CN115684456A - Method for controlling ion exchange chromatography and ion exchange chromatography - Google Patents
Method for controlling ion exchange chromatography and ion exchange chromatography Download PDFInfo
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- CN115684456A CN115684456A CN202210358612.2A CN202210358612A CN115684456A CN 115684456 A CN115684456 A CN 115684456A CN 202210358612 A CN202210358612 A CN 202210358612A CN 115684456 A CN115684456 A CN 115684456A
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- 238000000034 method Methods 0.000 title claims abstract description 32
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- 238000000926 separation method Methods 0.000 claims abstract description 108
- 239000007788 liquid Substances 0.000 claims abstract description 95
- 238000002347 injection Methods 0.000 claims abstract description 65
- 239000007924 injection Substances 0.000 claims abstract description 65
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 150000001413 amino acids Chemical class 0.000 claims description 37
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- 238000005406 washing Methods 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 abstract description 30
- 235000001014 amino acid Nutrition 0.000 description 33
- 230000009467 reduction Effects 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 238000007796 conventional method Methods 0.000 description 10
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
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- 238000001514 detection method Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Natural products CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 125000000899 L-alpha-glutamyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C(O[H])=O 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
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- 239000003957 anion exchange resin Substances 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
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- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
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- 235000018102 proteins Nutrition 0.000 description 2
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- 238000004904 shortening Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
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- 210000001124 body fluid Anatomy 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 238000005342 ion exchange Methods 0.000 description 1
- 229940071264 lithium citrate Drugs 0.000 description 1
- WJSIUCDMWSDDCE-UHFFFAOYSA-K lithium citrate (anhydrous) Chemical compound [Li+].[Li+].[Li+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O WJSIUCDMWSDDCE-UHFFFAOYSA-K 0.000 description 1
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- 238000001121 post-column derivatisation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 229950006389 thiodiglycol Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The present invention relates to a method for controlling ion exchange chromatography and ion exchange chromatography, which can improve the separation performance of a sample component and shorten the analysis time at the same time even when a single separation column is used. A control method for ion exchange chromatography, comprising a liquid sending unit, a sample injection unit, a separation column, and a control device, wherein the control device executes a specific time program so that the liquid sending of the 2 nd eluent is started simultaneously with or before the injection of the sample, and the mixing ratio of the eluents is changed and the eluents are sent to the liquid sending unit based on the specific time program.
Description
Technical Field
The present invention relates to a method for controlling ion exchange chromatography and ion exchange chromatography.
Background
As a method for analyzing a sample component in a sample, an ion exchange chromatography is known. Ion exchange chromatography is a method of separating and analyzing sample components having different properties by utilizing an interaction between the sample components in both a stationary phase composed of an ion exchanger such as an ion exchange resin and a mobile phase composed of a buffer solution or the like. The ion exchanger includes a cation exchanger such as sulfonic acid or carboxylic acid, which is obtained by chemically modifying an ion exchange group, and an anion exchanger such as quaternary ammonium or tertiary ammonium.
Examples of the sample to be analyzed by ion exchange chromatography include amino acids.
In the simultaneous analysis of amino acids in which a plurality of amino acids are simultaneously analyzed, the amino acids and amino acid analogs to be analyzed are roughly classified into about 20 kinds of protein hydrolysate amino acids and 40 or more kinds of physiological body fluid amino acids and amino acid analogs. These various amino acids can be simultaneously analyzed by mixing 2 or more buffers, adding a sample to the mixed buffer, passing the sample through a separation column, and detecting the sample.
In this method, an analytical method has been developed for the purpose of improving the separation performance and shortening the analysis time by improving the kind, buffer composition, flow rate, and the like of the ion exchange resin packed in the separation column. As an example thereof, there is patent document 1.
Patent document 1 describes the following technique: "in the simultaneous analysis of amino acid multi-components, a plurality of columns using packing materials having different chemical properties are arranged in series to perform the analysis, whereby high separation portions due to the properties of the packing materials can be effectively used for separation, thereby improving the separation performance and speeding up the analysis" (see the abstract of patent document 1).
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open No. 2008-249447
Disclosure of Invention
Technical problems to be solved by the invention
However, in the technique of patent document 1, although improvement in separation performance and speeding up of analysis are achieved, since a plurality of separation columns using a filler having different chemical characteristics are arranged in series, there is room for improvement in terms of column size and cost reduction.
In addition, for example, the analysis time for simultaneous analysis of amino acids is long, and further reduction of the analysis time is desired.
The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for controlling ion exchange chromatography and ion exchange chromatography, which can achieve both improvement in separation performance of a sample component and reduction in analysis time even when a single separation column is used in terms of column miniaturization and cost reduction.
Means for solving the problems
The ion exchange chromatography and the control method thereof of the present invention comprise: a liquid sending unit that sends two or more types of eluents into the flow path, the two or more types of eluents including a 1 st eluent and a 2 nd eluent that starts liquid sending for sample separation after the 1 st eluent; a sample injection section provided downstream of the liquid sending section and injecting the sample into the eluent in the flow path; a separation column provided downstream of the sample injection section and configured to separate a sample component in the sample; a detector for detecting the sample component separated by the separation column; and a control device that changes a mixing ratio of the eluents based on a specific time program and feeds the eluents to the liquid feeding unit, wherein the control device executes the specific time program so that liquid feeding of the 2 nd eluent is started simultaneously with or before injection of the sample.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, it is possible to provide a control method for ion exchange chromatography and ion exchange chromatography, which can achieve both improvement in separation performance of a sample component and reduction in analysis time even when a single separation column is used in terms of column miniaturization and cost reduction.
Technical problems, configurations, and effects other than those described above will be clarified by the following description of embodiments and examples.
Drawings
FIG. 1 is a schematic diagram of the apparatus configuration and flow path of an amino acid analyzer 100.
Fig. 2 is a schematic diagram of a time chart on a time program for controlling the operation of the amino acid analyzer 100.
FIG. 3 is chromatograms of examples and comparative examples.
Detailed Description
Embodiments of the present invention will be described below. The following description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the invention, its objects of application, or its uses.
< ion exchange chromatography >
The ion exchange chromatography of the present invention can be used as an analyzer for a sample that can be analyzed by the ion exchange chromatography in general. Specifically, ion exchange chromatography is an amino acid analyzer used for analysis of amino acid composition of proteins and peptides, analysis of amino acids or analogues thereof such as pharmaceuticals and physiological fluids, and the like; the HPLC system used for analysis of proteins, amines, organic acids, and the like is preferably an amino acid analyzer.
In the ion exchange chromatography of the present invention, the components of the sample are analyzed by a gradient elution method. In the present specification, the term "gradient" is used as a term including a stepwise gradient, a curved gradient, and a linear gradient.
The ion exchange chromatography of the present invention includes, as essential components, a liquid feeding unit, a sample injection unit, a separation unit, a detector, and a control device. The ion exchange chromatography may have other optional configurations in addition to these configurations. Specifically, for example, from the viewpoint of facilitating the detection of a sample component, devices such as a reagent for pre-column derivatization or post-column derivatization, a pump, a mixer, and a reaction column may be provided.
The liquid sending unit sends two or more kinds of eluents into the flow path, the two or more kinds of eluents including a 1 st eluent and a 2 nd eluent which starts liquid sending after the 1 st eluent. The liquid feeding unit can feed column cleaning liquid, conditioning liquid not used for separating a sample, and the like in addition to the eluent.
The eluent is a buffer such as a sodium citrate buffer, a lithium citrate buffer, or the like, which is introduced into the separation column in the solution feeding before injection step and the separation step described later. As the eluent, the column cleaning solution, and the conditioning solution, various liquids generally used in ion exchange chromatography can be used.
Of the two or more kinds of eluents, the 1 st eluent is an eluent first introduced into the separation column, and is introduced into the separation column at least in the injection liquid feed step. Therefore, the eluent of the 1 st eluent has a function of stabilizing the separation column in addition to a function as an eluent contributing to separation of the sample component. On the other hand, the 2 nd eluent may not have a function of stabilizing the separation column because it greatly contributes to the separation of the sample components as compared with the 1 st eluent.
The salt concentration of the 1 st eluent is preferably lower than the salt concentration of the 2 nd eluent. Thereby effectively stabilizing the separation column. Thus, the separation performance of the sample component by the remaining eluent including the 2 nd eluent can be improved. In the present specification, the "salt concentration" refers to the concentration of cations or the concentration of anions contained in the eluent. Specifically, the salt concentration of the 1 st eluent is preferably 0.05N or more and less than 0.2N, more preferably 0.12N or more and 0.19N or less. The salt concentration of the eluent 2 is preferably more than 0.16N and less than 1.2N, more preferably 0.2N or more and 1N or less.
The difference between the pH of the 1 st eluent and the pH of the 2 nd eluent is preferably within 0.5, more preferably within 0.3. In the case where the stationary phase of the separation column to be described later is a cation exchange resin, it is more preferable that the pH of the 1 st eluent is higher than the pH of the 2 nd eluent. Thereby effectively stabilizing the separation column. Thus, the separation performance of the sample component by the surplus eluent including the 2 nd eluent can be improved.
When the stationary phase of the separation column described later is a cation exchanger, the pH of the 1 st eluent and the 2 nd eluent is preferably around 3, specifically 2.5 to 3.5. In this case, when the eluent includes 3 or more kinds of eluents, the pH of the 3 rd eluent, which starts to be sent after the 2 nd eluent, is preferably higher than the pH of the 1 st eluent and the 2 nd eluent.
Specifically, the liquid feeding unit is constituted by, for example, a container for storing various liquids such as an eluent, a column cleaning liquid, and a control liquid, an electromagnetic valve for controlling the start/end of feeding of the various liquids in the container to the flow path or the flow rates of the various liquids, and a pump for controlling the flow rates of the various liquids. The electromagnetic valve may be provided in a flow path provided in correspondence with each container, in correspondence with each container. The pump may be disposed downstream of the above-described electromagnetic valve on the flow path. Specifically, for example, the flow paths corresponding to the respective containers may be joined downstream of the electromagnetic valve to form 1 flow path, and 1 pump may be provided in the flow path (low-pressure gradient elution). Further, a plurality of pumps may be provided in the flow path corresponding to each container so as to correspond to each container (high-pressure gradient elution).
The sample injection section is provided downstream of the liquid sending section, and is a mechanism for injecting the sample into the eluent in the channel. The sample injection unit may be a manual syringe or an automatic sampler, and is preferably an automatic sampler in view of controlling the injection timing and injection amount of the sample with high accuracy.
The separation section is provided downstream of the sample injection section and includes a separation column for separating a sample component in the sample. The separation section may include a mechanism for adjusting the temperature of the separation column in addition to the separation column. The separation column is not particularly limited, and a column generally used in ion exchange chromatography may be used. The stationary phase of the separation column may be a cation exchanger such as a cation exchange resin or an anion exchanger such as an anion exchange resin, preferably a cation exchanger, more preferably a cation exchange resin.
The detector is provided downstream of the separation column and is a device for detecting the sample component separated by the separation column. The detector is not particularly limited, and a detector commonly used in ion exchange chromatography, such as a conductivity detector, an ultraviolet-visible absorbance detector, a fluorescence photometry detector, and an electrochemical detector, can be used.
The control device is electrically connected or connected to at least an electromagnetic valve and a pump of the liquid feeding unit, a sample injection unit of the automatic sampler (in the case where the sample injection unit is present), a mechanism for adjusting the temperature of the container such as the liquid feeding unit and the column (in the case where the sample injection unit is present), and the like, and transmits a control signal to these units to control their operations. In addition, the control device is also electrically connected with the detector or connected with the detector in an online manner, and the detection result of the detector is obtained and output as a chromatogram and data. The control device is, for example, a device based on a known microcomputer, and includes: an input unit for inputting information from the outside, a storage unit for storing information, an arithmetic unit for performing various arithmetic operations based on various information, and an output unit such as a display unit for outputting information.
A time program for executing an analysis procedure described later is stored in the storage unit of the control device. The control device changes the mixing ratio of the two or more eluents based on a specific gradient elution time program included in the time program and sends the mixture to the liquid sending part.
< analytical procedure for ion exchange chromatography >
The analysis step of ion exchange chromatography includes a sample injection step, a separation step, a washing step, an optional adjustment step, and a liquid injection-prior-to-liquid step, and these steps are repeated as 1 step (method) for the number of times set by the user.
The sample injection step is a step of injecting a sample into the flow path by the sample injection section. Without intending to be limiting, a time program may create the sample injection step as a time zero.
The separation step is a step of separating the sample components in the sample in the separation column after the sample injection step. At least in the separation step, the liquid feeding of the eluent is performed based on a gradient elution time program among the time programs.
At the end of the separation step, the state of the separation column needs to be restored to the initial state before the sample injection and stabilized in order to perform the next process (method). The cleaning step, the sorting adjustment step, and the liquid feeding before injection step after the separation step are provided for this purpose.
First, in the washing step, a column washing liquid is fed to wash the separation column.
Then, in the liquid feeding before injection step, at least the 1 st eluent is fed before the sample injection step of the next process (method) to stabilize the separation column.
The cleaning step may be followed by a conditioning step before the liquid feeding step. In the conditioning step, a conditioning liquid that is not used for separating the sample is fed. This enables the separation column to be quickly returned to the initial state. Examples of the adjustment liquid include a low salt concentration aqueous solution, a low pH aqueous solution in the case where the stationary phase of the separation column is a cation exchanger, a high pH aqueous solution in the case where the stationary phase of the separation column is an anion exchange resin, and pure water.
< method for controlling ion exchange chromatography >
The ion exchange chromatography of the present invention has the following features.
The control device executes a time program so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample.
In the present embodiment, the time program includes a time table (timetable) corresponding to each of the sample injection step, the separation step, the washing step, the optional adjustment step, and the liquid injection-prior-to-liquid step. And the time program includes a gradient elution time program for changing a mixing ratio of the eluent and feeding the eluent to the liquid feeding portion.
For example, the gradient elution time program may be a program in which the liquid feeding of the 1 st eluent is started first, and then the liquid feeding of the 2 nd eluent is started.
In this case, the control device may execute the gradient elution time program so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample, preferably, in the middle of the liquid feeding step before the injection.
According to this aspect, since the gradient elution time program is started before the sample injection step, the analysis time can be shortened.
In addition, as explained below, according to the present embodiment, the separation performance of the separation column can be improved.
Even when the electromagnetic valve is opened to start the supply of the eluent from the container storing each eluent, a time lag occurs in accordance with the capacity of the channel from the container to the separation column before the eluent reaches the separation column. Further, after the sample is injected into the sample injection section, a time lag occurs in accordance with the volume of the flow path from the sample injection section to the separation column before the sample reaches the separation column. Thus, a difference occurs between the timing at which the sample reaches the separation column and the timing at which each eluent reaches the separation column.
When the 2 nd eluent starts to be supplied, a part of the 1 st eluent in the flow path from the confluence portion of the flow paths corresponding to the containers of the 1 st eluent and the 2 nd eluent to the separation column is replaced by the 2 nd eluent in a short time. Then, the sample is injected into the 1 st eluent, the mixed solution of the 1 st eluent and the 2 nd eluent or the 2 nd eluent according to the timing of starting the liquid feeding of the 2 nd eluent.
In the case where the sample has been injected into the mixed solution of the 1 st eluent and the 2 nd eluent or the 2 nd eluent, the 2 nd eluent reaches the separation column simultaneously with or before the sample reaches the separation column. As a result, the sample component can be developed by starting the development of the sample component from the mobile phase partially or entirely replaced with the 2 nd eluent at the same time as the sample reaches the separation column, thereby improving the separation performance of the sample component.
Further, even when the sample is injected into the 1 st eluent, since the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample, the 2 nd eluent reaches the separation column in a shorter time than before after the sample reaches the separation column. Thus, after the sample reaches the separation column, the mobile phase partially or entirely replaced with the 2 nd eluent rapidly starts to develop the sample component, and therefore, the separation performance of the sample component can be improved.
Thus, according to the present invention, both improvement of separation performance of sample components and reduction of analysis time can be achieved. In addition, since a single separation column is used, it can contribute to miniaturization and cost reduction of the column.
For example, the gradient elution time program may be the following program: first, the liquid feeding of the 2 nd eluent is started, and then the liquid feeding of other eluents (specifically, for example, the 3 rd eluent in the case where 3 or more kinds of eluents are used) is started.
In this case, the control device may execute the gradient elution time program simultaneously with or before the injection of the sample so that the liquid supply of the 2 nd eluent is started simultaneously with or before the injection of the sample. This can shorten the improvement of the separation performance of the sample components and the reduction of the analysis time.
In the case of using 3 or more kinds of eluents, the 3 rd eluent delivery of the eluent that starts after the 2 nd eluent delivery can be started before the sample injection step or simultaneously with the sample injection. The 3 rd eluent is more preferably fed after the sample injection step. This ensures high separation performance of sample components by 2 or more types of eluents from which liquid delivery is started after the 2 nd eluent.
Examples
The following describes an amino acid analyzer 100 (ion exchange chromatography) and a control method thereof according to an embodiment of the present invention with reference to the drawings.
In the following description, the names and abbreviations of amino acids to be analyzed are shown in table 1.
[ Table 1]
| Shorthand notation | Name of amino acid |
| Asp | Aspartic acid |
| Thr | Threonine |
| Ser | Serine |
| Glu | Glutamic acid |
| Pro | Proline |
FIG. 1 is a schematic diagram of the apparatus configuration and flow path of an amino acid analyzer 100 according to an embodiment of the present invention.
The amino acid analyzer 100 can be provided with 1 st to 4 th eluents 1 to 4, distilled water 5, and a column regenerating liquid 6. Any one of the liquids is selected by the electromagnetic valves 7A to 7F, and is sent by the eluent pump 9. The eluate is introduced into the separation column 13 after passing through the ammonia filtration column 11. An autosampler 12 (sample injection section) is provided downstream of the ammonia filtration 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 reaches the separation column 13 together with the eluent, and is separated by the separation column 13.
The amino acid analyzer 100 further includes a ninhydrin reagent 8 and a ninhydrin pump 10 for feeding the ninhydrin reagent 8. Each amino acid component separated in the separation column 13 and the ninhydrin reagent 8 fed from the ninhydrin pump 10 are mixed by the mixer 14, and reacted in the heated reaction column 15.
The amino acid (luhmann violet) developed by the reaction is continuously detected by the detector 16, and is output as a chromatogram and data by the data processing device 17 (control device) and recorded and stored.
The data processing device 17 controls the electromagnetic valves 7A to 7D of the eluent containers, the electromagnetic valve 7E of the distilled water container, the electromagnetic valve 7F of the column regenerating liquid container, the eluent pump 9, the ninhydrin pump 10, the autosampler 12, the containers for various liquids, the separation column 13, the reaction column 15, and other mechanisms (not shown). This control is mainly executed by a program stored in a storage unit (not shown) of the data processing device 17.
As the 1 st to 4 th eluents 1 to 4 and the column regeneration solution 6, sodium citrate buffer solutions shown in Table 2 were used. In table 2, the salt concentration is represented as Na concentration.
[ Table 2]
Sodium citrate buffer solution composition
| Name (R) | 1 st eluent | Eluent of No. 2 | 3 rd eluent | 4 th eluent | Column regenerating liquid |
| Na concentration [ N ]] | 0.16 | 0.2 | 0.2 | 1.2 | 0.2 |
| Sodium citrate (2H) 2 O)[g] | 6.19 | 7.74 | 13.31 | 26.67 | - |
| Sodium hydroxide [ g ]] | - | - | - | - | 8.00 |
| Sodium chloride [ g ]] | 5.66 | 7.07 | 3.74 | 54.35 | - |
| Citric acid (H) 2 O)[g] | 19.80 | 22.00 | 12.80 | 6.10 | - |
| Ethanol [ mL ]] | 130.0 | 20.0 | 4.0 | - | 100.0 |
| Benzyl alcohol [ mL ]] | - | - | - | 5.0 | - |
| Thiodiglycol [ mL ]] | 5.0 | 5.0 | 5.0 | - | - |
| 25％BRIJ-35[ml] | 4.0 | 4.0 | 4.0 | 4.0 | 4.0 |
| Total amount [ L ]] | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| Octanoic acid [ mL ]] | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| pH (nominal) | 3.3 | 3.2 | 4.0 | 4.9 | 13.0 |
The measurement conditions of the amino acid analyzer 100 used in the examples and comparative examples are shown in table 3.
[ Table 3]
Measurement method
| Separation column | #2620M 4.6mm I.D.×80mm |
| Ammonia filtering column | #2650L 4.6mm I.D.×60mm |
| Flow rate of eluent | 0.22mL/min |
| Temperature of separation column | 55℃ |
| Reaction reagent | Ninhydrin color development solution kit for Hitachi |
| Flow rate of reaction reagent | 0.30mL/min |
| Reaction temperature | 135℃ |
| Detection wavelength | VIS 440nm，570nm |
| Amount of sample injected | 20μL |
Fig. 2 is a schematic diagram of a time chart on the time program of the amino acid analyzer 100. In fig. 2, (a), (b), and (c) show the time courses of comparative example (conventional method), example 1, and example 2, respectively.
[ comparative example ]
In the analysis step of the conventional method, in order to stabilize the separation column 13, a liquid injection-prior-to-injection step of feeding at least the 1 st eluent 1 (liquid B1) to the separation column 13 is provided prior to the sample injection step of injecting the sample into the flow path by the auto-sampler 12. After the liquid feeding step before injection, the sample injection step is performed, and the process proceeds to the separation step. In the separation step, the mixture ratio is changed from the 1 st eluent (liquid B1) through the 2 nd eluent 2 (liquid B2), then the 3 rd eluent 3 (liquid B3) and the 4 th eluent 4 (liquid B4) based on the gradient elution time program, and is fed to the separation column 13.
That is, in the conventional method, the time program is executed so that the time of starting the gradient elution time program coincides with the time of injecting the sample.
[ example 1]
In example 1 of the present invention, the gradient elution time program is started in the liquid feeding before injection step before the sample injection step. In other words, the 1 st eluent 1 (B1 liquid) to be sent first by executing the gradient elution time program in the conventional method is also used as the 1 st eluent 1 (B1 liquid) to be sent in the injection-prior liquid sending step.
[ example 2]
In example 2 of the present invention, the start time of the gradient elution time program was set earlier than in example 1, and the liquid supply of the 2 nd eluent (liquid B2) was started in the middle of the injection liquid supply step.
In examples 1 and 2, the gradient elution time program was started before the sample injection step, as compared with the comparative example (conventional method), and thereby the analysis time required for 1 process was shortened as shown by symbol a in fig. 2.
Fig. 3 is a chromatogram of a comparative example (conventional method) and examples 1 and 2.
In the chromatograms (b) and (c) of examples 1 and 2 shown in the middle and lower stages, the elution time of Asp (aspartic acid) is earlier than that of the chromatogram (a) of the comparative example (conventional method) shown in the upper stage of fig. 3. In the comparative example (conventional method), the peaks of Glu/Pro (glutamic acid/proline) are close to each other, whereas in examples 1 and 2, the peaks are gradually separated from each other, and it is seen that the Glu/Pro separation degree is further improved. It can be seen from this that the separation performance of the sample components is improved in examples 1 and 2 as compared with the comparative example (conventional method).
In the amino acid analysis further containing a plurality of amino acid components, the decrease in elution time of Asp (aspartic acid) which is the first eluting component leads to a decrease in the overall analysis time. In examples 1 and 2, the elution time of Asp (aspartic acid) is shortened as described above, compared with the comparative example (conventional method). Therefore, in the amino acid analysis further containing a plurality of amino acid components, the analysis time can be further shortened from the above-described aspect.
As described above, according to the present invention, both improvement of separation performance of sample components and reduction of analysis time can be achieved. In addition, since a single separation column is used, it can contribute to miniaturization of the column and reduction in cost.
[ example 3]
From the viewpoint of shortening the analysis time, a method of rapidly transitioning the column to the initial state before sample injection by using a dedicated control solution instead of the eluent for separation may be considered.
The control solution is not used for separation of sample components, unlike the eluent. As the conditioning liquid, pure water such as a low salt concentration and/or low pH aqueous solution, distilled water, or the like can be used. In this case, for example, distilled water 5 is introduced into the separation column after the washing step and before the liquid feeding step.
The present invention is not limited to the above-described embodiments, and includes various modifications. For example, the above-described embodiments are described in detail to facilitate understanding of the present invention, and are not limited to having all of the described configurations. In addition, a part of the configuration of one embodiment may be replaced with the configuration of another embodiment, and the configuration of another embodiment may be added to the configuration of one embodiment. Further, some of the configurations of the embodiments may be added, deleted, or replaced with other configurations.
Description of the symbols
1, B1 st 1 st eluent
2, B2 nd 2 nd eluent
3,B3 No. 3 eluate
4,B4 th 4 th eluent
5. Distilled water (adjustment liquid)
6,B6 column regenerating liquid (column cleaning liquid)
7A,7B,7C,7D,7E,7F electromagnetic valve (liquid feeding part)
8. Ninhydrin reagent
9. Eluent pump (liquid sending part)
10. Ninhydrin pump
11. Ammonia filtering column
12. Automatic sample injector (sample injection part)
13. Separation column
14. Mixing device
15. Reaction column
16. Detector
17. Data processing device (control device)
Claims (10)
1. A method for controlling ion exchange chromatography, the ion exchange chromatography comprising:
a liquid sending unit that sends two or more types of eluents into the flow path, the two or more types of eluents including a 1 st eluent and a 2 nd eluent, the 2 nd eluent being an eluent that starts liquid sending for sample separation after the 1 st eluent;
a sample injection section provided downstream of the liquid sending section and injecting the sample into the eluent in the flow path;
a separation column provided downstream of the sample injection section and configured to separate a sample component in the sample;
a detector for detecting the sample component separated by the separation column; and
a control device for changing the mixing ratio of the eluent and sending the eluent to the liquid sending part based on a specific time program,
the control device executes the specific time program so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample.
2. The method for controlling ion exchange chromatography according to claim 1,
the specific time program includes a gradient elution time program for changing a mixing ratio of the eluent and feeding the eluent to the liquid feeding portion,
the control device executes the gradient elution time program before the injection of the sample so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample.
3. The method for controlling ion exchange chromatography according to claim 2, wherein the analyzing step of ion exchange chromatography comprises:
a sample injection step of injecting the sample into the flow path by the sample injection section;
a liquid feeding step of feeding at least the 1 st eluent before the sample injection step to stabilize the separation column; and
a separation step of separating the sample component of the sample in the separation column after the sample injection step, and executing the gradient elution time program in the middle of the liquid feeding step before injection.
4. The method for controlling ion exchange chromatography according to claim 3, wherein the analyzing step includes:
a washing step of sending a column washing liquid to wash the separation column after the separation step; and
and an adjustment step of feeding, after the washing step, an adjustment liquid that is not used for separating the sample.
5. The method of controlling ion exchange chromatography according to any one of claims 1 to 4, wherein the sample is injected into the flow path in a state where a part of the 1 st eluent in the flow path is replaced with the 2 nd eluent.
6. The method for controlling ion exchange chromatography according to any one of claims 1 to 4,
the eluent comprises a No. 3 eluent which starts to send liquid after the No. 2 eluent,
the 3 rd eluent starts to be delivered after the injection of the sample.
7. The method for controlling ion exchange chromatography according to any one of claims 1 to 4, wherein the ion exchange chromatography is an amino acid analyzer for analyzing an amino acid.
8. An ion exchange chromatography, comprising:
a liquid sending unit that sends two or more types of eluents into the flow path, the two or more types of eluents including a 1 st eluent and a 2 nd eluent, the 2 nd eluent being an eluent that starts liquid sending for sample separation after the 1 st eluent;
a sample injection section provided downstream of the liquid sending section and injecting the sample into the eluent in the flow path;
a separation column provided downstream of the sample injection section and configured to separate a sample component in the sample;
a detector for detecting the sample component separated by the separation column; and
a control device for changing the mixing ratio of the eluent and sending the eluent to the liquid sending part based on a specific time program,
the control device executes the time program so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample.
9. The ion exchange chromatography of claim 8,
the specific time program includes a gradient elution time program for changing a mixing ratio of the eluents and feeding the eluents to the liquid feeding portion,
the control unit executes the gradient elution time program before the injection of the sample so that the liquid feeding of the 2 nd eluent is started simultaneously with or before the injection of the sample.
10. Ion exchange chromatography according to claim 8 or 9, which is an amino acid analyzer for analyzing amino acids.
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| JP2021125668A JP2023020352A (en) | 2021-07-30 | 2021-07-30 | Method for controlling ion exchange chromatograph and ion exchange chromatograph |
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