, the polyisoprene mass spectrometry method disclosed herein can measure the molecular weight of polyisoprene by using an ionizing agent solution containing a transition metal compound, without using an organic matrix agent. Furthermore, the polyisoprene mass spectrometry method disclosed herein can ionize polyisoprene on a thin-layer chromatography plate. This allows the thin-layer chromatography plate used to separate polyisoprene to be used as is, facilitating measurement.

FIG. 1 shows a mass spectrum of Experimental Example 1. FIG. 10 is a diagram showing a mass spectrum of Experimental Example 2.

The following describes an embodiment of the disclosed method for mass spectrometry of polyisoprene. Note that the embodiment is not limited to the following, and various modifications and improvements that can be made by those skilled in the art are possible. The disclosed method for mass spectrometry of polyisoprene includes a separation step, a measurement plate preparation step, and a measurement step.

<Separation process>
This step is a step of separating polyisoprene contained in a sample by thin layer chromatography.

[sample]
The sample may be any liquid containing polyisoprene, such as a solution in which polyisoprene is dissolved in a solvent or a solution in which a product obtained by isoprene polymerization is dissolved in a solvent. Examples of solvents that can be used include tetrahydrofuran (THF), butanol, and toluene. The concentration of polyisoprene in the solution (sample) is preferably 2.5 mg/mL or more. The molecular weight of polyisoprene contained in the sample is preferably, for example, a number average molecular weight of 400 or more and 2500 or less. If the molecular weight is too large, it becomes difficult for the polyisoprene to be desorbed during measurement by LDI-TOFMS.

[Thin Layer Chromatography]
Thin layer chromatography separates components in a sample by attaching and drying a sample to a thin layer chromatography plate and then developing it with a solvent. Thin layer chromatography plates have a carrier that has an affinity for polyisoprene and that adequately adsorbs polyisoprene. Examples of the carrier include silica gel, alumina, and cellulose. Chemically modified silica gel in which an organic moiety is chemically bonded to the silanol group of silica gel may also be used. Examples of the organic moiety include an octadecyl group, an octyl group, and a dimethylsilyl group. As will be described later, as the molecular weight of polyisoprene increases, interactions with the carrier may make it difficult for polyisoprene to be released from the carrier during measurement by LDI-TOFMS. In such cases, it is desirable to change, for example, the type of carrier or the type of organic moiety chemically bonded to the silica gel.

The thin layer chromatography plate may have a support that supports the carrier. Examples of supports include glass plates, plastic sheets, and aluminum sheets. The support may be selected appropriately depending on the type of sample and developing solvent. The sample may be applied to the thin layer chromatography plate in a spot-like or planar manner using a capillary or micropipette. Alternatively, the sample may be applied by contacting one side of the thin layer chromatography plate with the sample. The developing solvent may be selected appropriately taking into account the solubility and separability of polyisoprene. Polar solvents such as water, acetone, dichloromethane, and methanol may be used alone or in combination.

<Measurement plate preparation process>
This step is a step of preparing a measurement plate by applying an ionizing agent solution containing a transition metal compound to polyisoprene separated on a thin layer chromatography plate.

The ionizing agent solution is a reagent used to ionize polyisoprene in the subsequent measurement step. The transition metal compound may be any compound capable of absorbing laser light, converting its energy into thermal energy, generating transition metal nanoparticles, and attaching cations to polyisoprene. Examples include silver compounds. Silver trifluoroacetate is particularly desirable. The solvent may be selected appropriately depending on the type of transition metal compound; for example, THF or methanol may be used. The ionizing agent solution may be prepared so that the concentration of the transition metal compound is between 5 mg/mL and 20 mg/mL. The ionizing agent solution may be applied dropwise to the polyisoprene on the thin-layer chromatography plate using a capillary or micropipette, or the thin-layer chromatography plate may be immersed in the ionizing agent solution.

In this process, it is not excluded to use a conventionally used organic matrix agent in addition to the ionizing agent solution as a reagent for ionizing polyisoprene. However, if an organic matrix agent is used in combination, it may adsorb to the support of the thin-layer chromatography plate, inhibiting the desorption and ionization of polyisoprene. Therefore, when preparing a measurement plate, it is preferable not to use an organic matrix agent.

The higher the molecular weight of polyisoprene, the greater its interaction with the support of the thin-layer chromatography plate. For example, when the support is silica gel having octadecyl groups (C ₁₈ H ₃₇ ), polyisoprene may be adsorbed to the octadecyl groups, making it difficult for the polyisoprene to be desorbed from the support during measurement by LDI-TOFMS in the subsequent step. If the polyisoprene is not sufficiently desorbed, accurate measurement becomes difficult. Therefore, from the perspective of suppressing the interaction with the support and accurately measuring polyisoprene with a large molecular weight, it is desirable to add a surfactant to the ionizing agent solution. Adding a surfactant inhibits the interaction between polyisoprene and the support and can promote desorption of polyisoprene. For example, the effect of adding a surfactant is most pronounced when the number-average molecular weight of the polyisoprene is 1,800 or more.

Examples of surfactants include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The surfactant may be dissolved in a solvent such as THF, methanol, or water and blended in the form of a solution prepared to the desired concentration. A surfactant concentration of 10 mg/mL or higher is preferred.

<Measurement process>
This step involves measuring the prepared measurement plate using a laser desorption/ionization time-of-flight mass spectrometer (LDI-TOFMS) to obtain a mass spectrum. Measurements using LDI-TOFMS can be performed using commonly used equipment and conditions. In LDI-TOFMS, the measurement plate is placed on the sample plate of the instrument and irradiated with a predetermined laser beam, thereby ionizing the polyisoprene and measuring its mass-to-charge ratio. The horizontal axis of the obtained mass spectrum represents m/z (the ratio of the ion mass m to the charge number z), and the vertical axis represents the relative peak intensity (relative abundance). From the obtained mass spectrum, the components with different molecular weights that make up the polyisoprene and their relative abundances are measured.

(1) Experimental Example 1
A solution containing polyisoprene (PI) with a number average molecular weight (Mn) of 800 was used as a sample, and measurement was carried out by TLC-LDI-TOFMS.

<Separation process>
Polyisoprene contained in the sample was separated by thin-layer chromatography. The sample used was a polyisoprene solution ("PSS-pio800" manufactured by PPS (Polymer Standard Services), polyisoprene concentration 2.5 mg/mL) in which polyisoprene (Mn = 800) was dissolved in THF. The thin-layer chromatography plate used was a product of Merck Ltd., "Silica Gel 60 RP-18 F254S Aluminum Sheet," which has an aluminum sheet support and an octadecyl-modified silica gel carrier, cut into a rectangular shape measuring 70 mm long and 25 mm wide. The developing solvent used was a mixed solvent of dichloromethane and methanol in a volume ratio of 7:3.

First, 5 μL of sample was dropped using a micropipette onto one of the short sides of the thin layer chromatography plate, depositing it in a dot shape 10 mm from the short side, and then drying. Next, the developing solvent was poured into the development tank, the lid was closed, and the plate was left to stand for 5 minutes. Next, the lid of the development tank was opened, and the thin layer chromatography plate was placed in with the short side to which the sample had been deposited facing downwards, and left to stand until the developing solvent had migrated to the specified height. After development was complete, the thin layer chromatography plate was removed and dried.

<Measurement plate preparation process>
A measurement plate was prepared by dropping 5 μL of the ionizing agent solution onto the polyisoprene on the thin-layer chromatography plate. The ionizing agent solution used was a silver trifluoroacetate (AgTFA) solution dissolved in THF. The AgTFA concentration in the AgTFA solution was 10 mg/mL.

<Measurement process>
The prepared measurement plate was attached to an ITO (indium tin oxide) glass substrate and measured using an LDI-TOFMS in positive mode using a spiral TOFMS device (JEOL Ltd., "JMS-S3000", Nd:YLF laser).

<Measurement results>
Figure 1 shows the mass spectrum obtained by measurement at a position 4.7 cm after thin-layer chromatography development. In Figure 1, the horizontal axis represents m/z, and the vertical axis represents relative peak intensity. As shown in Figure 1, peaks of polyisoprene with a degree of polymerization n of 6 to 11 were observed as peaks of ionized polyisoprene [PI+Ag ⁺ ] at m/z values of 560 to 920. This demonstrates that by using an ionizing agent solution containing AgTFA, polyisoprene can be ionized on a thin-layer chromatography plate and subjected to mass analysis.

(2) Experimental Example 2
A solution containing polyisoprene having a number average molecular weight (Mn) of 1820 was used as a sample, and measurement was carried out on a thin layer chromatography plate by LDI-TOFMS.

In Experiment 2, a measurement plate was prepared by dropping a sample onto a thin-layer chromatography plate and then dropping an ionizing agent solution onto it without spreading. The sample used was a polyisoprene solution (PSS-pio2.1k, manufactured by PPS, polyisoprene concentration 10 mg/mL) in which polyisoprene (Mn = 1820) was dissolved in THF. First, 5 μL of the sample was dropped onto the same thin-layer chromatography plate as in Experiment 1, 10 mm from one of the short edges, using a micropipette. The plate was then dried. Next, 5 μL of the ionizing agent solution was dropped onto the polyisoprene on the thin-layer chromatography plate to prepare the measurement plate. The ionizing agent solution was a mixture of the same AgTFA solution (AgTFA concentration 10 mg/mL) as in Experiment 1 and a CHAPS solution (CHAPS concentration 40 mg/mL) in which the surfactant CHAPS was dissolved in THF.

The prepared measurement plate was attached to an ITO glass substrate and measured using an LDI-TOFMS. Measurements were performed in positive mode using the same spiral TOFMS device as in Experimental Example 1.

<Measurement results>
The obtained mass spectrum is shown in Figure 2. In Figure 2, the horizontal axis represents m/z, and the vertical axis represents relative peak intensity. As shown in Figure 2, peaks of polyisoprene with a degree of polymerization n of 13 to 27 were observed as peaks of ionized polyisoprene [PI+Ag ⁺ ] at m/z values of 1000 to 2000. Thus, it was confirmed that by using an ionizing agent solution containing AgTFA and CHAPS, high-molecular-weight polyisoprene with a number-average molecular weight of 1800 or more can be ionized on a thin-layer chromatography plate and subjected to mass analysis.

The mass spectrometry method for polyisoprene disclosed herein allows accurate measurement of the molecular weight of polyisoprene even when the sample contains impurities. This method is useful, for example, for analyzing artificially synthesized polyisoprene and polystyrene decomposition products.

Claims (6)

a separation step of separating polyisoprene contained in the sample by thin layer chromatography;
a measurement plate preparation step of applying an ionizing agent solution containing a transition metal compound and a surfactant to the polyisoprene separated on the thin layer chromatography plate to prepare a measurement plate;
a measuring step of measuring the measurement plate using a laser desorption ionization time-of-flight mass spectrometer to obtain a mass spectrum;
A method for mass spectrometry of polyisoprene, comprising: The method for mass spectrometry of polyisoprene described in claim 1, wherein no organic matrix agent is used when preparing the measurement plate. The mass spectrometry method for polyisoprene according to claim 1 or claim 2, wherein the transition metal compound includes a silver compound. The method for mass spectrometry of polyisoprene described in claim 3, wherein the silver compound comprises silver trifluoroacetate. The thin layer chromatography plate has a support and a carrier,
5. The method for mass spectrometry of polyisoprene according to claim 1, wherein the carrier comprises silica gel modified with an octadecyl group. 6. The method for mass spectrometry of polyisoprene according to claim 1 , wherein the surfactant comprises 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS).