Analytical methods

Abstract

Provided is an analysis method for performing a first analysis step and a second analysis step in parallel, in which the first step is a step for introducing a non-derivatized first sample 132 into a first column 44 together with a first mobile phase 54 and analyzing a component contained in the first sample 132, and the second step is a step for introducing a derivatized second sample 137 into a second column 84 together with a second mobile phase 94 and analyzing a component contained in the second sample 137. In the analysis method, inosinic acid and guanylic acid are contained in the first sample 132, and the component contained in the first sample 132 is separated in the first column 44 that has a length of 185 mm or longer to obtain a chromatogram in which peaks respectively corresponding to inosinic acid and guanylic acid are separated at a resolution of 1.5 or more in the first analysis step.

Description

[Technical field]

[0001] The present invention relates to an analytical method. [Background technology]

[0002] For example, the liquid chromatograph disclosed in Patent Document 1 below is configured to be capable of simultaneously performing analytical processing using a plurality of columns. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 183663 Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have considered using a liquid chromatograph as described above to quickly analyze both the freshness and spoilage of foods such as meat (including fish). The freshness of food can be measured by analyzing ATP-related substances including inosinic acid. Meanwhile, the spoilage of food can be measured by analyzing histamine. When analyzing histamine and specific amino acids including glutamic acid (hereinafter referred to as "histamine-amino acids"), they are derivatized and then detected by a detector.

[0005] When analyzing both ATP-related substances and histamine and amino acids, it is necessary to use different analytical systems for the ATP-related substances that are not derivatized and the histamine and amino acids that are derivatized. In this case, it is possible to prepare an analytical device for analyzing ATP-related substances and another for analyzing histamine and amino acids, but this would cause problems such as space for installing the devices. Therefore, it is possible to use a liquid chromatograph as described above and analyze ATP-related substances in one of the two columns and histamine and amino acids in the other column.

[0006] In the course of such investigations, the inventors of the present application have attempted to simultaneously analyze guanylic acid, which is an example of an umami component along with inosinic acid and glutamic acid contained in foods, along with ATP-related substances and histamine / amino acids. In this case, it is possible to perform the analysis using three columns by setting up a separate column for analyzing guanylic acid, but it is more efficient to use the same column for the analysis of ATP-related substances that are not derivatized.

[0007] However, since the retention time of inosinic acid contained in ATP-related substances is close to that of guanylic acid, it was found to be difficult to obtain a chromatogram in which the peaks corresponding to inosinic acid and guanylic acid were separated.

[0008] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide an analytical method capable of separating peaks corresponding to inosinic acid and guanylic acid contained in a first sample when analyzing components contained in a first underivatized sample and a second derivatized sample in parallel. [Means for solving the problem]

[0009] A first aspect of the present invention is an analytical method for performing in parallel a first analysis step, in which an underivatized first sample is introduced into the first column together with a first mobile phase and components contained in the first sample are analyzed, and a second analysis step, in which a derivatized second sample is introduced into the second column together with a second mobile phase and components contained in the second sample are analyzed, using a first column and a second column each composed of an ODS column, wherein the first sample contains inosinic acid and guanylic acid, and in the first analysis step, the components contained in the first sample are separated by the first column having a length of 185 mm or more, thereby obtaining a chromatogram in which peaks corresponding to inosinic acid and guanylic acid are separated with a resolution of 1.5 or more. Effect of the Invention

[0010] According to the present invention, when the components contained in a first non-derivatized sample and a second derivatized sample are analyzed in parallel, the peaks corresponding to inosinic acid and guanylic acid contained in the first sample can be separated. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a block diagram showing an example of the configuration of a liquid chromatograph according to the present embodiment. [Diagram 2] FIG. 2 is a schematic diagram showing an example of the configuration of an autosampler according to the present embodiment. [Diagram 3] FIG. 2 is a block diagram showing an example of the electrical configuration of the liquid chromatograph of the present embodiment. [Figure 4] FIG. 11 is a schematic diagram for explaining a dispensing step of the present embodiment. [Diagram 5] FIG. 11 is a schematic diagram for explaining a dispensing step of the present embodiment. [Figure 6] FIG. 11 is a schematic diagram for explaining a dispensing step of the present embodiment. [Figure 7] FIG. 11 is a schematic diagram for explaining a dispensing step of the present embodiment. [Figure 8] FIG. 2 is a schematic diagram for explaining a first analysis step and a second analysis step of the present embodiment. [Figure 9] FIG. 1 is a diagram showing a first chromatogram in a comparative example. [Figure 10] FIG. 10 is an enlarged view of a portion of the first chromatogram in FIG. [Figure 11] FIG. 2 is a diagram showing a first chromatogram in an example. [Figure 12] FIG. 13 is a diagram showing another first chromatogram in an example. [Figure 13] FIG. 13 is a diagram showing an example of a UV spectrum in an embodiment corresponding to FIGS. 11 and 12. [Figure 14] FIG. 2 is a block diagram specifically showing the electrical configuration of the liquid chromatograph of the present embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Liquid chromatograph configuration 1 is a block diagram showing an example of the configuration of a liquid chromatograph 10 of this embodiment. As shown in FIG. 1, the liquid chromatograph 10 includes a first analysis system 20 and a second analysis system 60.

[0013] The first analytical system 20 includes a container 22, a flow path 24, a first pump 26, a flow path 28, a container 30, a flow path 32, a second pump 34, a flow path 36, a first mixer 38, a flow path 40, a first switching valve 102, a flow path 42, a first column 44, a flow path 46, and a first detector 48, etc.

[0014] The second analytical system 60 includes a container 62, a flow path pipe 64, a third pump 66, a flow path pipe 68, a container 70, a flow path pipe 72, a fourth pump 74, a flow path pipe 76, a second mixer 78, a flow path pipe 80, a second switching valve 110, a flow path pipe 82, a second column 84, a flow path pipe 86, and a second detector 88, etc.

[0015] In the first analytical system 20, the upstream end of the flow path pipe 24 is inserted into the container 22, and the downstream end is connected to a first pump 26. The upstream end of the flow path pipe 28 is connected to the first pump 26, and the downstream end is connected to a first mixer 38.

[0016] The upstream end of the flow path pipe 32 is inserted into the container 30, and the downstream end is connected to the second pump 34. The upstream end of the flow path pipe 36 is connected to the second pump 34, and the downstream end is connected to the first mixer 38.

[0017] The upstream end of the flow path pipe 40 is connected to the first mixer 38, and the downstream end is connected to the first switching valve 102. The upstream end of the flow path pipe 42 is connected to the first switching valve 102, and the downstream end is connected to the first column 44. The upstream end of the flow path pipe 46 is connected to the first column 44, and the downstream end is connected to the first detector 48.

[0018] Furthermore, the container 22 contains a first liquid 50, and the container 30 contains a second liquid 52. Specifically, the first liquid 50 and the second liquid 52 are solvents that constitute a mobile phase, and the second liquid 52 has a higher content of an organic solvent than the first liquid 50.

[0019] In the second analytical system 60, the upstream end of the flow path pipe 64 is inserted into the container 62, and the downstream end is connected to a third pump 66. The upstream end of the flow path pipe 68 is connected to the third pump 66, and the downstream end is connected to a second mixer 78.

[0020] The upstream end of the flow path pipe 72 is inserted into the container 70, and the downstream end is connected to a fourth pump 74. The upstream end of the flow path pipe 76 is connected to the fourth pump 74, and the downstream end is connected to a second mixer 78.

[0021] The upstream end of the flow path pipe 80 is connected to the second mixer 78, and the downstream end is connected to the second switching valve 110. The upstream end of the flow path pipe 82 is connected to the second switching valve 110, and the downstream end is connected to the second column 84. The upstream end of the flow path pipe 86 is connected to the second column 84, and the downstream end is connected to the second detector 88.

[0022] Furthermore, the container 62 contains a third liquid 90, and the container 70 contains a fourth liquid 92. The third liquid 90 and the fourth liquid 92 are specifically solvents that constitute the mobile phase, and the fourth liquid 92 has a higher content of organic solvent than the third liquid 90.

[0023] The first switching valve 102 and the second switching valve 110 constitute a part of the autosampler 100. The autosampler 100 is shared by the first analytical system 20 and the second analytical system 60.

[0024] In the first analytical system 20, for example, when the first pump 26 and the second pump 34 are driven, the first liquid 50 and the second liquid 52 are mixed in the first mixer 38 to generate a first mobile phase 54, and the first mobile phase 54 is discharged from the first mixer 38 to the flow path tube 40. That is, in this case, the first mobile phase 54 contains the first liquid 50 and the second liquid 52.

[0025] Also, for example, when only the first pump 26 is driven, the second liquid 52 is not introduced into the first mixer 38, and the first mobile phase 54 containing only the first liquid 50 is discharged from the first mixer 38 to the flow path tube 40.

[0026] Furthermore, when only the second pump 34 is driven, the first liquid 50 is not introduced into the first mixer 38, and the first mobile phase 54 containing only the second liquid 52 is discharged from the first mixer 38 to the flow path pipe 40.

[0027] Therefore, in the first analytical system 20, by appropriately driving the first pump 26 and the second pump 34 and adjusting the flow rates of the first liquid 50 and the second liquid 52, it is possible to adjust the mixing ratio of the first liquid 50 and the second liquid 52 in the first mobile phase 54 and perform gradient analysis.

[0028] A sample is injected into the first mobile phase 54 using the first switching valve 102 of the autosampler 100. Furthermore, the first mobile phase 54 into which the sample has been injected passes through the first column 44 and is then introduced into the first detector 48, whereby a chromatogram including peaks corresponding to each component in the sample separated in the process of passing through the first column 44 is obtained.

[0029] In the second analysis system 60, similarly to the first analysis system 20, the mixing ratio of the third liquid 90 and the fourth liquid 92 can be adjusted to perform gradient analysis by appropriately driving the third pump 66 and the fourth pump 74. The second mobile phase 94, in which the mixing ratio of the third liquid 90 and the fourth liquid 92 has been adjusted, is led out from the second mixer 78 to the flow path pipe 80.

[0030] In addition, a sample is injected into the second mobile phase 94 using the second switching valve 110 of the autosampler 100. Furthermore, the second mobile phase 94 into which the sample has been injected is introduced into the second detector 88 after passing through the second column 84, thereby obtaining a chromatogram including peaks corresponding to each component in the sample separated in the process of passing through the second column 84.

[0031] 2. Autosampler Configuration Fig. 2 is a schematic diagram showing an example of the configuration of the autosampler 100 of this embodiment. As shown in Fig. 2, in addition to the above-mentioned first switching valve 102 and second switching valve 110, the autosampler 100 includes a first sample loop 104, a first injection port 106, an opening / closing valve 108, a second sample loop 112, a second injection port 114, a third switching valve 116, a washing port 118, a metering pump 120, a needle loop 122, and a needle 124.

[0032] The first switching valve 102 is a six-way valve having six ports A1 to A6. Port A1 is connected to a first injection port 106. Port A2 is connected to an open / close valve 108. Port A3 is connected to a port A6 via a first sample loop 104. Port A4 is connected to a flow path tube 40. Port A5 is connected to a flow path tube 42.

[0033] Furthermore, the rotor seal (not shown) of the first switching valve 102 has three flow paths that connect adjacent ports to each other. The first switching valve 102 switches between a first state and a second state by the rotation of the rotor seal. The combinations of ports that are connected to each other differ between the first state and the second state.

[0034] When the first switching valve 102 is in the first state, as shown in FIG. 2, the port A1 and the port A2 are connected, the port A3 and the port A4 are connected, and the port A5 and the port A6 are connected.

[0035] That is, when the first switching valve 102 is in the first state, the flow path tube 40 is connected to the flow path tube 42 via ports A4, A3, the first sample loop 104, A6, A5, etc. Also, the first injection port 106 is connected to the opening and closing valve 108 via ports A1, A2, etc.

[0036] On the other hand, when the first switching valve 102 is in the second state, the port A2 and the port A3 are connected, the port A4 and the port A5 are connected, and the port A6 and the port A1 are connected.

[0037] That is, when the first switching valve 102 is in the second state (see FIG. 5), the flow path pipe 40 is connected to the flow path pipe 42 via ports A4 and A5, etc. Also, the first injection port 106 is connected to the opening / closing valve 108 via ports A1, A6, the first sample loop 104, A3, A2, etc.

[0038] The second switching valve 110 is a six-way valve having six ports B1 to B6. Port B1 is connected to port B4 via a second sample loop 112. Port B2 is connected to the flow path 80. Port B3 is connected to the flow path 82. Port B5 is connected to a second injection port 114. Port B6 is connected to a drainage flow path.

[0039] Furthermore, the rotor seal (not shown) of the second switching valve 110 has three flow paths connecting adjacent ports to each other, and similar to the first switching valve 102, the rotor seal rotates to switch between the first state and the second state.

[0040] When the second switching valve 110 is in the first state, as shown in FIG. 2, the ports B1 and B2 are connected, the ports B3 and B4 are connected, and the ports B5 and B6 are connected.

[0041] That is, when the second switching valve 110 is in the first state, the flow path 80 is connected to the flow path 82 via the port B2, the port B1, the second sample loop 112, the port B4, the port B3, etc. Also, the second injection port 114 is connected to the drainage flow path via the port B5, the port B6, etc.

[0042] On the other hand, when the second switching valve 110 is in the second state (see FIG. 7), the ports B2 and B3 are connected, the ports B4 and B5 are connected, and the ports B6 and B1 are connected.

[0043] That is, when the second switching valve 110 is in the second state, the flow path pipe 80 is connected to the flow path pipe 82 via ports B2 and B3, etc. In addition, the second injection port 114 is connected to the drainage flow path via ports B5, B4, the second sample loop 112, B1, B6, etc.

[0044] The third switching valve 116 is a rotary valve having five ports C1 to C5. The port C1 is connected to a cleaning port 118. The port C2 is disposed outside the autosampler 100 and is connected to a container 126 that contains a cleaning liquid 128. The port C3 is connected to the suction side of the metering pump 120. The port C4 is connected to the outlet side of the metering pump 120. The port C5 is connected to a needle 124 via a needle loop 122.

[0045] In addition, the rotor seal (not shown) of the third switching valve 116 has a flow path that connects any one of the ports C1, C2, C3, and C4 to the port C5, and a flow path that connects adjacent ports to each other.

[0046] In the third switching valve 116, the rotor seal can rotate to switch between a first state, a second state, a third state, a fourth state, a fifth state, or a sixth state, and the first to sixth states each have a different combination of ports that are connected to each other.

[0047] When the third switching valve 116 is in the first state, the port C3 and the port C5 are connected as shown in Fig. 2. That is, when the third switching valve 116 is in the first state, the suction side of the metering pump 120 is connected to the needle 124 via the port C3, the port C5, the needle loop 122, etc.

[0048] When the third switching valve 116 is in the second state, the port C4 and the port C1 are connected to each other. That is, when the third switching valve 116 is in the second state, the blowing side of the metering pump 120 is connected to the cleaning port 118 via the port C4, the port C1, etc.

[0049] When the third switching valve 116 is in the third state, the port C2 and the port C3 are connected to each other. That is, when the third switching valve 116 is in the third state, the suction side of the metering pump 120 is connected to the container 126 via the port C3, the port C2, etc.

[0050] When the third switching valve 116 is in the fourth state (see FIG. 5), the port C4 and the port C5 are connected. That is, when the third switching valve 116 is in the fourth state, the blowing side of the metering pump 120 is connected to the needle 124 via the port C4, the port C5, the needle loop 122, etc.

[0051] When the third switching valve 116 is in the fifth state, the port C1 and the port C5 are connected to each other. That is, when the third switching valve 116 is in the fifth state, the washing port 118 is connected to the needle 124 via the port C1, the port C5, the needle loop 122, etc.

[0052] When the third switching valve 116 is in the sixth state, the port C2 and the port C5 are connected to each other. That is, when the third switching valve 116 is in the sixth state, the container 126 is connected to the needle 124 via the port C2, the port C5, the needle loop 122, etc.

[0053] Although not shown in the drawings, the autosampler 100 is provided with an electrically controllable movement mechanism that movably supports the needle 124. The first switching valve 102, the opening / closing valve 108, the second switching valve 110, the third switching valve 116, the metering pump 120, etc. are electrically controllable.

[0054] 3. Electrical configuration of liquid chromatograph Fig. 3 is a block diagram showing an example of the electrical configuration of the liquid chromatograph 10 of this embodiment. As shown in Fig. 3, the liquid chromatograph 10 includes a control unit 200, an operation control circuit 210, a display control circuit 212, a display unit 214, an operation detection circuit 216, an operation unit 218, and the like, in addition to the first pump 26, the second pump 34, the first detector 48, the third pump 66, the fourth pump 74, the second detector 88, the autosampler 100, and the like.

[0055] Furthermore, the first detector 48, the second detector 88, the control unit 200, the operation control circuit 210, the display control circuit 212, and the operation detection circuit 216 are electrically connected to each other via wiring 208 such as a bus.

[0056] Furthermore, the operation control circuit 210 is electrically connected to the first pump 26, the second pump 34, the third pump 66, the fourth pump 74, and the autosampler 100. Furthermore, the display control circuit 212 is electrically connected to a display unit 214, and the operation detection circuit 216 is electrically connected to an operation unit 218.

[0057] The control unit 200 is responsible for overall control of the liquid chromatograph 10. The control unit 200 includes a CPU (Central Processing Unit) 202. The control unit 200 also includes a RAM (Random Access Memory) 204 and a storage unit 206 that the CPU 202 can directly access.

[0058] The CPU 202 controls each component of the liquid chromatograph 10. The RAM 204 is used as a work area and a buffer area for the CPU 202. The storage unit 206 is a non-volatile memory, and for example, a hard disk drive (HDD) or a solid state drive (SSD) is used as the storage unit 206.

[0059] The storage unit 206 stores a control program for controlling each component of the liquid chromatograph 10, execution data required for executing the control program, and the like. The storage unit 206 may be configured to include the RAM 204.

[0060] The operation control circuit 210 generates voltages for driving the first pump 26, the second pump 34, the third pump 66, the fourth pump 74, the autosampler 100, etc., and applies the voltages to these as appropriate.

[0061] The display control circuit 212 includes a GPU (Graphics Processing Unit) and a VRAM (Video Random Access Memory), and under the direction of the CPU 202, the GPU generates display images in the VRAM for displaying various screens on the display unit 214 and outputs the generated display images to the display unit 214.

[0062] The display unit 214 is a general-purpose display device such as an LCD (Liquid Crystal Display) or an EL (Electro-Luminescence) display.

[0063] The operation detection circuit 216 outputs a signal or data corresponding to an operation on the operation unit 218 to the control unit 200 , specifically, to the CPU 202 .

[0064] The operation unit 218 includes hardware keys (operation keys). Examples of the operation keys include a power key. The operation unit 218 also includes an input device. Examples of the input device include a keyboard and a mouse.

[0065] Furthermore, the input device may include a touch panel. In this case, the touch panel is provided on the display surface of the display unit 214. The touch panel and the display unit 214 may be formed integrally.

[0066] 4. Operation of Liquid Chromatograph The dispensing step can be performed at any timing in the liquid chromatograph 10. For example, when a predetermined operation on the operation unit 218 is detected, the dispensing step is performed.

[0067] The dispensing step is a step of aspirating and discharging a sample. In the present embodiment, during the dispensing step, the first mobile phase 54 is introduced into the first column 44 and the second mobile phase 94 is introduced into the second column 84.

[0068] 4 to 7 are schematic diagrams for explaining the dispensing process of this embodiment. As shown in Fig. 4 to 7, a container 130 for containing a first sample 132 and a container 134 for containing a second sample 136 are set in advance in the autosampler 100.

[0069] In this embodiment, the second sample 136 is derivatized using the derivatization reagent 133 contained in the container 131. Specifically, the derivatization reagent 133 is injected from the container 131 into an empty container 135, and the second sample 136 is injected from the container 134, so that the derivatized second sample 137 is contained in the container 135. One example of the derivatization is fluorescent derivatization. The type of the derivatization reagent 133 is not particularly limited, and may be a mixture of multiple types (for example, two types) of derivatization reagents, or may be one type of derivatization reagent. On the other hand, the first sample 132 is not derivatized.

[0070] After the derivatization of the second sample 136 is started as described above, a first suction operation is performed to suction the first sample 132. When the first suction operation is performed, the needle 124 is inserted into the container 130, as shown in Fig. 4. When the first suction operation is performed, the first switching valve 102, the second switching valve 110, and the third switching valve 116 are set to the first state.

[0071] In the first suction operation, the metering pump 120 is controlled to introduce the first sample 132 into the needle loop 122 via the needle 124. The first sample 132 introduced into the needle loop 122 is stored in the needle loop 122.

[0072] After the first suction operation is performed, a first discharge operation is then performed to discharge the first sample 132. When the first discharge operation is performed, the needle 124 is inserted into the first injection port 106, as shown in Fig. 5. When the first discharge operation is performed, the first switching valve 102 is switched from the first state to the second state, and the third switching valve 116 is switched from the first state to the fourth state. Furthermore, when the first discharge operation is performed, the opening / closing valve 108 is opened.

[0073] In the first dispensing operation, the metering pump 120 is controlled to introduce the first sample 132 in the needle loop 122 into the first sample loop 104 via the first injection port 106. Furthermore, when the first dispensing operation is performed, the open / close valve 108 is closed so that the first sample 132 in the first sample loop 104 does not flow out. As a result, the first sample 132 introduced into the first sample loop 104 is stored in the first sample loop 104.

[0074] After the first discharge operation is performed, a cleaning operation is then performed to clean the needle 124. Although not shown in the drawings, when the cleaning operation starts, the third switching valve 116 switches from the fourth state to the third state, whereby the suction side of the metering pump 120 is connected to the container 126 via ports C3 and C2, etc., and the cleaning liquid 128 in the container 126 is sucked in.

[0075] In addition, when the cleaning liquid 128 in the container 126 is sucked up, the third switching valve 116 switches from the third state to the second state, and the blowing side of the metering pump 120 is connected to the cleaning port 118 via ports C4 and C1, etc., and the cleaning liquid 128 is introduced into the cleaning port 118.

[0076] Furthermore, when the cleaning liquid 128 is introduced into the cleaning port 118, the needle 124 is inserted into the cleaning port 118, and the needle 124 is washed. In this embodiment, after the derivatization reagent 133 and the second sample 136 are injected into the empty container 135, the first suction operation, the first discharge operation, and the cleaning operation are performed as described above, so that time can be utilized to efficiently derivatize the second sample 136.

[0077] After the washing operation is performed in this manner, a second suction operation is then performed to suction the derivatized second sample 137. When the second suction operation is performed, the needle 124 is inserted into the container 135, as shown in Fig. 6. When the second suction operation is performed, the third switching valve 116 is switched from the second state to the first state.

[0078] In the second suction operation, the metering pump 120 is controlled to introduce the derivatized second sample 137 into the needle loop 122 via the needle 124. The derivatized second sample 137 introduced into the needle loop 122 is stored in the needle loop 122.

[0079] After the second suction operation is performed, a second discharge operation is then performed to discharge the derivatized second sample 137. When the second discharge operation is performed, the needle 124 is inserted into the second injection port 114, as shown in Fig. 7. When the second discharge operation is performed, the second switching valve 110 is switched from the first state to the second state, and the third switching valve 116 is switched from the first state to the fourth state.

[0080] In the second discharge operation, the metering pump 120 is controlled, and the derivatized second sample 137 in the needle loop 122 is introduced into the second sample loop 112 via the second injection port 114. At this time, the drainage flow path is connected to the blowing side of the metering pump 120 via ports B6 and C4, and the port C3 connected to the suction side of the metering pump 120 is sealed. As a result, the derivatized second sample 137 introduced into the second sample loop 112 is stored in the second sample loop 112.

[0081] In this embodiment, after the dispensing step is performed in this manner, the first analysis step and the second analysis step are performed in parallel. The first analysis step is a step of introducing the first sample 132 together with the first mobile phase 54 into the first column 44 and analyzing the components contained in the first sample 132. The second analysis step is a step of introducing the derivatized second sample 137 together with the second mobile phase 94 into the second column 84 and analyzing the components contained in the second sample 137. Note that the term "parallel" is not limited to the case where the first analysis step and the second analysis step are started simultaneously, but is a concept that includes the case where at least a part of the first analysis step and the second analysis step are performed simultaneously. Therefore, at least one of the start timing and the end timing of the first analysis step and the second analysis step does not have to be simultaneous.

[0082] 8 is a schematic diagram for explaining the first and second analysis steps of this embodiment. When the first and second analysis steps are performed, the first switching valve 102 and the second switching valve 110 are switched from the second state to the first state as shown in FIG.

[0083] When the first switching valve 102 is switched from the second state to the first state, the first sample 132 in the first sample loop 104 is introduced into the first mobile phase 54 introduced from the flow path tube 40. Therefore, the first sample 132 is introduced into the first column 44 together with the first mobile phase 54.

[0084] Furthermore, when the second switching valve 110 is switched from the second state to the first state, the derivatized second sample 137 in the second sample loop 112 is introduced into the second mobile phase 94 introduced from the flow path tube 80. Therefore, the derivatized second sample 137 is introduced into the second column 84 together with the second mobile phase 94.

[0085] When the first sample 132 passes through the first column 44, each component in the first sample 132 is separated, and each separated component is detected by the first detector 48. In addition, when each component is detected by the first detector 48, a first chromatogram is obtained based on the detection result.

[0086] On the other hand, when the derivatized second sample 137 passes through the second column 84, each component in the second sample 137 is separated, and each separated component is detected by the second detector 88. In addition, when each component is detected by the second detector 88, a second chromatogram is obtained based on the detection result.

[0087] The introduction of the first mobile phase 54 into the first column 44 and the introduction of the second mobile phase 94 into the second column 84 are continued until both the first analysis step and the second analysis step are completed. The degree of resolution of each component in the first chromatogram varies depending on the shape and type of the first column 44, and the degree of resolution of each component in the second chromatogram varies depending on the shape and type of the second column 84.

[0088] Furthermore, the configuration of the autosampler 100 is not particularly limited as long as it can perform the first and second analysis steps in parallel as described above. For example, the autosampler 100 may be configured so that the first sample 132 is introduced into the first column 44 according to a total volume injection method, and the derivatized second sample 137 is introduced into the second column 84 according to a loop injection method.

[0089] 4. Working Example The following describes an embodiment in which the first sample 132 and the second sample 136 are analyzed using the above-described liquid chromatograph 10. In this embodiment, the analysis conditions were examined in a case where both the freshness and spoilage of food such as meat (including fish) are analyzed using a single liquid chromatograph 10, and umami components contained in the food are also analyzed using the liquid chromatograph 10 at the same time.

[0090] The freshness of food can be measured by analyzing ATP-related substances. ATP-related substances include nucleic acid-related substances such as hypoxanthine (Hx), inosinic acid (IMP), inosine (HxR), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). The first sample 132 contains at least inosinic acid (IMP) among the six ATP-related substances as described above. In addition to the ATP-related substances, the first sample 132 also contains guanylic acid (GMP), which is an example of an umami component contained in food. Note that the first sample 132 is pretreated.

[0091] Food spoilage can be measured by analyzing histamine (Him). Other analytes may include specific amino acids known as nutritional components (including umami components). Specifically, specific amino acids include aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), histidine (His), threonine (Thr), β-alanine (β-Ala), arginine (Arg), alanine (Ala), taurine (Tau), anserine (Ans), carnosine (Car), tyrosine (Tyr), valine (Val), methionine (Met), cystine (Cys), tryptophan (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), etc. The second sample 136 contains histamine (Him) and at least one of 24 specific amino acid components. The second sample 136 is pretreated. The pretreatment applied to the second sample 136 may be the same as the pretreatment applied to the first sample 132.

[0092] ODS (Octal Decyl Silyl) columns were used as the first column 44 and the second column 84. The ODS column is a column packed with a packing material in which octadecylsilyl groups are chemically bonded to a silica gel carrier. The first column 44 has an inner diameter of 3.0 mm, and the particle diameter of the particles to be separated is 3 μm. On the other hand, the second column 84 has an inner diameter of 3.0 mm, and the particle diameter of the particles to be separated is 2.7 μm.

[0093] The flow rate of the first mobile phase 54 in the first column 44 was 0.8 mL / min, and the first column 44 was used in a state heated to 30° C. Furthermore, 5 μL of the first sample 132 was injected into the first mobile phase 54. As the first detector 48, a photodiode array detector with a measurement wavelength of 260 nm was used.

[0094] The flow rate of the second mobile phase 94 in the second column 84 was 0.8 mL / min, which was the same as the flow rate of the first mobile phase 54 in the first column 44. The second column 84 was used in a state heated to 35° C. Furthermore, 1 μL of the derivatized second sample 137 was injected into the second mobile phase 94. A fluorescence detector was used as the second detector 88. More specifically, a multichannel detector having a channel for detecting fluorescence at 450 nm with an excitation wavelength of 350 nm and a channel for detecting fluorescence at 305 nm with an excitation wavelength of 266 nm was used as the fluorescence detector.

[0095] The length of the second column 84 is 100 mm. When the first column 44 was also 100 mm long like the second column 84, the first chromatogram obtained contained peaks that could not be separated with sufficient resolution. Therefore, the case where the first column 44 with a length of 100 mm was used will be described below as a comparative example.

[0096] Fig. 9 is a diagram showing a first chromatogram in a comparative example, and Fig. 10 is a diagram showing an enlarged portion of the first chromatogram in Fig. 9.

[0097] In FIG. 9, multiple peaks appear corresponding to ATP-related substances and the like contained in the first sample 132. Among these multiple peaks, the highest peak that appears near the retention time of 2.3 min is enlarged and shown in FIG. 10. This peak can be divided into a peak corresponding to inosinic acid (IMP) and a peak corresponding to guanylic acid (GMP), as additionally shown in FIG. 10. That is, since the retention times of inosinic acid and guanylic acid are close in the first chromatogram, the peak corresponding to inosinic acid and the peak corresponding to guanylic acid cannot be sufficiently separated, and appear as a single peak.

[0098] 9 and 10, the resolution between the peaks corresponding to inosinic acid and guanylic acid can be calculated using the following formula (1): In formula (1), R is the resolution, t1 is the retention time of guanylic acid, t2 is the retention time of inosinic acid, W1 is the peak width of guanylic acid, and W2 is the peak width of inosinic acid.

number

[0099] Using the above formula (1), the degree of resolution between the peak corresponding to inosinic acid and the peak corresponding to guanylic acid was calculated to be 1.1. In other words, when a first column 44 having a length of 100 mm is used, the peak corresponding to inosinic acid and the peak corresponding to guanylic acid are hardly separated. In addition, since the detection wavelengths of inosinic acid and guanylic acid are the same, they cannot be separated by wavelength.

[0100] Therefore, in this embodiment, the peak corresponding to inosinic acid and the peak corresponding to guanylic acid are sufficiently separated from each other by using a first column 44 having a length equal to or greater than a predetermined threshold value, i.e., the degree of separation between the peak corresponding to inosinic acid and the peak corresponding to guanylic acid is set to 1.5 or more.

[0101] The following describes the above-mentioned predetermined threshold value related to the length of the first column 44. When the widths of adjacent peaks are equal, the resolution is expressed by the following formula (2): In formula (2), R is the resolution, N is the number of theoretical plates, α is the separation factor, and k is the retention factor.

[0102]

number

[0103] According to the above formula (2), the resolution is proportional to the square root of the number of theoretical plates. Therefore, if the resolution is to be improved from R to R1, that is, multiplied by R1 / R, the number of theoretical plates is increased by (R1 2 ) / (R 2 ) needs to be multiplied.

[0104] In addition, since the length of the column is proportional to the number of theoretical plates, the length L of the column corresponding to the resolution R is (R1 2 ) / (R 2 ) to calculate the length L1 of the column required to achieve the resolution R1. Therefore, the length L1 of the column is expressed by the following formula (3).

[0105]

number

[0106] As in the above-mentioned Comparative Example, when the length of the first column 44 was 100 mm, the resolution between the peak corresponding to inosinic acid and the peak corresponding to guanylic acid in the first chromatogram obtained using the first column 44 was 1.1. Moreover, the resolution required to sufficiently separate the peak corresponding to inosinic acid and the peak corresponding to guanylic acid is 1.5 or more.

[0107] Therefore, in the above formula (3), if L is 100, R is 1.1, and R1 is 1.5, the length L1 of the first column 44 required to obtain a resolution of 1.5 is calculated to be 185 mm. Therefore, in the embodiment, the length of the first column 44 is 185 mm or more.

[0108] For example, when the length of the first column 44 is 250 mm, first chromatograms as shown in Figs. 11 and 12 are obtained. Fig. 11 is a diagram showing a first chromatogram in an example. Fig. 12 is a diagram showing another first chromatogram in an example. The first chromatogram shown in Fig. 11 corresponds to a case where a standard solution is mixed and separated in the first column 44, and the first chromatogram shown in Fig. 12 corresponds to a case where a sample extracted from tuna is separated in the first column 44. Note that the standard solution here is a solution in which the same components as those contained in the first sample 132 are mixed in equal amounts (50 µmol / L each).

[0109] As shown in Fig. 11, in the first chromatogram obtained when the standard solution is mixed and separated in the first column 44, the peak corresponding to inosinic acid (IMP) and the peak corresponding to guanylic acid (GMP) are completely separated. In the first chromatogram using the actual sample shown in Fig. 12, the peak corresponding to inosinic acid (IMP) and the peak corresponding to guanylic acid (GMP) are also completely separated, although the peak heights are different. In the first chromatograms shown in Figs. 11 and 12, the degree of resolution between the peak corresponding to inosinic acid and the peak corresponding to guanylic acid was calculated to be 1.9 using the above formula (1).

[0110] FIG. 13 shows the basis for the fact that the peak with a retention time of about 5.7 min in the first chromatogram shown in FIG. 12 corresponds to guanylic acid. FIG. 13 shows an example of a UV spectrum in the examples of FIG. 11 and FIG. 12. In FIG. 13, spectrum A of the peak corresponding to guanylic acid (GMP) with a retention time of about 5.7 min in the example of FIG. 11 and spectrum B of the peak with a retention time of about 5.7 min in the example of FIG. 12 are shown on the same graph. As shown in FIG. 13, spectrum A and spectrum B are almost the same. That is, it can be seen that the peak with a retention time of about 5.7 min in the first chromatogram shown in FIG. 11 corresponds to guanylic acid.

[0111] In this manner, by separating the components contained in the first sample 132 using the first column 44 having a length of 185 mm or more, a first chromatogram is obtained in which the peak corresponding to inosinic acid (IMP) and the peak corresponding to guanylic acid (GMP) are separated with a resolution of 1.5 or more.

[0112] In the above embodiment, the gradient step is performed during the first analysis step by controlling the driving of the first pump 26 and the second pump 34. That is, the first analysis step includes a gradient step. In the gradient step during the first analysis step, the mixing ratio of the first liquid 50 and the second liquid 52 is changed to gradually increase the ratio of the organic solvent in the first mobile phase 54.

[0113] The first liquid 50 is a mixture of water and acetonitrile in a volume ratio of 100:1, and the second liquid 52 is a mixture of water and acetonitrile in a volume ratio of 80:20. Both the first liquid 50 and the second liquid 52 contain 0.15 mol / L phosphoric acid and 0.225 mol / L triethylamine.

[0114] In the gradient step in the first analysis step, the ratio of the first liquid 50 in the first mobile phase 54 is gradually decreased and the ratio of the second liquid 52 is gradually increased. Specifically, only the first liquid 50 is introduced as the first mobile phase 54 until 5 minutes have elapsed since the start of the first analysis step, and thereafter the ratio of the second liquid 52 in the first mobile phase 54 is gradually increased. As a result, the ratio of the second liquid 52 in the first mobile phase 54 becomes 24% when 19 minutes have elapsed since the start of the first analysis step. Since the retention time is long in the first column 44 having a length of 185 mm or more, the content ratio of the organic solvent in the second liquid 52 is increased as described above, and the ratio of the second liquid 52 is increased at a high gradient, so that the first mobile phase 54 can be introduced into the first column 44 at high pressure.

[0115] Also, during the second analysis step performed in parallel with the first analysis step, the gradient step is performed by controlling the driving of the third pump 66 and the fourth pump 74. That is, the second analysis step includes a gradient step. In the gradient step, the mixing ratio of the third liquid 90 and the fourth liquid 92 is changed to gradually increase the ratio of the organic solvent in the second mobile phase 94.

[0116] The third liquid 90 is a phosphate buffer solution of pH 6.5 containing 20 mmol / L phosphoric acid, and the fourth liquid 92 is a mixture of acetonitrile, methanol, and water in a volume ratio of 45:40:15.

[0117] In the gradient step in the second analysis step, the ratio of the third liquid 90 in the second mobile phase 94 is gradually decreased, and the ratio of the fourth liquid 92 is gradually increased. Specifically, the ratio of the fourth liquid 92 in the second mobile phase 94 is gradually increased from 5% to 13% until 8 minutes have elapsed since the start of the second analysis step. Thereafter, the ratio of the fourth liquid 92 in the second mobile phase 94 is gradually increased from 13% to 25% until 15 minutes have elapsed since the start of the second analysis step. Thereafter, the ratio of the fourth liquid 92 in the second mobile phase 94 is further gradually increased from 25% to 52% until 21.5 minutes have elapsed since the start of the second analysis step.

[0118] The gradient process can shorten the time required to complete elution of each component without deteriorating the resolution of the peaks of components with short retention times among the components contained in the first sample 132 or the derivatized second sample 137. In other words, it is possible to prevent the time required to complete elution of each component contained in the first sample 132 or the derivatized second sample 137 from becoming longer as the length of the first column 44 or the second column 84 increases.

[0119] Furthermore, the first and second analysis steps each include a washing step, which is carried out after the gradient step.

[0120] The washing step in the first analysis step is a step in which only the second liquid 52 is introduced into the first column 44 as the first mobile phase 54 for a period from 19.10 min to 25.00 min from the start of the first analysis step, to wash the inside of the first column 44. Thereafter, only the first liquid 50 is introduced into the first column 44 as the first mobile phase 54 for a period from 25.10 min to 32.00 min from the start of the first analysis step.

[0121] The washing step in the second analysis step is a step in which only the fourth liquid 92 is introduced into the second column 84 as the second mobile phase 94 for a period from 21.51 min to 27.50 min from the start of the second analysis step, to wash the inside of the second column 84. Thereafter, only the third liquid 90 is introduced into the second column 84 as the second mobile phase 94 for a period from 27.51 min to 32.00 min from the start of the second analysis step.

[0122] According to the cleaning process, impurity components are not accumulated in the first column 44 or the second column 84, and therefore it is possible to prevent the load on the first column 44 or the second column 84 from increasing. In other words, it is possible to prevent the first column 44 or the second column 84 from deteriorating.

[0123] In addition, in such a liquid chromatograph 10, as described above, the introduction of the first mobile phase 54 into the first column 44 and the introduction of the second mobile phase 94 into the second column 84 are continued until both the first analysis step and the second analysis step are completed, so that even if each component contained in the first sample 132 has already been detected by the first detector 48, the first mobile phase 54 is continuously introduced into the first column 44 until each component contained in the derivatized second sample 137 is detected by the second detector 88. In the first analysis step, each component contained in the first sample 132 is detected by the first detector 48 during the period from the start of the first analysis step until 19 minutes have elapsed.

[0124] In this way, the analysis of each component contained in the first sample 132 is completed before the analysis of each component contained in the derivatized second sample 137 is completed. Therefore, by performing the cleaning step during the first analysis step for a relatively long period of time before both the first analysis step and the second analysis step are completed, the first column 44 having a length of 185 mm or more can be sufficiently cleaned.

[0125] 5. Specific Electrical Configuration of Liquid Chromatograph 14 is a block diagram specifically showing the electrical configuration of the liquid chromatograph 10 of this embodiment. Note that in FIG. 14, the storage unit 206 is omitted from the illustration.

[0126] The RAM 204 stores execution data that has been read out in advance from the storage unit 206. When data acquired using devices, sensors, and the like is stored in the storage unit 206, the acquired data is temporarily stored in the RAM 204.

[0127] In the example shown in FIG. 14, the RAM 204 stores control data 220, first chromatogram data 222, second chromatogram data 224, display image data 226, and operation data 228.

[0128] The control data 220 is various data necessary to control the operation of the liquid chromatograph 10. The first chromatogram data 222 is data corresponding to the first chromatogram. The second chromatogram data 224 is data corresponding to the second chromatogram. The display image data 226 is data corresponding to a display image. The operation data 228 is data representing the operation state of the operation unit 218.

[0129] Although not shown in the figure, the RAM 204 stores a control program previously read from the storage unit 206, and when the CPU 202 executes the control program, the control unit 200 functions as a dispensing processing unit 230, a first analysis processing unit 232, a second analysis processing unit 238, a first acquisition processing unit 240, a second acquisition processing unit 242, a display processing unit 244, and an operation detection processing unit 246. The first analysis processing unit 232 also includes a gradient processing unit 234 and a cleaning processing unit 236. Although not shown in the figure, the second analysis processing unit 238 also includes a gradient processing unit and a cleaning processing unit.

[0130] The dispensing processing unit 230 performs a dispensing step by controlling the autosampler 100 using the control data 220. The first analysis processing unit 232 performs a first analysis step by controlling the first pump 26, the second pump 34, and the autosampler 100 using the control data 220.

[0131] The gradient processing unit 234 performs a gradient process by controlling the first pump 26 and the second pump 34 using the control data 220. After the gradient process is performed, the cleaning processing unit 236 performs a cleaning process by controlling the second pump 34 using the control data 220.

[0132] The second analytical processing unit 238 controls the third pump 66, the fourth pump 74, and the autosampler 100 to perform the second analytical step.

[0133] The first acquisition processing unit 240 acquires first chromatogram data 222 based on the signal from the first detector 48, and stores the data in the RAM 204. The second acquisition processing unit 242 acquires second chromatogram data 224 based on the signal from the second detector 88, and stores the data in the RAM 204.

[0134] The display processing unit 244 outputs the display image data 226 to the display unit 214. The operation detection processing unit 246 detects operations on the operation unit 218, and stores the operations in the RAM 204 as operation data 228 in chronological order.

[0135] 6. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0136] (Item 1) An analytical method according to one embodiment comprises: An analytical method for performing, in parallel, a first analysis step of introducing a non-derivatized first sample into the first column together with a first mobile phase and analyzing components contained in the first sample, and a second analysis step of introducing a derivatized second sample into the second column together with a second mobile phase and analyzing components contained in the second sample, using a first column and a second column each composed of an ODS column, the method comprising: the first sample contains inosinic acid and guanylic acid; In the first analysis step, components contained in the first sample may be separated using the first column having a length of 185 mm or more to obtain a chromatogram in which peaks corresponding to inosinic acid and guanylic acid are separated with a resolution of 1.5 or more.

[0137] According to the analytical method described in paragraph 1, while analyzing the components contained in the first non-derivatized sample and the second derivatized sample in parallel, a chromatogram can be obtained in which the peaks corresponding to inosinic acid and guanylic acid contained in the first sample are sufficiently separated. In other words, the peak corresponding to guanylic acid, which is an example of an umami component contained in food, can be separated from the peak corresponding to inosinic acid without providing a separate column.

[0138] (Item 2) In the analysis method according to item 1, The first analysis step may include a gradient step in which a ratio of an organic solvent in a first mobile phase introduced into the first column is gradually increased by changing a mixing ratio of a first liquid to a second liquid having a higher content of an organic solvent than the first liquid.

[0139] According to the analytical method described in paragraph 2, it is possible to shorten the time until the elution of each component is completed without deteriorating the resolution of the peaks of components with short retention times among the components contained in the first sample. In other words, it is possible to prevent the time until the elution of each component contained in the first sample is completed from increasing as the length of the first column increases.

[0140] (Item 3) In the analysis method according to item 2, The first analysis step may include a washing step of washing the inside of the first column by introducing only the second liquid into the first column after the gradient step.

[0141] According to the analytical method described in paragraph 3, since impurity components are not accumulated in the first column, it is possible to prevent the load on the first column from increasing, that is, it is possible to prevent the first column from being deteriorated.

[0142] Furthermore, if the analysis of each component contained in the first sample is completed before the analysis of each component contained in the derivatized second sample is completed, the first column can be sufficiently washed by performing the washing step during the first analysis step for a relatively long period of time until both the first analysis step and the second analysis step are completed. [Explanation of symbols]

[0143] 44 Column 1 50 1st liquid 52 2nd liquid 54 1st mobile phase 84 Column 2 94 Second mobile phase 132 First sample 136 Second sample 137 Derivatized second sample 222 First chromatogram data 232 First Analysis Processing Unit 234 Gradient Processing Section 236 Cleaning Processing Section 238 Second Analysis Processing Unit 240 First acquisition processing unit

Claims

1. An analytical method for performing, in parallel, a first analysis step of introducing a non-derivatized first sample into the first column together with a first mobile phase and analyzing components contained in the first sample, and a second analysis step of introducing a derivatized second sample into the second column together with a second mobile phase and analyzing components contained in the second sample, using a first column and a second column each composed of an ODS column, the method comprising: the first sample contains inosinic acid and guanylic acid; In the first analysis step, a chromatogram is obtained in which peaks corresponding to inosinic acid and guanylic acid are separated with a resolution of 1.5 or more by separating components contained in the first sample using the first column having a length of 185 mm or more.

2. 2. The analytical method according to claim 1, wherein the first analysis step includes a gradient step of gradually increasing a ratio of the organic solvent in the first mobile phase introduced into the first column by changing a mixing ratio of a first liquid to a second liquid having a higher content of the organic solvent than the first liquid.

3. 3. The analytical method according to claim 2, wherein the first analysis step includes a washing step of washing the inside of the first column by introducing only the second liquid into the first column after the gradient step.

4. The analytical method described in claim 1, wherein the length of the first column is longer than the length of the second column.

5. The introduction of the first mobile phase into the first column and the introduction of the second mobile phase into the second column are continued until both the first analysis step and the second analysis step are completed; The analytical method according to claim 3 , wherein the washing step is performed during the first analytical step until both the first analytical step and the second analytical step are completed.

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