Crystalline graphite substrate and measuring device using crystalline graphite substrate

Abstract

This crystalline graphite substrate is a crystalline graphite substrate having a carbon purity of 95% or greater, in which: a surface roughness Ra thereof is 1 μm or greater and 8 μm or less; and the full width at half maximum (FWHM) of an X-ray diffraction peak corresponding to interlayer spacing of stacked hexagonal network structures in which six-membered rings of carbon atoms are continuous is 3.5 degrees or greater and 9 degrees or less.

Description

Crystalline graphite substrate and measuring device using said crystalline graphite substrate

[0001] The present disclosure relates to a crystalline graphite substrate, and in particular to a measurement device that uses the crystalline graphite substrate in a portion where a measurement target is ionized in laser desorption ionization mass spectrometry, which is widely used to analyze molecules such as proteins, peptides, and organic compounds.

[0002] Mass spectrometry using matrix-assisted laser desorption ionization (MALDI) mass spectrometry is performed by ionizing relatively high molecular weight substances such as proteins (generally with a molecular weight of 10,000 or more) and peptides (generally with a molecular weight of 1,000 or more). However, it is usually difficult to detect molecules with a mass of 500 Da or less. In other words, in this method, when the analyte is ionized by a laser, instead of the analyte receiving the laser energy directly, a matrix pre-mixed with the analyte converts the laser energy into thermal energy, and this thermal energy is used as the ionization energy for the analyte, thereby enabling ionization without destroying the analyte.

[0003] However, the amount of ionization (or ionization efficiency) that can be achieved without destroying the analyte varies depending on factors such as the type of analyte and the type of matrix. Therefore, optimization of the matrix selection, laser irradiation conditions, preparation conditions, etc., is required for each analyte. Furthermore, although the assumption is that the matrix is ​​mixed with the liquid analyte and then dried to achieve uniform concentration, this may not be achieved depending on the drying conditions and the physicochemical properties of the analyte and matrix. Furthermore, the matrix used to mix the analyte generates cluster-like noise in the low molecular weight region, making it difficult to detect low molecular weight compounds using a matrix-based MALDI-MS method.

[0004] In order to solve the above problems, measurement methods without using a matrix have been devised, and various measurement substrates have been proposed. Patent Document 1 shows an example in which a highly oriented graphite sheet is used as a measurement substrate, and the surface has irregularities of 10 nm to 1 μm, thereby suppressing a decrease in detection accuracy. Patent Document 2 shows that efficient ionization can be achieved by forming a graphite thin film on the measurement substrate. Furthermore, Patent Document 3 shows that efficient ionization can be achieved by using a graphene sheet or carbon nanowalls.

[0005] Japanese Patent Publication No. 2014-232055 Japanese Patent No. 5013421 Japanese Patent No. 5406621

[0006] Synthesis and properties of pyrolytic graphite (IEEJ Trans, FM, Vol. 123, No. 11, pp. 1115-1123, 2003)

[0007] A crystalline graphite substrate according to one embodiment of the present disclosure has a carbon purity of 95% or more, a surface roughness Ra of 1 μm or more and 8 μm or less, and an FWHM (Full Width at Half Maximum) of a peak in X-ray diffraction corresponding to a layer spacing of stacked hexagonal network structures in which six-membered rings of carbon atoms are continuous is 3.5 degrees or more and 9 degrees or less.

[0008] A method for producing a crystalline graphite substrate according to one embodiment of the present disclosure includes a heating step of heating a polyimide film to 2500°C or higher in an oxygen-free state to remove hydrogen, oxygen, and nitrogen, and then recrystallizing the remaining carbon atoms to form a crystalline graphite substrate having a layered structure in which a hexagonal network structure in which six-membered rings of carbon atoms are continuously stacked; and a pressurizing step of applying a pressure of 0.5 MPa to 10 MPa to the resulting crystalline graphite substrate.

[0009] 1 is an SEM photograph showing the surface state of a crystalline graphite substrate according to embodiment 1. FIG. 2 is a schematic diagram showing the crystalline structure of the crystalline graphite substrate according to embodiment 1. FIG. 3 is a schematic diagram showing an example of the distribution of a measurement object before (a) and after (b) pressing to homogenize the surface state of the crystalline graphite substrate. FIG. 4 is a plan view (a) and a cross-sectional view (b) showing the configuration of a measurement device in which the crystalline graphite substrate according to embodiment 1 is used as a portion on which a measurement object is placed. FIG. 5 is Table 1 showing evaluation results in Examples and Comparative Examples. FIG. 6 is a graph showing the relationship between apparent density and detection intensity of a crystalline graphite substrate. FIG. 7 is a graph showing the relationship between detection intensity and FWHM of an X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure in which six-membered rings of carbon atoms are stacked in a crystalline graphite substrate. FIG. 8 is a graph showing the relationship between surface roughness Ra of a crystalline graphite substrate and detection intensity.

[0010] An object of the present disclosure is to provide a crystalline graphite substrate that can be used in a measurement device for laser desorption ionization mass spectrometry, which does not require a matrix and can minimize deterioration in detection accuracy.

[0011] A crystalline graphite substrate according to a first aspect is a crystalline graphite substrate having a carbon purity of 95% or more, a surface roughness Ra of 1 μm or more and 8 μm or less, and an FWHM of a peak in X-ray diffraction corresponding to a layer spacing of stacked hexagonal network structures in which six-membered rings of carbon atoms are successively formed, of 3.5 degrees or more and 9 degrees or less.

[0012] The crystalline graphite substrate according to a second aspect may be the crystalline graphite substrate according to the first aspect, having a thickness of 0.05 mm or more and 3 mm or less.

[0013] The crystalline graphite substrate according to a third aspect may be the crystalline graphite substrate according to the first or second aspect, and may have an apparent density of 0.5 or more and 1.5 or less.

[0014] A measuring device according to a fourth aspect is a measuring device for placing a measurement object in laser desorption ionization mass spectrometry, and uses the crystalline graphite substrate according to any one of the first to third aspects as a portion for placing the measurement object to be ionized, and when the thickness is 0.5 mm or less, the measurement object is held in place by applying a tensile force from the periphery.

[0015] A method for producing a crystalline graphite substrate according to the fifth aspect includes a heating step of heating a polyimide film to 2500°C or higher in an oxygen-free state to remove hydrogen, oxygen, and nitrogen, and then recrystallizing the remaining carbon atoms to form a crystalline graphite substrate having a layered structure in which a hexagonal network structure in which six-membered rings of carbon atoms are continuously stacked; and a pressurizing step of applying a pressure of 0.5 MPa to 10 MPa to the crystalline graphite substrate.

[0016] A sixth aspect of the method for producing a crystalline graphite substrate is the same as the fifth aspect, except that in the heating step, the heating rate up to 1300°C is 7°C / min to 13°C / min, the heating rate from 1300°C to 2200°C is 4.9°C / min to 9.1°C / min, and the heating rate above 2200°C is 3.5°C / min to 6.5°C / min.

[0017] A crystalline graphite substrate according to one embodiment of the present disclosure has high thermal conductivity in the basal plane. Due to this characteristic, when used in a measurement device for placing a measurement target in laser desorption / ionization mass spectrometry, the thermal energy generated by laser irradiation is diffused more quickly, resulting in uniform thermal energy at the laser irradiation location. Furthermore, the substrate has a homogeneous surface condition, with a surface roughness within the above-described range and an X-ray diffraction peak FWHM corresponding to the layer spacing of the stacked hexagonal mesh structure within the above-described range. The measurement target is placed in the recessed portion of the surface, allowing efficient propagation of thermal energy. As a result, the ionization reaction of the measurement target is uniform, preventing a decrease in detection accuracy and widening the detection range from low to high molecular weight regions. Furthermore, since no matrix material is required, advantages such as a shortened measurement preparation time and no need for matrix training are obtained.

[0018] Hereinafter, a crystalline graphite substrate according to an embodiment, a method for manufacturing the same, and a measuring device for use in laser desorption ionization mass spectrometry using the crystalline graphite substrate will be described with reference to the accompanying drawings.

[0019] (Embodiment 1) <Crystalline Graphite Substrate> Fig. 1 is an SEM photograph showing the surface of a crystalline graphite substrate according to embodiment 1. Fig. 2 is a schematic diagram showing the crystal structure of the crystalline graphite substrate according to embodiment 1.

[0020] The crystalline graphite substrate according to the first embodiment is a crystalline graphite substrate having a carbon purity of 95% or more, preferably 99% or more. The crystalline graphite substrate has a surface roughness Ra of 1 μm or more and 8 μm or less. The surface roughness Ra may be 3 μm or more and 6 μm or less. The FWHM of a peak in X-ray diffraction corresponding to the layer spacing of stacked hexagonal network structures in which six-membered rings of carbon atoms are continuous is 3.5 degrees or more and 9 degrees or less. It may be 5 degrees or more and 8 degrees or less.

[0021] This crystalline graphite substrate has flatness specified by the surface roughness Ra and FWHM, and therefore, when used in a measurement device on which a measurement object is placed in laser desorption ionization mass spectrometry, it can convert laser energy into thermal energy and efficiently propagate the thermal energy to the measurement object.

[0022] The crystalline graphite substrate may have a thickness of 0.05 mm or more and 3 mm or less, and an apparent density of 0.5 to 1.5 or less, or 0.7 to 1.2 or less.

[0023] As shown in FIG. 1, the crystalline graphite substrate has an uneven surface, specifically, scale-like convex portions and concave portions.

[0024] As shown in FIG. 2 , the crystalline graphite substrate according to the first embodiment has a layered structure of stacked hexagonal mesh structures, each of which is made up of six-membered rings of carbon atoms firmly connected by covalent bonds. In other words, the crystalline graphite substrate has a multilayer structure including a plurality of hexagonal mesh layers stacked at intervals. Each hexagonal mesh layer includes a plurality of six-membered rings bonded by covalent bonds. Each six-membered ring includes six carbon atoms. This crystalline graphite substrate has a carbon purity of 95% or more, preferably 99% or more. As will be described later, this crystalline structure is responsible for the FWHM of 9 degrees or less for the peak in X-ray diffraction corresponding to the spacing between the stacked layers of the hexagonal mesh structures.

[0025] 3A and 3B are schematic diagrams showing an example of the distribution of the measurement object before and after pressing to homogenize the surface of a crystalline graphite substrate. This pressing allows the substrate to have an apparent density of 0.5 to 1.5, a surface roughness Ra of 1 to 8 μm, and a layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms.

[0026] 4A and 4B are a plan view and a cross-sectional view showing the configuration of a measuring device 10 in which a crystalline graphite substrate 13 according to embodiment 1 is used in a portion on which a measurement object 11 is placed. This measuring device 10 uses a crystalline graphite substrate 13 in a portion on which the measurement object 11 is placed, and also uses a metal plate or a resin plate as a holding material 14 for reinforcement and improved handling. The basal plane of the crystalline graphite substrate 13 is set so as to be parallel to a surface 12 of the measuring device 10 on which the measurement object 11 is placed.

[0027] If the thickness of the crystalline graphite substrate is small, the surface may not be able to maintain flatness, so if the thickness is 0.5 mm or less, it is sufficient to apply a tensile force from the periphery to hold the substrate in place, thereby maintaining the flatness of the surface of the crystalline graphite substrate.

[0028] In laser desorption ionization mass spectrometry, for example, about 0.5 μl of a solution containing the measurement object 11 is dropped onto the crystalline graphite substrate 13 of this measurement device 10, and the solution is evaporated and dried, so that the measurement object 11 is fixed on the crystalline graphite substrate 13. The measurement object 11 is ionized by irradiating it with a laser, and the components are determined.

[0029] <Method for Manufacturing Crystalline Graphite Substrate> The crystalline graphite substrate according to the first embodiment can be manufactured by the following method.

[0030] (1) A specific resin material (e.g., polyimide) is heated to 2500°C or higher in an oxygen-free state to remove hydrogen, oxygen, and nitrogen, and then the remaining carbon atoms are recrystallized by heating to form layers of stacked hexagonal carbon atom networks (heating step) (see, for example, Non-Patent Document 1). In this case, the surface condition of the resulting crystalline graphite substrate can be changed by changing the heating rate or maintaining the temperature at a high temperature. Heating to 3000°C or higher is also possible.

[0031] (2) After the heating step, the resulting crystalline graphite substrate is pressed at, for example, 2 MPa to homogenize the surface (pressing step). The pressing pressure may be, for example, 0.5 MPa to 10 MPa.

[0032] In the first embodiment, taking into consideration the different temperatures at which hydrogen, oxygen, and nitrogen desorb in the heating step, the heat treatment may be performed in argon gas at a temperature increase rate of, for example, 10°C / min (7°C / min to 13°C / min) up to 1300°C, a temperature increase rate of, for example, 7°C / min (4.9°C / min to 9.1°C / min) from 1300°C to 2200°C, and a temperature increase rate of, for example, 5°C / min (3.5°C / min to 6.5°C / min) from 2200°C onward. The temperature increase rate is preferably controlled within ±30%. Although two temperatures, 1300°C and 2200°C, are cited as the temperatures at which the temperature increase rate is changed, the temperature increase rate does not need to be strictly controlled at these temperatures. For example, the temperature may be changed within a range of about 100°C within a ±50°C range around these temperatures. Furthermore, the temperature increase rate is not limited to a change involving numerical jumps, and may be changed gradually, for example, continuously. Furthermore, the rate of temperature rise does not need to be kept constant, but may be varied within the above range.

[0033] The following describes how the mass spectrometry measurement substrate functions in the measurement device according to this embodiment. In a laser desorption / ionization mass spectrometer, which is the target of the measurement device according to this embodiment, a measurement object is placed on the measurement device and irradiated with a laser, and the measurement object is ionized by the generated thermal energy. This basic mechanism is widely known as matrix-assisted laser desorption / ionization mass spectrometry or soft laser desorption / ionization mass spectrometry. The generated ions of the measurement object are released and accelerated from the measurement device, travel a predetermined distance between electrodes within the device, and then are detected by a detector. By measuring this time of flight, the mass of the generated ions can be determined, allowing the components to be identified. Note that there are also methods for measuring the mass of ions other than time-of-flight measurement.

[0034] In matrix-assisted laser desorption / ionization mass spectrometry using a matrix material, laser energy is converted into thermal energy in the matrix, raising the temperature of the irradiation point. During this temperature rise, the measurement object sublimes from a solid to a gas, and at the same time, some of the H contained in the matrix material is released. ＋ A method in which ions (= protons) move to the object to be measured, thereby ionizing the object to be measured.

[0035] On the other hand, in the measurement device for use in laser desorption ionization mass spectrometry according to the present disclosure, the laser energy is absorbed by a crystalline graphite substrate on which the measurement object is placed and converted into thermal energy, thereby raising the temperature at the irradiation point and sublimating the measurement object. ＋ In negative ion mode, ions are added to the object to be measured, and H ＋ The ionization of the object to be measured occurs as a result of the desorption of the ions.

[0036] The measurement conditions were as follows, and the comparison was made in negative mode (negative ion mode). However, similar results can also be obtained in positive mode (positive ion mode).

[0037] Measurement target: histidine Measurement target amount: 50 pmol / 0.5 μl-spot Laser desorption ionization mass spectrometer: AutoFlex III (manufactured by Bruker Daltonics) Measurement range: 0-2000 m / z Laser irradiation conditions: Wavelength 355 nm (Nd:YAG), 100 shots / pixel To be used as a measurement device, the detection intensity Intensity / pixel needs to be 2000 or more, preferably 4000 or more. Examples and comparative examples are shown below. Figure 5 is Table 1, which lists the evaluation results for the examples and comparative examples.

[0038] Example 1: A polyimide film was heat-treated in argon gas at a heating rate of 10°C / min up to 1300°C, 7°C / min from 1300°C to 2200°C, and 5°C / min from 2200°C and above, up to 3000°C, and then pressed at 3 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 1.04, a surface roughness of 4.2 μm, and an FWHM of 5.99° for the X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms. This crystalline graphite substrate, measuring 40 mm x 20 mm x 80 μm thick, was used as a measurement device to place a measurement object to be ionized and measured using a matrix-assisted laser desorption / ionization mass spectrometer under the conditions described above. The resulting intensity per pixel was 7130. The obtained detection intensity is relatively high, and it is clear that the device can be used sufficiently as a measurement device.

[0039] Example 2: A polyimide film was heat-treated in argon gas at a heating rate of 10°C / min up to 1300°C, 8°C / min from 1300°C to 2200°C, and 4°C / min from 2200°C and above, up to 3000°C, and then pressed at 1 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 0.75, a surface roughness of 3.7 μm, and an FWHM of 7.64° for the X-ray diffraction peak corresponding to the interlayer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms. This crystalline graphite substrate, measuring 40 mm x 20 mm x 79 μm thick, was used as a measurement device to place the object to be ionized and measured using a matrix-assisted laser desorption / ionization mass spectrometer under the conditions described above. The resulting intensity / pixel was 3600. The obtained detection intensity is relatively high, and it is clear that the device can be used as a measurement device.

[0040] Comparative Example 1: A polyimide film was heat-treated in argon gas at a heating rate of 10°C / min up to 1300°C, 4°C / min from 1300°C to 2200°C, and 8°C / min from 2200°C and above, up to 3000°C, and then pressed at 3 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 0.70, a surface roughness of 5 μm, and an FWHM of 11.5° for the X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms. This crystalline graphite substrate, measuring 40 mm x 20 mm x 100 μm thick, was used as a measurement device for placing a measurement object to be ionized, and was measured using a matrix-assisted laser desorption / ionization mass spectrometer under the conditions described above. The resulting intensity / pixel was 600. The detected intensity obtained was relatively low and insufficient, and it was found that the device could not be used as a measuring device.

[0041] (Comparative Example 2) A polyimide film was heat-treated in argon gas at a heating rate of 10°C / min up to 1300°C, 10°C / min from 1300°C to 2200°C, and 3°C / min from 2200°C and above, up to 3000°C, and then pressed at 3 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 0.46, a surface roughness of 5.2 μm, and an FWHM of 12.8° for the X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms. This crystalline graphite substrate, measuring 40 mm x 20 mm x 182 μm thick, was used as a measurement device to place the object to be ionized and measured using a matrix-assisted laser desorption / ionization mass spectrometer under the conditions described above. The resulting intensity per pixel was 20. The detected intensity obtained was relatively low and insufficient, and it was found that the device could not be used as a measuring device.

[0042] (Comparative Example 3) A polyimide film was heat-treated in argon gas at a heating rate of 6°C / min up to 1300°C, 4°C / min from 1300°C to 2200°C, and 3°C / min from 2200°C and above, up to 3000°C, and then pressed at 3 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 2.13, a surface roughness of 0.8 μm, and an FWHM of 0.84 degrees for the X-ray diffraction peak corresponding to the interlayer spacing of a hexagonal network structure of continuous six-membered carbon rings. This crystalline graphite substrate, measuring 40 mm x 20 mm x 1 mm thick, was measured under the aforementioned conditions using a matrix-assisted laser desorption / ionization mass spectrometer as a measurement device for placing a measurement object to be ionized. The resulting intensity / pixel was 90. The detected intensity obtained was relatively low and insufficient, and it can be seen that the device cannot be used as a measuring device.

[0043] (Comparative Example 4) A polyimide film was heat-treated in argon gas at a heating rate of 14°C / min up to 1300°C, 10°C / min from 1300°C to 2200°C, and 8°C / min from 2200°C and above, up to 3000°C, and then pressed at 10 MPa to obtain a crystalline graphite substrate. This crystalline graphite substrate had an apparent density of 0.53, a surface roughness of 8.47 μm, and an FWHM of 11.1° for the X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms. This crystalline graphite substrate, measuring 40 mm x 20 mm x 154 μm thick, was used as a measurement device to place the object to be ionized and measured using a matrix-assisted laser desorption / ionization mass spectrometer under the conditions described above. The resulting intensity / pixel was 1600. The detected intensity obtained was relatively low and insufficient, and it can be seen that the device cannot be used as a measuring device.

[0044] Fig. 6 is a graph showing the relationship between apparent density and detected intensity of a crystalline graphite substrate. Fig. 7 is a graph showing the relationship between FWHM and detected intensity of an X-ray diffraction peak corresponding to the layer spacing of a hexagonal network structure of continuous six-membered rings of carbon atoms of a crystalline graphite substrate. Fig. 8 is a graph showing the relationship between surface roughness Ra of a crystalline graphite substrate and detected intensity.

[0045] 6 to 8, the evaluation results including Examples 1 and 2 and Comparative Examples 1 to 4 are plotted.

[0046] As shown in FIG. 6, the apparent density of a crystalline graphite substrate having a detection intensity of 2000 or more is 0.5 or more and 1.5 or less.

[0047] As shown in FIG. 7, the FWHM of the X-ray diffraction peak corresponding to the layer spacing of the stacked hexagonal network structure of continuous six-membered rings of carbon atoms of the crystalline graphite substrate, with a detection intensity of 2000 or more, is 3.5 degrees or more and 9 degrees or less.

[0048] As shown in FIG. 8, the surface roughness Ra of the crystalline graphite substrate having a detection intensity of 2000 or more is 1 μm or more and 8 μm or less.

[0049] The crystalline graphite substrate according to the present disclosure can be used in a measurement device on which a measurement target is placed in laser desorption ionization mass spectrometry, thereby eliminating the need for a matrix and minimizing the decrease in detection accuracy.

[0050] 10 Measuring device 11 Measurement object 12 Surface of crystalline graphite substrate 13 Crystalline graphite substrate 14 Holding material

Claims

1. A crystalline graphite substrate having a carbon purity of 95% or more, a surface roughness Ra of 1 μm or more and 8 μm or less, and a FWHM (Full Width at Half Maximum) of a peak in X-ray diffraction corresponding to a layer spacing of a hexagonal network structure in which six-membered rings of carbon atoms are successively stacked, of 3.5 degrees or more and 9 degrees or less.

2. The crystalline graphite substrate according to claim 1, having a thickness of 0.05 mm or more and 3 mm or less.

3. The crystalline graphite substrate according to claim 1, having an apparent density of 0.5 or more and 1.5 or less.

4. A measuring device for placing an object to be measured in laser desorption ionization mass spectrometry, the measuring device using the crystalline graphite substrate according to any one of claims 1 to 3 as a portion for placing the object to be measured to be ionized, and holding the object by applying a tensile force from all sides if the thickness is 0.5 mm or less.

5. A method for producing a crystalline graphite substrate, comprising: a heating step of heating a polyimide film to 2500°C or higher in an oxygen-free state to remove hydrogen, oxygen, and nitrogen, and then recrystallizing the remaining carbon atoms to form a crystalline graphite substrate having a layered structure of a hexagonal network structure in which six-membered rings of carbon atoms are continuously stacked; and a pressurizing step of applying a press pressure of 0.5 MPa to 10 MPa to the obtained crystalline graphite substrate.

6. The method for producing a crystalline graphite substrate as recited in claim 5, wherein the heating step is carried out at a heating rate of 7°C / min to 13°C / min up to 1300°C, at a heating rate of 4.9°C / min to 9.1°C / min from 1300°C to 2200°C, and at a heating rate of 3.5°C / min to 6.5°C / min above 2200°C.

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