JP7740928B2 - Method for controlling an ion exchange chromatograph and an ion exchange chromatograph - Google Patents

Description

This disclosure relates to a method for controlling an ion exchange chromatograph and an ion exchange chromatograph.

Ion exchange chromatography is a well-known method for analyzing sample components in a sample. Ion exchange chromatography utilizes interactions that occur between sample components and two phases: a stationary phase made of an ion exchanger such as an ion exchange resin, and a mobile phase made of a buffer solution, to separate and analyze sample components with different properties. Ion exchangers have chemically modified ion exchange groups, and include cation exchangers such as sulfonic acid and carboxylic acid, and anion exchangers such as quaternary ammonium and tertiary ammonium.

Examples of samples that can be analyzed using ion exchange chromatography include amino acids.

In simultaneous amino acid analysis, which analyzes a wide variety of amino acids, the amino acids and amino acid analogs analyzed can be broadly divided into approximately 20 types of protein hydrolyzed amino acids and over 40 types of biological fluid amino acids and amino acid analogs. These diverse 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.

In this technique, analytical methods have been developed with the goal of improving separation and shortening analysis time by devising the type of ion exchange resin packed into the separation column, the buffer composition, the flow rate, etc. Patent Document 1 is an example of this.

Patent Document 1 describes a technology that states, "In simultaneous analysis of multiple amino acids, multiple columns using packing materials with different chemical properties are arranged in series, enabling separation that takes advantage of the high-resolution areas of each packing material, thereby improving separation performance and speeding up analysis" (see abstract).

Japanese Patent Application Laid-Open No. 2008-249447

However, while the technology in Patent Document 1 can improve separation performance and speed up analysis, it requires arranging multiple separation columns in series using packing materials with different chemical properties, leaving room for improvement in terms of making the columns more compact and reducing costs.

In addition, simultaneous amino acid analysis, for example, takes a long time to analyze, so there is a demand for further shortening the analysis time.

This disclosure was made in consideration of the technical challenges described above, and provides an ion exchange chromatograph and a control method for an ion exchange chromatograph that can simultaneously improve the separation performance of sample components and shorten the analysis time, even when using a single separation column from the perspective of column compactness and cost reduction.

The ion exchange chromatograph and control method thereof disclosed herein include an ion exchange chromatograph and control method comprising: a liquid delivery unit that delivers two or more eluent solutions, including a first eluent and a second eluent that begins delivery after the first eluent for sample separation, to a flow path; a sample injection unit that is located downstream of the liquid delivery unit and delivers the sample into the eluent solution in the flow path; a separation column that is located downstream of the sample injection unit and delivers sample components in the sample; a detector that detects the sample components separated by the separation column; and a control device that changes the mixing ratio of the eluent solutions and delivers the eluent solution to the liquid delivery unit based on a predetermined time program, wherein the control device executes the predetermined time program to start delivery of the second eluent simultaneously with or before injection of the sample.

This disclosure provides an ion exchange chromatograph control method and an ion exchange chromatograph that can simultaneously improve the separation performance of sample components and shorten the analysis time, even when using a single separation column in order to make the column more compact and reduce costs.

Other issues, configurations, and advantages will become clearer from the following description of the embodiments and examples.

1 is a schematic diagram of the device configuration and flow paths of an amino acid analyzer 100. FIG. 1 is a schematic diagram of a timetable on a time program that controls the operation of an amino acid analyzer 100. FIG. Chromatograms of Examples and Comparative Examples.

Embodiments of the present disclosure are described below. The following description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the present disclosure, its applications, or its uses.

<Ion exchange chromatography>
The ion exchange chromatograph according to the present disclosure can be generally used as an analyzer for samples that can be analyzed by ion exchange chromatography. Specific examples of the ion exchange chromatograph include an amino acid analyzer used for analyzing the amino acid composition of proteins and peptides, amino acids and their related substances in pharmaceuticals, biological fluids, etc., and an HPLC system used for analyzing proteins, amines, organic acids, etc., and is preferably an amino acid analyzer.

The ion exchange chromatograph disclosed herein analyzes sample components using gradient elution. Note that, in this specification, the term "gradient" is used to encompass stepwise gradients, curved gradients, and linear gradients.

The ion exchange chromatograph according to the present disclosure essentially comprises a liquid delivery unit, a sample injection unit, a separation unit, a detector, and a control device. In addition to these components, the ion exchange chromatograph may also comprise any other components. Specifically, for example, to facilitate the detection of sample components, it may also comprise devices such as pre-column or post-column derivatization reagents, pumps, mixers, and reaction columns.

The liquid delivery unit delivers two or more types of eluent, including a first eluent and a second eluent, which begins to be delivered after the first eluent, to the flow path. In addition to the eluent, the liquid delivery unit may also be capable of delivering column wash liquid, adjustment liquid not used in sample separation, etc.

The eluent is a buffer solution such as sodium citrate buffer or lithium citrate buffer, which is introduced into the separation column in the pre-injection liquid transfer process and separation process described below. Liquids commonly used in ion exchange chromatography can be used as the eluent, column wash solution, and conditioning solution.

Of the two or more eluents, the first eluent is the first eluent introduced into the separation column, and is introduced into the separation column at least during the pre-injection liquid transfer step. Therefore, the first eluent not only serves as an eluent that contributes to the separation of sample components, but also serves to stabilize the separation column. On the other hand, the second eluent contributes more to the separation of sample components than the first eluent, and does not necessarily have to stabilize the separation column.

The salt concentration of the first eluent is preferably lower than the salt concentration of the second eluent. This effectively stabilizes the separation column, thereby improving the separation performance of sample components using the remaining eluent, including the second eluent. In this specification, "salt concentration" refers to the concentration of cations or anions contained in the eluent. Specifically, the salt concentration of the first eluent is preferably 0.05N or more and less than 0.2N, and more preferably 0.12N or more and 0.19N or less. The salt concentration of the second eluent is preferably more than 0.16N and less than 1.2N, and more preferably 0.2N or more and 1N or less.

Furthermore, the difference in pH between the first eluent and the second eluent is preferably within 0.5, and more preferably within 0.3. When the stationary phase of the separation column described below is a cation exchange resin, it is more preferable that the pH of the first eluent be higher than the pH of the second eluent. This effectively stabilizes the separation column. This in turn improves the separation performance of the sample components using the remaining eluent, including the second eluent.

When the stationary phase of the separation column described below is a cation exchanger, the pH of the first and second eluents is preferably around 3, specifically 2.5 or higher and 3.5 or lower. In this case, when the eluent contains three or more types of eluents, the pH of the third eluent, which begins to be pumped after the second eluent, is preferably higher than the pH of the first and second eluents.

Specific examples of the liquid delivery unit include containers for storing various liquids, such as eluent, column wash liquid, and adjustment liquid; solenoid valves for starting and stopping delivery of each liquid in the containers to the flow path or adjusting the flow rate of each liquid; and pumps for adjusting the flow rate of each liquid. The solenoid valves can be provided on the flow paths corresponding to each container. The pumps can be provided on the flow paths downstream of the solenoid valves. Specifically, for example, the flow paths corresponding to each container can merge downstream of the solenoid valves to form a single flow path, and a single pump can be provided on that flow path (low-pressure gradient elution). Alternatively, multiple pumps can be provided on the flow paths corresponding to each container (high-pressure gradient elution).

The sample injection unit is located downstream of the liquid delivery unit and is a means for injecting a sample into the eluent in the flow path. The sample injection unit may be a manual injector or an automatic autosampler, but an autosampler is preferred from the perspective of accurately controlling the injection timing and injection amount of the sample.

The separation section is located downstream of the sample injection section and includes a separation column for separating sample components in the sample. In addition to the separation column, the separation section may also include a means for adjusting the temperature of the separation column. The separation column is not particularly limited, and columns commonly used in ion exchange chromatography can 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, but a cation exchanger is preferred, and a cation exchange resin is more preferred.

The detector is a device installed downstream of the separation column to detect the sample components separated by the separation column. There are no particular limitations on the detector, and detectors commonly used in ion exchange chromatography, such as electrical conductivity detectors, ultraviolet-visible absorbance detectors, fluorescence detectors, and electrochemical detectors, can be used.

The control device is electrically or online connected to at least the solenoid valves and pumps of the liquid delivery unit, the sample injection unit in the case of an autosampler, and, if present, the means for adjusting the temperature of containers in the liquid delivery unit and each column, and sends control signals to these to control their operation. The control device is also electrically or online connected to the detector, and acquires the detector's detection results and outputs them as chromatograms and data. The control device is, for example, a well-known microcomputer-based device, and includes an input unit for inputting information from outside, a memory 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.

The control device's memory stores a time program for executing the analysis process described below. Based on a predetermined gradient elution time program included in the time program, the control device changes the mixing ratio of two or more eluents and sends the eluents to the eluent delivery unit.

<Ion exchange chromatography analysis process>
The analysis process of an ion exchange chromatograph comprises a sample injection process, a separation process, a cleaning process, an optional adjustment process, and a pre-injection liquid transfer process, and these processes are treated as one method and are repeated a number of times set by the user.

The sample injection process is a process in which a sample is injected into the flow path by the sample injection unit. Although not intended to be limiting, the time program can be created with the sample injection process as time zero.

The separation process is a process that follows the sample injection process, in which the sample components are separated in the separation column. At least in the separation process, the eluent is delivered based on the gradient elution time program in the time program.

Once the separation process is complete, the separation column must be returned to its initial state, i.e., the state before sample injection, and stabilized for the next method. The post-separation washing process, optional adjustment process, and pre-injection liquid delivery process are provided for this purpose.

First, in the washing process, column washing solution is pumped through the separation column to wash it.

Then, in the pre-injection liquid delivery step, at least the first eluent is delivered to stabilize the separation column before the sample injection step of the next method.

In addition, an adjustment step may be performed after the cleaning step and before the pre-injection liquid delivery step. In the adjustment step, an adjustment liquid that will not be used in sample separation is delivered. This allows the separation column to quickly return to its initial state. Examples of adjustment liquids that can be used include a low-salt aqueous solution, a low-pH aqueous solution if the stationary phase of the separation column is a cation exchanger, a high-pH aqueous solution if the stationary phase of the separation column is an anion exchange resin, and pure water.

<Method of controlling ion exchange chromatography>
The ion exchange chromatograph of the present disclosure is characterized by the following:

The control device executes a time program to start the delivery of the second eluent simultaneously with or before the injection of the sample.

In this embodiment, the time program includes time tables corresponding to the sample injection process, separation process, washing process, optional adjustment process, and pre-injection liquid delivery process. The time program also includes a gradient elution time program that changes the mixing ratio of the eluent and delivers the eluent to the liquid delivery section.

For example, a gradient elution time program can be configured to start the delivery of the first eluent first and the delivery of the second eluent second.

In this case, the control device may execute a gradient elution time program prior to the injection of the sample, preferably during the pre-injection liquid delivery process, so that delivery of the second eluent begins simultaneously with or before the injection of the sample.

With this configuration, the gradient elution time program begins execution prior to the sample injection process, thereby shortening the analysis time.

Furthermore, as explained below, this configuration can improve the separation performance of the separation column.

Even when the solenoid valves are opened and the eluents begin to flow from the containers holding each eluent, there is a time lag corresponding to the volume of the flow path from the container to the separation column before the eluent reaches the separation column. Furthermore, there is a time lag corresponding to the volume of the flow path from the sample injection unit to the separation column after the sample is injected into the sample injection unit before the sample reaches the separation column. This results in a difference between the time the sample reaches the separation column and the time each eluent reaches the separation column.

When the flow of the second eluent begins, a portion of the first eluent in the flow path from the junction of the flow paths corresponding to the containers for the first and second eluent to the separation column is eventually replaced with the second eluent. Then, depending on the timing of the start of the flow of the second eluent, the sample will be injected into the first eluent, a mixture of the first and second eluent, or the second eluent.

When the sample is injected into a mixture of the first and second eluents, or into the second eluent, the second eluent reaches the separation column simultaneously with or before the sample reaches the separation column. This allows the sample components to begin developing in the mobile phase, partially or completely replaced by the second eluent, simultaneously with the sample's arrival at the separation column, thereby improving the separation performance of the sample components.

In addition, even when the sample is injected into the first eluent, the flow of the second eluent begins simultaneously with or before the injection of the sample, so the second eluent reaches the separation column in a shorter time than before after the sample reaches the separation column. As a result, after the sample reaches the separation column, the development of the sample components begins quickly using the mobile phase, some or all of which has been replaced with the second eluent, thereby improving the separation performance of the sample components.

In this way, the present disclosure enables both improved separation performance of sample components and shorter analysis times. Furthermore, the use of a single separation column contributes to more compact columns and reduced costs.

For example, the gradient elution time program may be configured to first start the delivery of the second eluent, and then start the delivery of other eluents, specifically, for example, the third eluent if there are three or more types of eluents.

In this case, the control device can execute a gradient elution time program simultaneously with or prior to sample injection so that the delivery of the second eluent begins simultaneously with or before sample injection. This allows for both improved separation performance of sample components and shorter analysis times.

When three or more types of eluents are used, the delivery of the third eluent, which begins after the second eluent, may begin before the sample injection step or simultaneously with sample injection. It is more preferable to begin delivery of the third eluent after the sample injection step. This ensures high separation performance of sample components using the multiple eluents that begin delivery after the second eluent.

The following describes an amino acid analyzer 100 (ion exchange chromatograph) and its control method according to an embodiment of the present disclosure, using the drawings.

In the following explanation, the names and abbreviations of the amino acids to be analyzed follow the notation in Table 1.

Figure 1 is a schematic diagram of the device configuration and flow paths of an amino acid analyzer 100 according to an embodiment of the present disclosure.

The amino acid analyzer 100 can be equipped with first eluent 1 to fourth eluent 4, distilled water 5, and column regenerant 6. One of these liquids is selected by solenoid valves 7A to 7F and delivered by eluent pump 9. The eluent passes through an ammonia filter column 11 and then enters separation column 13. An autosampler 12 (sample injection section) is provided downstream of the ammonia filter column 11 and upstream of the separation column 13, and the autosampler 12 injects the amino acid sample into the eluent in the flow path. The injected amino acid sample reaches separation column 13 along with the eluent and is separated in separation column 13.

The amino acid analyzer 100 also includes a ninhydrin reagent 8 and a ninhydrin pump 10 for delivering the ninhydrin reagent 8. The amino acid components separated in the separation column 13 are mixed in a mixer 14 with the ninhydrin reagent 8 delivered by the ninhydrin pump 10, and reacted in a heated reaction column 15.

The amino acids colored by the reaction (Ruhemann purple) are continuously detected by the detector 16, and the data is output as a chromatogram and data by the data processing device 17 (control device), which are then recorded and stored.

The data processing device 17 controls the solenoid valves 7A-7D for each eluent container, the solenoid valve 7E for the distilled water container, the solenoid valve 7F for the column regenerant container, the eluent pump 9, the ninhydrin pump 10, the autosampler 12, the temperature adjustment means (not shown) for each liquid container, the separation column 13, and the reaction column 15. This control is primarily performed by a program stored in the memory unit (not shown) of the data processing device 17.

The sodium citrate buffer solutions shown in Table 2 were used as the first eluent 1 to the fourth eluent 4 and the column regenerant 6. Note that in Table 2, salt concentrations are shown as Na concentrations.

The measurement conditions for the amino acid analyzer 100 used in the examples and comparative examples are as shown in Table 3.

Figure 2 is a schematic diagram of the timetable on the time program of the amino acid analyzer 100. (a), (b), and (c) in Figure 2 show the time programs for the Comparative Example (conventional method), Example 1, and Example 2, respectively.

[Comparative Example]
In the analysis process of the conventional method, a pre-injection liquid delivery step is provided in which at least a first eluent 1 (liquid B1) is delivered to the separation column 13 in order to stabilize the separation column 13 before a sample injection step into the flow path by the autosampler 12. After the pre-injection liquid delivery step, the process proceeds to a sample injection step and then to a separation step. In the separation step, the first eluent (liquid B1), second eluent 2 (liquid B2), third eluent 3 (liquid B3), and fourth eluent 4 (liquid B4) are delivered to the separation column 13 at varying mixing ratios based on a gradient elution time program.

In other words, in conventional methods, the start of the gradient elution time program is executed to coincide with the time of sample injection.

[Example 1]
In Example 1 according to the present disclosure, the gradient elution time program is started in advance in the pre-injection liquid delivery step prior to the sample injection step. In other words, the first eluent 1 (liquid B1) that is first delivered by executing the gradient elution time program in the conventional method also serves as the first eluent 1 (liquid B1) delivered in the pre-injection liquid delivery step.

[Example 2]
In Example 2 of the present disclosure, the start time of the gradient elution time program is made earlier than in Example 1, and the delivery of the second eluent (liquid B2) is started in the middle of the pre-injection delivery process.

Compared to the comparative example (conventional method), in Examples 1 and 2, the gradient elution time program is started prior to the sample injection step, thereby shortening the analysis time required for one method, as shown by symbol A in Figure 2.

Figure 3 shows chromatograms for the comparative example (conventional method) and Examples 1 and 2.

Compared to the chromatogram (a) of the Comparative Example (conventional method) shown in the top row of Figure 3, the chromatograms (b) and (c) of Examples 1 and 2 shown in the middle and bottom rows show an earlier elution time for Asp (aspartic acid). Furthermore, while the Glu/Pro (glutamic acid/proline) peaks are close together in the Comparative Example (conventional method), the peaks are gradually separated in Examples 1 and 2, demonstrating improved Glu/Pro resolution. Thus, it can be seen that the separation performance of sample components is improved in Examples 1 and 2 compared to the Comparative Example (conventional method).

Furthermore, in amino acid analysis involving a wider variety of amino acid components, shortening the elution time of Asp (aspartic acid), the first eluting component, leads to a reduction in the overall analysis time. Compared to the comparative example (conventional method), in Examples 1 and 2, the elution time of Asp (aspartic acid) is shortened as described above. Therefore, in amino acid analysis involving a wider variety of amino acid components, it is possible to further shorten the analysis time from the above perspective as well.

As described above, the present disclosure can improve the separation performance of sample components while shortening the analysis time. Furthermore, the use of a single separation column contributes to a more compact column and cost reduction.
[Example 3]
From the viewpoint of shortening the analysis time, a method of quickly restoring the column to the initial state before sample injection by using a dedicated conditioning solution instead of an eluent for separation may be considered.

Unlike the eluent, the conditioning liquid is not used to separate the sample components. The conditioning liquid can be a low-salt and/or low-pH aqueous solution, or pure water such as distilled water. In this case, for example, distilled water 5 is introduced into the separation column after the washing step and before the pre-injection liquid transfer step.

Note that the present disclosure is not limited to the above-described examples and includes various modifications. For example, the above-described examples have been described in detail to clearly explain the present disclosure, and are not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one example with the configuration of another example, or to add the configuration of another example to the configuration of one example. Furthermore, it is possible to add, delete, or replace part of the configuration of each example with other configurations.

1, B1 First eluent 2, B2 Second eluent 3, B3 Third eluent 4, B4 Fourth eluent 5 Distilled water (adjusted solution)
6. B6 Column regeneration solution (column washing solution)
7A, 7B, 7C, 7D, 7E, 7F Solenoid valve (liquid feeding part)
8 Ninhydrin reagent 9 Eluent pump (liquid delivery section)
10 Ninhydrin pump 11 Ammonia filter column 12 Autosampler (sample injection section)
13 Separation column 14 Mixer 15 Reaction column 16 Detector 17 Data processing device (control device)

Claims (6)

a liquid delivery unit that delivers two or more types of eluents, including a first eluent and a second eluent that starts to be delivered after the first eluent for sample separation, to the flow path;
a sample injection unit provided downstream of the liquid delivery unit and configured to inject the sample into the eluent in the flow channel;
a separation column provided downstream of the sample injection section for separating sample components in the sample;
a detector for detecting sample components separated by the separation column;
a control device that causes the eluent to be delivered to the delivery unit based on a predetermined time program;
A method for controlling an ion exchange chromatograph comprising:
The ion exchange chromatographic analysis step includes:
a sample injection step in which the sample is injected into the flow channel by the sample injection unit;
a pre-injection liquid delivery step of delivering at least the first eluent to stabilize the separation column before the sample injection step;
a separation step of separating sample components of the sample in the separation column after the sample injection step;
a washing step of washing the separation column by supplying a column washing solution after the separation step;
and an adjusting step of feeding an adjusting solution that is not used for separating the sample after the washing step,
the predetermined time program includes a stepwise gradient elution time program for changing the mixing ratio of the eluent and delivering the eluent to the eluent delivery unit,
A method for controlling an ion exchange chromatograph, in which the control device executes the stepwise gradient elution time program prior to the injection of the sample during the pre-injection liquid delivery process so as to start the delivery of the second eluent simultaneously with or before the injection of the sample. 2. A method for controlling an ion exchange chromatograph according to claim 1 , comprising:
A method for controlling an ion exchange chromatograph, wherein the sample is injected into the flow path in a state where a portion of the first eluent in the flow path has been replaced with the second eluent. 3. A method for controlling an ion exchange chromatograph according to claim 1 or 2 , comprising:
the eluent includes a third eluent that is started to be delivered after the second eluent,
A method for controlling an ion exchange chromatograph, wherein the third eluent begins to be delivered after the sample is injected. A method for controlling an ion exchange chromatograph according to any one of claims 1 to 3 , comprising:
The ion exchange chromatograph is an amino acid analyzer for analyzing amino acids. a liquid delivery unit that delivers two or more types of eluents, including a first eluent and a second eluent that starts to be delivered after the first eluent for sample separation, to the flow path;
a sample injection unit provided downstream of the liquid delivery unit and configured to inject the sample into the eluent in the flow channel;
a separation column provided downstream of the sample injection section for separating sample components in the sample;
a detector for detecting sample components separated by the separation column;
a control device that causes the eluent to be delivered to the delivery unit based on a predetermined time program;
An ion exchange chromatograph comprising:
The ion exchange chromatographic analysis step includes:
a sample injection step in which the sample is injected into the flow channel by the sample injection unit;
a pre-injection liquid delivery step of delivering at least the first eluent to stabilize the separation column before the sample injection step;
a separation step of separating sample components of the sample in the separation column after the sample injection step;
a washing step of washing the separation column by supplying a column washing solution after the separation step;
and an adjusting step of feeding an adjusting solution that is not used for separating the sample after the washing step,
the predetermined time program includes a stepwise gradient elution time program for changing the mixing ratio of the eluent and delivering the eluent to the eluent delivery unit,
the predetermined time program includes time tables corresponding to the sample injection step, the separation step, the cleaning step, the adjustment step, and the pre-injection liquid delivery step,
The control device executes the stepwise gradient elution time program prior to the injection of the sample during the pre-injection liquid delivery process so as to start the delivery of the second eluent simultaneously with or before the injection of the sample. 6. The ion exchange chromatograph of claim 5 ,
An ion exchange chromatograph is an amino acid analyzer used to analyze amino acids.