Amino acid analysis method and liquid chromatography apparatus

Abstract

To provide an amino acid analysis method and a liquid chromatograph device capable of improving separation performance of threonine, serine, glycine and alanine.SOLUTION: A method for analyzing amino acids using a liquid chromatograph device equipped with a cation-exchange column includes a step of separating threonine, serine, glycine, and alanine by circulating a sample containing threonine, serine, glycine, and alanine as amino acids with an eluent in a cation-exchange column. The column temperature for separating threonine and serine is made higher than the column temperature for separating glycine and alanine.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present disclosure relates to an amino acid analysis method and a liquid chromatograph device.

Background Art

[0002] As a method for analyzing sample components in a sample, a liquid chromatography method is known. In the liquid chromatography method, generally, during the separation process of sample components by a separation column, the temperature of the separation column is increased to improve the separation performance.

[0003] For example, in Patent Document 1, a sample containing a plurality of types of amino acids is passed through a separation column heated with a temperature gradient including a temperature range of 100°C or higher, and the amino acids in the sample are separated and analyzed with high accuracy in a shorter time.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] By the way, in the prior art such as Patent Document 1, there is no disclosure regarding the improvement of separation performance focusing on amino acid components that elute at the initial stage of analysis with a short retention time, particularly threonine, serine, glycine, and alanine.

[0006] Therefore, in the present disclosure, in an amino acid analysis method and a liquid chromatograph device, it is an object to improve the separation performance of threonine, serine, glycine, and alanine.

Means for Solving the Problems

[0007] An amino acid analysis method according to one embodiment of the present disclosure is a method for analyzing amino acids using a liquid chromatograph apparatus equipped with a cation exchange column, comprising the step of passing a sample containing threonine, serine, glycine, and alanine as amino acids through the cation exchange column together with an eluent to separate the threonine, serine, glycine, and alanine, characterized in that the column temperature when separating the threonine and serine is higher than the column temperature when separating the glycine and alanine.

[0008] Furthermore, a liquid chromatograph apparatus according to one embodiment of the present disclosure comprises: a liquid delivery unit for delivering an eluent into a flow channel; a sample injection unit provided downstream of the liquid delivery unit for injecting a sample containing threonine, serine, glycine, and alanine into the eluent in the flow channel; a cation exchange column provided downstream of the sample injection unit for separating sample components in the sample; a temperature adjustment means for adjusting the column temperature of the cation exchange column; and a control means for controlling the temperature adjustment means, wherein the control means controls the temperature adjustment means such that the column temperature when separating threonine and serine is higher than the column temperature when separating glycine and alanine. [Effects of the Invention]

[0009] According to this disclosure, the separation performance of threonine, serine, glycine, and alanine can be improved in amino acid analysis methods and liquid chromatographs. [Brief explanation of the drawing]

[0010] [Figure 1] A schematic diagram of the device configuration and flow path of the amino acid analyzer 100 of Example 1. [Figure 2] A schematic diagram of the timetable in the time program that controls the operation of the amino acid analyzer 100 in Example 1. [Figure 3] Chromatogram of Example 1. [Figure 4]A schematic diagram of the timetable in the time program that controls the operation of the amino acid analyzer 100 in Example 2. [Figure 5] Chromatogram of Example 2. [Figure 6] Chromatogram of Example 3. [Figure 7] Chromatogram of Comparative Example 1. [Figure 8] Chromatogram of Comparative Example 2. [Modes for carrying out the invention]

[0011] Embodiments of the present disclosure will be described in detail below with reference to the drawings. The following description of preferred embodiments is illustrative in nature and is not intended to limit the present disclosure, its applications, or its uses in any way.

[0012] <Liquid Chromatography System> A liquid chromatograph is a device that separates the target components of a sample using a separation column while a mobile phase (eluent) is delivered, and then detects the components as they flow in the order they were separated using a detector such as a spectrophotometer to analyze the components of the sample. Examples of liquid chromatographs according to this disclosure include high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC), and although not intended to limit the scope, HPLC is preferred.

[0013] The separation mode of the liquid chromatograph apparatus according to this disclosure is an ion exchange mode for separating ionic components in a sample. Ion exchange chromatography is a method of separating and analyzing sample components with different properties by utilizing the interaction that occurs between sample components in two phases: a stationary phase consisting of an ion exchange material such as an ion exchange resin and a mobile phase consisting of a buffer solution. Generally, ion exchange materials include cation exchange materials such as sulfonic acids and carboxylic acids, and anion exchange materials such as quaternary ammonium and tertiary ammonium, depending on the properties of the chemically modified ion exchange groups. The ion exchange material of the liquid chromatograph apparatus according to this disclosure is a cation exchange material, and in this specification, a column packed with a cation exchange material as the stationary phase is referred to as a cation exchange column or simply a separation column.

[0014] The liquid chromatograph apparatus according to this disclosure is for analyzing samples containing amino acids, particularly samples containing at least threonine, serine, glycine, and alanine as components to be separated. Specifically, the liquid chromatograph apparatus according to this disclosure is, for example, an amino acid analyzer used for analyzing the amino acid composition of proteins and peptides, and for analyzing amino acids and related substances in pharmaceuticals and biological fluids, or an HPLC system used for analyzing proteins, amines, organic acids, etc., and is preferably an amino acid analyzer.

[0015] The sample analyzed by the liquid chromatograph apparatus according to this disclosure may contain other amino acids, etc., as long as it contains at least threonine, serine, glycine, and alanine. For example, in simultaneous amino acid analysis, the amino acids and amino acid analogs to be analyzed can be broadly classified into approximately 20 types of protein hydrolysate amino acids and 40 or more types of biological fluid amino acids and amino acid analogs. These various amino acids can be analyzed simultaneously by mixing multiple buffer solutions, adding the sample to the mixed buffer solution, and passing it through a separation column for detection.

[0016] The liquid chromatograph device according to the present disclosure may be a device that analyzes sample components by gradient elution. In this case, multiple types of eluents are used as the mobile phase for separation. Note that in this specification, the term "gradient" is used as a term including stepwise gradient, curved gradient, and linear gradient.

[0017] The liquid chromatograph device according to the present disclosure includes, for example, a liquid feeding unit, a sample injection unit, a separation unit, and a detector in order from the upstream side of the flow path, and also includes a control device that is connected to each unit and controls these operations. In addition to these configurations, the liquid chromatograph device may include any other arbitrary configurations. Specifically, for example, from the viewpoint of facilitating the detection of sample components, the device may include devices such as reagents for pre-column derivatization or post-column derivatization, pumps, mixers, cartridge-type reactors, etc.

[0018] [Liquid feeding unit] The liquid feeding unit feeds the mobile phase into the flow path. Specifically, the liquid feeding unit feeds at least a single eluent as the mobile phase for separation. In particular, when using the gradient elution method, the liquid feeding unit feeds two or more types of eluents into the flow path. Further, the liquid feeding unit may be capable of feeding a mobile phase that is not used for sample separation, such as a column cleaning liquid for cleaning the separation column or an adjustment liquid for adjusting the state of the separation column, as the mobile phase. Note that column cleaning may be expressed as column regeneration, and in this specification, the column cleaning liquid is also referred to as a column regeneration liquid.

[0019] The liquid delivery unit is specifically composed of, for example, containers for storing each mobile phase such as an eluent, a column washing liquid, and an adjustment liquid, solenoid valves for starting / ending the liquid delivery of each mobile phase in each container or adjusting the flow rate of each liquid to a flow path, and pumps for delivering the mobile phase in each container to the flow path and adjusting the flow rate of each mobile phase. The solenoid valves can be provided corresponding to each container on the flow path provided corresponding to each container. The pump can be provided downstream of the solenoid valve on the flow path. Specifically, for example, the flow paths corresponding to each container may merge downstream of the solenoid valve to form one flow path, and one pump may be provided on this flow path (low-pressure gradient elution). Also, a plurality of pumps corresponding to each container may be provided on the flow paths corresponding to each container (high-pressure gradient elution).

[0020] The eluent is not limited, and each liquid generally used in the analysis of amino acids by liquid chromatography can be used.

[0021] Specifically, for example, as the eluent, an aqueous solution containing an alkali metal salt of a polybasic acid and / or a buffer solution can be adopted. Examples of the polybasic acid include inorganic polybasic acids such as sulfuric acid, selenic acid, phosphoric acid, and diphosphoric acid, and organic polybasic acids such as citric acid, sulfosalicylic acid, and fluorophthalic acid. Examples of the alkali metal include lithium, sodium, potassium, etc. Note that the eluent preferably contains at least one selected from the group consisting of sodium citrate buffer solution, lithium citrate buffer solution, and sodium sulfate aqueous solution from the viewpoint of improving the separation performance of amino acids.

[0022] Note that the pH of the eluent is not intended to be limited, but can be, for example, 2 or more and 5 or less, and preferably 2.5 or more and 5 or less. When using the gradient elution method, it is preferable to gradually increase the pH of two or more types of eluents. The difference between the pH of the eluent delivered first and the pH of the eluent delivered next is preferably within 0.5, and more preferably within 0.3.

[0023] Furthermore, the cation concentration, i.e., salt concentration, contained in the eluent is not limited, but can be, for example, 0.05 N or more and less than 0.2 N, and preferably 0.12 N or more and 0.19 N or less. When using the gradient elution method, it is preferable to gradually increase the salt concentration of the eluent.

[0024] Furthermore, the eluent may contain an organic solvent, mainly composed of the aforementioned aqueous solution and / or buffer solution, from the viewpoint of improving the separation performance of amino acids. Specific examples of organic solvents include alcohols such as ethanol and benzyl alcohol, and acetonitrile.

[0025] The flow rate of the eluent is not limited and can be any flow rate commonly used in liquid chromatography. Furthermore, from the viewpoint of speeding up the analysis, the flow rate of the eluent can be, for example, greater than 0.40 mL / min, preferably between 0.50 mL / min and 2.0 mL / min.

[0026] The column washing solution is not limited to any specific type; any solution commonly used in the analysis of amino acids by liquid chromatography can be used.

[0027] The preparation solution is not limited and any solution commonly used in liquid chromatography can be used. Specifically, examples of preparation solutions include low-salt aqueous solutions, low-pH aqueous solutions, and pure water such as distilled water. The salt concentration of the preparation solution is preferably lower than that of the eluent. Furthermore, from the viewpoint of rapidly transitioning the salt concentration of the separation column to its initial state, it is more preferable to use pure water as the preparation solution. In addition, when using the low-pH aqueous solution as the preparation solution, it is preferable that the pH of the low-pH aqueous solution is lower than that of the eluent.

[0028] [Sample injection section] The sample injection unit is located downstream of the liquid delivery unit in the flow path and is a means for injecting a sample containing the four components described above into the mobile phase flowing through the flow path. The sample injection unit may be a manual injector or an automatic autosampler, but an autosampler is preferred from the viewpoint of accurately controlling the injection timing and amount of the sample.

[0029] [Separation section] The separation unit is located downstream of the sample injection unit in the flow path and comprises a separation column for separating sample components from the sample, and a temperature control device (temperature control means) provided on the separation column for adjusting the temperature of the separation column (column temperature) during the separation of sample components by the separation column.

[0030] As for the separation column, as mentioned above, any column packed with a cation exchanger as the stationary phase is not particularly limited, and columns commonly used in liquid chromatography can be used.

[0031] The temperature control device is not particularly limited as long as it is a device that can adjust the temperature of the separation column, and can be any known device such as a heater, Peltier element, or heat pump.

[0032] The temperature of the separation column can be adjusted, for example, between 20°C and 150°C, although this is not intended to be limiting. Furthermore, the difference between the first column temperature (when separating threonine and serine) and the second column temperature (when separating glycine and alanine) can be set to, for example, 10°C or more, although this is not intended to be limiting.

[0033] [Detector] The detector is installed downstream of the separation column in the flow path and is a device for detecting sample components separated by the separation column. The detector is not particularly limited, and detectors commonly used in liquid chromatography, such as electrical conductivity detectors, ultraviolet-visible absorbance detectors, fluorescence detectors, and electrochemical detectors, can be used.

[0034] [Control device] The control device is at least a device that controls the temperature control device. In addition to the temperature control device, the control device may also control other parts such as the liquid delivery unit and the sample injection unit.

[0035] Specifically, the control device is electrically connected wirelessly or via wired connections to, for example, the solenoid valves and pumps of the liquid delivery unit, the sample injection unit in the case of an autosampler, the temperature control device, etc., and sends control signals to these to control their operation. The control device is also electrically connected wirelessly or via wired connections to, for example, a detector, and acquires the detection results of the detector and outputs them as a chromatogram and data. The control device is, for example, a device based on a well-known microcomputer and includes an input unit for receiving information from the outside, a storage unit for storing information, a calculation unit for performing various calculations based on various information, and an output unit such as a display unit for outputting information.

[0036] The control unit's memory unit stores a time program for executing the analysis process described later. The time program includes a timetable corresponding to each step in the analysis process described later. When using the gradient elution method, the time program includes a gradient elution time program for changing the mixing ratio of the eluents and sending the eluents to the liquid delivery unit. In other words, in a liquid chromatograph apparatus that performs analysis using the gradient elution method, the control unit changes the mixing ratio of two or more eluents based on the gradient elution time program and sends them to the liquid delivery unit.

[0037] <Analysis process using a liquid chromatography system> The analysis process of a liquid chromatograph includes, for example, a sample injection step, a separation step, and a pre-injection liquid delivery step, and these steps are repeated a number of times set by the user as one method. The analysis process may also include a washing step, in which a column washing solution is delivered to wash the separation column, and an adjustment step, in which a conditioning solution is delivered to adjust the condition of the separation column, after the separation step and before the pre-injection liquid delivery step.

[0038] The sample injection step is a process in which a sample containing the four components described above is injected into the eluent in the flow path by the sample injection unit. The time program may be created with the sample injection step as time zero, although this is not intended to be limiting.

[0039] The separation step is a process in which, after the sample injection step, the sample components of the sample flowing with the eluent, particularly the four components mentioned above, are separated in the separation column. When using the gradient elution method, the eluent is delivered at least during the separation step based on the gradient elution time program described above.

[0040] Furthermore, in the separation process, as will be described later, the temperature of the separation column is adjusted by a temperature control device from the viewpoint of improving the separation performance of the separation column.

[0041] Once the separation process is complete, it is necessary to stabilize the separation column by returning its temperature, salt concentration, pH, and other conditions to their initial state, i.e., the state before sample injection, for the next method. The pre-injection fluid delivery process after the separation process is provided for this purpose. In the pre-injection fluid delivery process, the separation column is stabilized by delivering, for example, the first eluent, before the sample injection process of the next method.

[0042] <Amino Acid Analysis Method> The amino acid analysis method described herein is characterized by setting the column temperature (also referred to as the "first temperature") used when separating threonine and serine ("group 1") to be higher than the column temperature (also referred to as the "second temperature") used when separating glycine and alanine ("group 2").

[0043] In other words, the control means controls the temperature adjustment means by executing a time program configured such that the first temperature is higher than the second temperature.

[0044] When separating threonine, serine, glycine, and alanine using a cation exchange column, the retention time for the first group is shorter than that for the second group, and the first group elutes before the second group. Therefore, for example, the control means can execute a time program configured to lower the column temperature after the first group has eluted to allow the second group to elute.

[0045] When separating the above four components using a cation exchange column, as described above, these four components have short retention times and elute early in the analysis. Therefore, if the column temperature is kept constant or gradually increased, it is difficult to clearly separate these four components. More specifically, even if one attempts to separate the second group while maintaining a temperature sufficient to separate the first group, the peaks of glycine and alanine overlap, making it difficult to clearly separate them.

[0046] Herein, the inventors of the present invention, after diligent study, have found that when separating the above four components using a cation exchange column, the first group elutes before the second group, while the column temperature suitable for separating the first group is higher than the column temperature suitable for separating the second group. This phenomenon is thought to be due to the different response performance of the retention time of each component to changes in column temperature. That is, the difference in retention time of threonine and serine is thought to be larger at higher column temperatures, while the difference in retention time of glycine and alanine is thought to be larger at lower column temperatures. Therefore, by setting the first and second temperatures to column temperatures that allow for the separation of the first and second groups, respectively, separating the first group at the first temperature, and then lowering the column temperature to the second temperature to separate the second group, the separation performance of the four components can be improved.

[0047] Furthermore, when an organic solvent is included in the eluent, it is preferable to set the concentration of the organic solvent in the eluent higher when separating the first group than when separating the second group, for example, by gradient elution. In other words, it is preferable to separate the second group by reducing the concentration of the organic solvent in the eluent after separating the first group. In this case, the control means should execute a time program configured in this way and further control the liquid delivery unit. This further improves the separation performance of amino acids, especially the second group.

[0048] When separating the first group, the concentration of the organic solvent in the eluent can be, for example, 5% to 20%, preferably 10% to 15%, although this is not intended to be a limitation.

[0049] Furthermore, the concentration of the organic solvent in the eluent when separating the second group is not limited as long as it is lower than the concentration of the organic solvent in the eluent when separating the first group, but specifically, for example, it can be less than 10%, preferably less than 5%. [Examples]

[0050] The following describes, with reference to the drawings, an amino acid analyzer 100 (liquid chromatograph) and an amino acid analysis method according to an embodiment of this disclosure.

[0051] [Example 1] Figure 1 is a schematic diagram of the apparatus configuration and flow path of the amino acid analyzer 100 according to Example 1 of this disclosure. The amino acid analyzer 100 is an ion exchange chromatograph system that utilizes a post-column derivatization method with ninhydrin.

[0052] The amino acid analyzer 100 can be equipped with the following mobile phases: the first to fourth eluents 1 to 4, distilled water 5 as a conditioning solution, and column regeneration solution 6 (also referred to as "solution B1" to "solution B6," respectively). From these, one of the solutions is selected by solenoid valves 7A to 7F and delivered by the mobile phase pump 9. The eluents pass through the ammonia filter column 11 and are then introduced into the separation column 13. An autosampler 12 (sample injection unit) is provided downstream of the ammonia filter column 11 and upstream of the separation column 13, and the amino acid sample is injected into the eluent in the flow path by the autosampler 12. The injected amino acid sample, along with the eluents, reaches the separation column 13 and is separated in the separation column 13.

[0053] The amino acid analyzer 100 also includes a ninhydrin reagent 8 and a ninhydrin pump 10 for delivering the ninhydrin reagent 8. Each amino acid component separated in the separation column 13 is mixed with the ninhydrin reagent 8 delivered by the ninhydrin pump 10 in the mixer 14 and reacted in the heated reactor 15.

[0054] The amino acids (Luhemann purple) that develop color due to the reaction are continuously detected by the detector 16, output as a chromatogram and data by the data processing device 17 (control device), and recorded and stored.

[0055] Separation column 13 is a sulfonic acid-based strong cation exchange column (packing material base: polystyrene resin, particle size: 3 μm).

[0056] The separation column 13 is equipped with a temperature control device 13A for adjusting the temperature of the separation column 13, allowing the column temperature to be freely heated to increase or cooled to decrease. Each mobile phase container and reactor 15, etc., may also be equipped with a temperature control device (not shown) for adjusting their temperatures.

[0057] Detector 16 is a visible absorbance spectrophotometer with a main wavelength of 570 nm, and is also capable of detecting at 440 nm, which is the target wavelength for proline and the like.

[0058] The data processing device 17 controls the solenoid valves 7A to 7F of each container that stores each mobile phase of liquid B1 to liquid B6, the mobile phase pump 9, the ninhydrin pump 10, the autosampler 12, the temperature control device 13A, and, if installed, a temperature control device (not shown) that adjusts the temperature of each mobile phase container and reactor 15, etc. This control is mainly performed by a time program stored in the memory unit (not shown) of the data processing device 17.

[0059] In Example 1, the amino acid sample used was prepared by dissolving four components—threonine, serine, glycine, and alanine—in water. In the following description, the names and abbreviations of the amino acids to be analyzed will follow those shown in Table 1.

[0060] [Table 1]

[0061] Furthermore, a single first eluent 1 was used as the eluent. The compositions of the first eluent 1 and column regeneration solution 6 are shown in Table 2. Note that in Table 2, salt concentration is shown as Na concentration.

[0062] [Table 2]

[0063] As shown in Table 2, the first eluent 1 was a sodium citrate buffer containing 13% by volume of ethanol as an organic solvent.

[0064] The detailed configuration and measurement conditions of the amino acid analyzer 100 are shown in Table 3.

[0065] [Table 3]

[0066] Figure 2 shows an example of a timetable on the time program of the amino acid analyzer 100. As shown in Figure 2, when the sample is injected into the flow path by the autosampler 12 during the sample injection process, the separation process begins. During the separation process, the first eluent (solution B1) is delivered to the separation column 13. This separates the sample components. Once all the sample components to be analyzed have been eluted and the separation process is complete, the washing process begins, and the column regeneration solution 6 (solution B6) is delivered to wash the separation column 13. After the washing process is complete, the pre-injection delivery process begins. The pre-injection delivery process is the process of delivering, for example, solution B1 to the separation column 13 before the sample injection process. This allows the separation column 13 to be stabilized before sample injection.

[0067] In Example 1, during the separation process, the column temperature was set to a first temperature of 60°C from the start of the separation process. Next, after confirming the elution of threonine and serine, the column temperature was lowered to a second temperature of 40°C.

[0068] Figure 3 shows an example of the chromatogram from Example 1. As shown in Figure 3, it was found that by setting the column temperature to the first temperature during the separation of the first group and lowering the column temperature to the second temperature during the separation of the second group, good separation performance could be obtained for both the first and second groups.

[0069] [Example 2] Except for the configuration described below, the analysis was performed using the same procedure and conditions as in Example 1.

[0070] As shown in Figure 4, in the separation step of Example 2, the first eluent 1 (Solution B1) and the second eluent 2 (Solution B2) were made available to the separation column 13 based on a gradient elution time program, with their mixing ratios being varied.

[0071] For the first eluent 1, a lithium citrate buffer solution was used, with "sodium citrate" replaced by "lithium citrate" in Table 2. For the second eluent 2, a buffer solution was used in which the ethanol concentration of the first eluent 1 was reduced to 0% by volume.

[0072] In Example 2, in addition to the column temperature control of Example 1, the first eluent 1 was introduced from the start of the separation process, and the ethanol concentration in the eluent was set to 13% by volume. Next, after confirming the elution of threonine and serine, the second eluent 2 was introduced in place of the first eluent 1, and the ethanol concentration in the eluent was set to 0% by volume.

[0073] Figure 5 shows an example of the chromatogram from Example 2. As shown in Figure 5, it was found that, in addition to controlling the column temperature, reducing the concentration of ethanol in the eluent during the separation of the second group further improved the separation performance of the four components, especially the second group.

[0074] [Example 3] Except for the configuration described below, the analysis was performed using the same procedure and conditions as in Example 2.

[0075] The first temperature was set to a temperature higher than 60°C (approximately 80°C), and the second temperature was set to a temperature higher than 40°C (approximately 60°C). In addition, the flow rate of the eluent was increased by approximately 10% from 0.40 mL / min to 0.44 mL / min.

[0076] In Example 3, the column temperature was increased overall compared to Examples 1 and 2. This reduces the viscosity of the eluent and suppresses the increase in pressure, making it possible to increase the flow rate.

[0077] Figure 6 shows an example of the chromatogram from Example 3. Comparing Figure 5 and Figure 6, it can be seen that the retention time of the four components has been shortened while the separation performance of the four components has been maintained. In other words, by increasing the column temperature overall and increasing the flow rate, it is possible to shorten the retention time of the four components while maintaining the good separation performance of the first and second groups, thereby achieving both high separation and high speed in analysis.

[0078] [Comparative Example 1] The analysis was performed using the same procedure and conditions as in Example 1, except that the column temperature was kept constant at 40°C.

[0079] Figure 7 shows an example of the chromatogram for Comparative Example 1. As shown in Figure 7, when the column temperature is kept constant at 40°C, the peaks of threonine and serine in particular overlap, and it was found that sufficient separation performance could not be obtained.

[0080] [Comparative Example 2] The analysis was performed using the same procedure and conditions as in Example 1, except that the column temperature was kept constant at 60°C.

[0081] Figure 8 shows an example of the chromatogram for Comparative Example 2. As shown in Figure 8, it was found that when the column temperature is kept constant at 60°C, the peaks of glycine and alanine in particular overlap in the second group, and sufficient separation performance cannot be obtained.

[0082] [summary] As is clear from the comparison of Figures 3, 7, and 8, the first group elutes earlier than the second group, while the column temperature suitable for separating the first group is higher than the column temperature suitable for separating the second group. This is thought to be because the difference in retention times for threonine and serine becomes larger at higher column temperatures, while the difference in retention times for glycine and alanine becomes larger at lower column temperatures. As shown in Figure 3, by setting the column temperature to the first temperature when separating the first group and lowering it to the second temperature when separating the second group, the separation performance of the four components is improved even when using a single eluent.

[0083] Furthermore, as shown in Figure 5, reducing the concentration of the organic solvent in the eluent during the separation of the second group compared to the separation of the first group is also effective in improving separation performance.

[0084] In Examples 1 and 2, the column temperature is lowered once after the separation of the first group before separating the second group, which may increase the analysis time required for separating the second group. In this regard, as shown in Figure 6, in Example 3, the flow rate of the eluent can be increased by raising the column temperature overall. Therefore, even if the separation of the second group takes some time, it is possible to achieve high separation while speeding up the analysis overall.

[0085] This disclosure is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail for the purpose of explaining this disclosure clearly, and are not necessarily limited to having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations. [Explanation of Symbols]

[0086] 1,B1 First eluent 2,B2 Second eluent 3. Third eluent 4. Fourth eluent 5. Distilled water (adjusted solution) 6. B6 Column regeneration solution (column washing solution) 7A,7B,7C,7D,7E,7F Solenoid valve (liquid sending part) 8. Ninhydrin Reagent 9. Mobile phase pump (liquid delivery section) 10 Ninhydrin pump 11 Ammonia filter column 12. Autosampler (sample injection section) 13 Separation column 14 Mixer 15 Reactors 16 detectors 17. Data Processing Device (Control Device)

Claims

1. A method for analyzing amino acids using a liquid chromatograph equipped with a cation exchange column, The cation exchange column is subjected to a step of passing a sample containing threonine, serine, glycine, and alanine as amino acids together with an eluent to separate the threonine, serine, glycine, and alanine. The column temperature used when separating threonine and serine is set higher than the column temperature used when separating glycine and alanine. The eluent contains an organic solvent, The concentration of the organic solvent in the eluent when separating the threonine and serine is set higher than the concentration of the organic solvent in the eluent when separating the glycine and alanine. A method for analyzing amino acids characterized by the following features.

2. The amino acid analysis method according to claim 1, The retention times of threonine and serine are shorter than the retention times of glycine and alanine. After the threonine and serine have been eluted, the column temperature is lowered to elute the glycine and alanine. A method for analyzing amino acids characterized by the following features.

3. A method for analyzing amino acids according to claim 1 or claim 2, The eluent comprises at least one selected from the group consisting of sodium citrate buffer, lithium citrate buffer, and aqueous sodium sulfate solution. A method for analyzing amino acids characterized by the following features.

4. A method for analyzing amino acids according to any one of claims 1 to 3, When the column temperature used to separate the threonine and serine is designated as the first temperature, and the column temperature used to separate the glycine and alanine is designated as the second temperature, the difference between the first temperature and the second temperature is 10°C or more. A method for analyzing amino acids characterized by the following features.

5. A liquid delivery unit that delivers the eluent into the flow path, A sample injection unit is provided downstream of the liquid delivery unit and injects a sample containing threonine, serine, glycine, and alanine into the eluent of the flow path, A cation exchange column is provided downstream of the sample injection section for separating the sample components in the sample, A temperature control means for adjusting the column temperature of the cation exchange column, Control means for controlling the temperature adjustment means, A liquid chromatograph apparatus comprising, The control means controls the temperature adjustment means such that the column temperature when separating the threonine and serine is higher than the column temperature when separating the glycine and alanine. The eluent contains an organic solvent, The control means further controls the liquid delivery unit so that the concentration of the organic solvent in the eluent when separating threonine and serine is higher than the concentration of the organic solvent in the eluent when separating glycine and alanine. A liquid chromatograph apparatus characterized by the following features.

6. A liquid chromatograph apparatus according to claim 5, The retention times of threonine and serine are shorter than the retention times of glycine and alanine. The control means controls the temperature adjustment means to lower the column temperature after the threonine and serine have been eluted, in order to elute the glycine and alanine. A liquid chromatograph apparatus characterized by the following features.

7. A liquid chromatograph apparatus according to claim 5 or claim 6, When the column temperature used to separate the threonine and serine is designated as the first temperature, and the column temperature used to separate the glycine and alanine is designated as the second temperature, the difference between the first temperature and the second temperature is 10°C or more. A liquid chromatograph apparatus characterized by the following features.

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