Liquid chromatograph and method for controlling a liquid chromatograph

Abstract

To speed up measurement by liquid chromatography.SOLUTION: A control unit 121 of a liquid chromatograph is configured to inject a standard sample and measure the same, and measure the column pressure, adjust the setting of the mixing ratio of the eluent and the change timing of the mixing ratio based on the measurement result of the standard sample, and control to adjust the setting of the flow velocity of the eluent according to the column pressure measured above.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a liquid chromatograph using a gradient elution method.

Background Art

[0002] In chromatography, there is known a technique in which a constant velocity gradient program is converted into a constant pressure gradient program in order to shorten the measurement time, and the liquid feed pump is controlled according to the converted constant pressure gradient program (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, it has been found that when using a constant pressure gradient program converted from a constant velocity gradient program as described above, the measurement content is likely to be affected by various measurement conditions, and thus an appropriate retention time may not be obtained.

[0005] In view of the above points, an object of the present invention is to achieve high-speed measurement by a liquid chromatograph and easily obtain an appropriate retention time.

Means for Solving the Problems

[0006] To achieve the above object, the present invention A control method for a liquid chromatograph that performs measurements by supplying an eluent, The process involves injecting a standard sample and performing measurements, as well as measuring the column pressure. The process involves adjusting the column temperature and the timing of changes in the column temperature based on the measurement results using the above standard sample, The process involves adjusting the flow rate of the eluent according to the measured column pressure, It has, The measurement results using the above standard sample are characterized by being a determination of whether an appropriate retention time or degree of separation has been obtained.

[0007] Also, It is a liquid chromatograph, A pump for delivering the eluent, A sample injection unit for injecting the sample into the eluent delivered from the above-mentioned pump, The sample injection unit above supplies the eluent containing the sample, and a separation column separates the target component in the sample. A detector for analyzing the target component separated by the above separation column, A control unit controls the measurement operation of the pump, the sample injection unit, the separation column, and the detector, and processes the data. Equipped with, The control unit is characterized by performing flow rate adjustment, which adjusts the flow rate of the eluent to satisfy a predetermined upper pressure limit of the eluent according to the temperature of the separation column and the timing of changes in the temperature of the separation column, and measurement control, which performs a measurement using a standard sample when the flow rate adjustment is performed and determines whether an appropriate retention time or separation degree has been obtained based on the measurement using the standard sample.

[0008] Thereby, Column temperature and column temperature while the change timing of these is appropriately maintained, the flow rate of the eluent according to these is controlled, so that high-speed analysis satisfying a predetermined upper limit pressure can be enabled while easily maintaining the retention time.

Advantages of the Invention

[0009] According to the present invention, it is possible to easily maintain an appropriate retention time or degree of separation while achieving high-speed measurement by liquid chromatography. The degree of separation is an index for quantifying the degree of separation between two peaks, whether good or bad. The meaning of appropriately expressing the retention time in this specification refers to obtaining a good degree of separation.

Brief Description of the Drawings

[0010] [Figure 1] It is a schematic configuration diagram of a liquid chromatography system. [Figure 2] It is an explanatory diagram showing an example of a gradient elution program generation process. [Figure 3] It is an explanatory diagram showing an example of a gradient elution program.

Modes for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described in detail based on the drawings.

[0012] (Schematic Configuration of Chromatograph) As shown in FIG. 1, for example, a liquid chromatograph includes a plurality of eluents 101…, and one or more of them are selected and sent by a liquid delivery pump 102 to an autosampler 103 (sample injection unit) so that a sample can be injected. The injected sample is separated into target components by a separation column (not shown) installed in a column thermostat 104 for maintaining a constant temperature.

[0013] The components separated by the separation column are detected by a detector 108, and thus analysis of the target analysis species is performed. In the case of the post-column derivatization method, the components separated by the separation column are mixed with a derivatization reagent 105 sent by a liquid delivery pump 106, react and develop color when passing through a reaction device 107, and are detected by a detector 108, so that analysis of analysis species such as amino acids is performed. In the case of amino acid analysis, for example, ninhydrin is used as the derivatization reagent.

[0014] The control unit 121 includes a measurement operation control unit 121a, a calculation unit 121b, a storage unit 121c, a processing unit 121d, and a display unit 121e, and is configured to control the operations of each part of the liquid chromatograph and process the detection results. Also, although not particularly limited, for example, it is configured to generate (function as a generation device) a program for an analysis operation (gradient elution program of an eluent) and control the operations of each part based on this. More specifically, for example, the measurement operation control unit 121a is configured to set and control the hardware of each part of the liquid chromatograph according to a method. The calculation unit 121b is configured to perform calculation processing of a pressure profile by a theoretical formula (flow rate, viscosity as a function of temperature, system delay volume), retention prediction of a standard sample, and closed-loop calculation thereof. The storage unit 121c is configured to store past pressure profiles, standard sample chromatograms, time programs, etc. The processing unit 121d is configured to perform waveform processing of the chromatograms of the obtained standard and unknown samples. Also, various information is displayed on the display unit 121e of the control unit 121.

[0015] (Explanation of terms) This section explains the main meanings of terms used in this specification. Note that this explanation is primarily for the purpose of facilitating understanding and is not necessarily intended to be strictly limited.

[0016] "Gradient elution" is a common operation in which solute is eluted while continuously changing the composition of the mobile phase (eluent 101).

[0017] "Temperature gradient elution" is an operation in which the column temperature is changed using a time program. Basically, it follows van't Hoof's equation, and the retention time changes with temperature.

[0018] "Flow gradient elution" is an operation in which the flow rate is changed using a time program. When the flow rate changes, ideally the retention volume does not change, but the retention time does.

[0019] "Temperature-flow gradient elution" is an operation that comprehensively changes not only the mobile phase composition but also the column temperature and flow rate using a time program. However, in the context of this invention, it is not essential that all three variables change; the term "temperature-flow gradient elution" can also be used in a broader sense if even one of the variables—mobile phase composition, temperature, or flow rate—changes over time.

[0020] (Example of creating a time program 1) The following describes an example in which a time program is generated while the control unit 121 controls the operation of the chromatograph, with reference to Figure 2. Note that the following operations are not necessarily all performed by the control unit 121; the time program may be read from an external source, or some parts may be set manually.

[0021] (S101) First, a time program for switching the mobile phase (eluent 101) is set in the instrument according to the separation state of each target analyte. That is, a so-called general gradient elution program is set. This may be a stepwise elution or a gradient elution. In addition, this time program may include control of the column temperature according to the separation state of each target analyte, so that temperature gradient elution can be performed.

[0022] (S102) Based on the above time program, the measurement operation is performed. In this measurement operation, a standard sample is injected and the column pressure is measured.

[0023] (S103) A chromatogram is generated using the injected standard sample.

[0024] (S104) Based on the above chromatogram, it is determined whether an appropriate retention time or resolution has been obtained.

[0025] (S105) If the retention time is not appropriate, the time program is adjusted and set in the instrument so that the mixing ratio of the eluent (timing of changes in the mixing ratio) and the column temperature are adjusted. These adjustments may be performed manually or automatically using a predetermined algorithm based on a knowledge database or the like.

[0026] Based on the time program adjusted as described above, the processes from (S102) onward are repeated.

[0027] (S106) If the retention time in (S104) above is deemed appropriate, it is determined whether the column pressure measured at the same time as the measurement using the standard sample is appropriate, that is, whether the column pressure does not exceed the upper limit pressure and is within a predetermined pressure range that is higher than a predetermined lower limit pressure set to speed up the measurement. Here, the upper limit pressure of the column pressure is preferably set with a certain margin, such as 90% or 80%, relative to the actual upper limit pressure of the column installed in the column thermostat 104 or the pump 102, etc. This is to take into account that the flow resistance of the column will vary to some extent. Also, there may usually be a period of time when the flow rate is constant. Once it is determined that the column pressure is appropriate, the time program is saved or output, and thereafter the measurement operation for the actual analysis is performed.

[0028] (S107) If it is determined in (S106) above that the column pressure is not appropriate, the time program is adjusted and set in the apparatus so that the flow rate of the eluent is adjusted. That is, for example, if it is lower than the lower limit pressure, the measurement time can be shortened by increasing the flow rate of the eluent to approach the upper limit pressure. Here, the flow rate can be specifically determined as follows, for example, with the upper limit pressure when the eluent 101 is delivered to the separation column being ΔPmax (Pa).

[0029] In other words, the calculation unit 121b determines a flow velocity program that shows the time change of the flow velocity, more precisely the linear velocity u0 (mm / s), of the eluent 101. Specifically, based on Kozeny Carman's equation, ΔP=(η·u0·L) / Kv, It can be calculated using the formula u0 = (ΔPmax·Kv) / (L·η).

[0030] Here, Kv(m^2) is column permeability. L(mm) is the column length. η(Pa·s) represents the viscosity of the mixture as the mixing ratio of the eluent 101 changes over time. Alternatively, the flow rate may be determined using the time-varying viscosity based on the temperature program of the separation column (eluent 101). That is, for example, when performing gradient elution using an aqueous solution and an organic solvent as eluents, the viscosities of each liquid differ, and their viscosity is temperature-dependent. Therefore, the flow rate u0 is determined based on the change in pressure value due to the mixing ratio and temperature during measurement.

[0031] Here, various converted or equivalent values ​​may be used, such as the flow rate per unit time F = Seff·u0 instead of the flow velocity u0. The same applies to other values. Here, Seff is the effective cross-sectional area through which the liquid can pass.

[0032] Furthermore, the flow velocity calculated as described above may be corrected based on the actually measured column pressure and the flow velocity at that time.

[0033] Note that the actual pressure profile has a certain delay time from the time program. Regarding mobile phase switching, the internal volume from the mobile phase mixing point to the column is called the system delay volume or due volume. The flow rate is switched with a delay equal to the due volume.

[0034] Furthermore, there is a delay time associated with switching column temperatures. This is a phenomenon similar to the transient response when heat is conducted from a Peltier element to the column, for example. On the other hand, in the case of changes in flow rate, unlike the previous two variables (mobile phase composition and column temperature), the pressure usually responds almost immediately, so the delay time can be ignored.

[0035] (S108) Based on the time program adjusted as described above, the measurement operation is performed while the standard sample is introduced and the column pressure is measured, as in (S102) above.

[0036] (S109) The column pressure in the above measurement operation is determined to be appropriate in the same way as in (S106). If it is determined to be inappropriate, the process from (S107) onwards is repeated to adjust the flow rate of the eluent.

[0037] (S110) If the column pressure is determined to be appropriate in (S109) above, a chromatogram is generated using the standard sample injected in the measurement operation in (S108).

[0038] (S111) Based on the chromatogram above, it is determined whether an appropriate retention time has been obtained, similar to (S104) above. If appropriate, the time program is saved or output, and the measurement operation for the actual analysis is then performed.

[0039] (S112) On the other hand, if the retention time is not appropriate, the time program is adjusted and set in the instrument to adjust the mixing ratio of the eluent (timing of changes in the mixing ratio) and the column temperature, as in (S105) above, and the process from (S102) onwards is repeated. In other words, if the retention time becomes inappropriate due to the adjustment of the flow rate of the eluent, it is adjusted again, and the measurement operation with the standard sample is repeated, so that the retention time is maintained appropriately while speeding up the measurement.

[0040] (Example 2 of generating a time program) In addition to switching the eluent 101 as described above, a time program may be generated that synchronizes the flow rate of the derivatization reagent 105 used in the case of amino acid analysis.

[0041] In other words, for example, amino acid analysis is performed by mixing each amino acid separated in the separation column with the derivatization reagent 105, and then reacting and producing a color change as it passes through the reaction apparatus 107, which is then detected by the detector 108. Since the number of ninhydrin molecules in the derivatization reagent is in excess of the number of amino acid molecules of the analyte, even if the number of ninhydrin molecules being reacted fluctuates somewhat, in principle, the number of molecules of the reaction product, Rue Hemmans Purple, does not change, and therefore the peak height does not change.

[0042] However, as mentioned above, if the flow rate and mixing ratio change moment by moment due to the flow rate program and mixing ratio program, the absorbance and fluorescence intensity of the mixture itself, rather than the reaction products, will be affected, causing fluctuations in the detection results of absorbance detection and fluorescence detection. This can lead to fluctuations in the baseline.

[0043] On the other hand, the absorbance of the mixture also varies depending on the amount of derivatization reagent 105. For example, if the flow rate of derivatization reagent 105 decreases, the absorbance will decrease, and if the flow rate of derivatization reagent 105 increases, the absorbance will increase.

[0044] Therefore, within an acceptable range for the optimal mixing amount of the derivatization reagent 105, the flow rate of the derivatization reagent 105 can be controlled according to at least one of the flow rate of the eluent 101 and the mixing ratio. More specifically, by, for example, coordinating the flow rates of the liquid delivery pumps 102 and 106 so that the flow rate ratio remains constant, changes in the absorbance of the mixture can be offset, and the baseline of the detector 108 can be stabilized.

[0045] (Example of a generated time program) An example of a time program generated in the manner described above is explained with reference to Figure 3.

[0046] In Figure 3, the mobile phases B1 and B2 are switched using a stepwise elution method, with a time difference of 0.1 minutes. Specifically, B1 is 100% until 20.0 minutes, and then from 20.1 minutes onwards, it is switched in a stepwise manner to 50% B1 and 50% B2.

[0047] The flow rate of the liquid delivery pump 102 is denoted as Flow1. Here, considering the system delay volume, even if the mobile phase composition is switched to 20.0 min, the flow rate is delayed until it switches to 35.0 min. Here, a flow rate linear gradient elution is performed from 35.0 min to 37.0 min over a period of 2 minutes. An asterisk (*) is used to indicate that this is a gradient linear gradient elution. Similarly, the stepwise mobile phase composition change from 40.0 to 40.1 min is reflected in the flow rate linear gradient elution from 60.0 min to 62.0 min. An asterisk (*) is also used here.

[0048] The temperature in the time program is switched in a stepwise manner. It starts at 40°C with a flow rate of 0.5 mL / min at the beginning of 0.0 min, and switches to 30°C at 2.0 min. However, the actual temperature exhibits a temperature profile similar to a transient response. Therefore, in order to make the flow rate change follow this temperature change, the program is cut off at 5.0 min and the flow rate is reduced linearly in a gradient manner towards 0.45 mL / min at 7.0 min. This is why an asterisk (*) is added to indicate a gradient. Here, the viscosity of the eluent increases as the column temperature decreases, so the flow rate is kept high to prevent the pressure from rising. The column temperature at 50°C at 20.0 min is also switched in a stepwise manner, but no particular flow rate change is set for this. Such cases are also acceptable.

[0049] This invention requires the conversion of the analytical method based on a constant pressure, that is, approximately constant pressure. This is a method transfer for so-called constant pressure. The indicator that is generally preserved before and after this conversion, especially in the variable flow velocity region, is the retained volume rather than the retaining time. For example, the volume can be calculated by integrating the constantly changing flow velocity along the time axis. If the origin of the retaining time and the origin of the retained volume are synchronized, the retained volume will also change as an integral value moment by moment.

[0050] The challenge here is that, before the conversion, the mixing ratio and column temperature switching time for gradient elution were specified using a timetable. However, when the flow rate becomes variable, the time axis is no longer an absolute reference. Therefore, after the conversion, instead of a timetable, the mixing ratio, column temperature, and flow rate must be specified using a program that could be called a volume table based on the retaining volume, which is an integral value. Due to circumstances that necessitate specifying parameters by volume rather than time, it is desirable to adjust the analytical method using the actual chromatogram obtained by injecting standard substances. Upon closer examination, both the time profile of the mixing ratio change in gradient elution and the transient response time profile of the column temperature can be understood using the time axis. For this reason, the replacement of the time axis with the volume axis has a significant impact on understanding these phenomena, and this is also why it is desirable to measure time-response phenomena in real-world conditions. However, it should be added that if the time programs for mixing ratio and flow rate can be described by polynomial functions with time as a variable, they can be easily integrated over time.

[0051] As described above, the pressure of the mobile phase (eluent) is proportional to its viscosity and also to its flow rate. Furthermore, the viscosity of the mobile phase depends on its composition and is a function of the column temperature. In addition, it undergoes time changes in response to mobile phase switching, etc. Therefore, by implementing flow rate control that takes these factors into account, column protection against pressure can be achieved while enabling high-speed analysis.

[0052] Furthermore, by checking the variation in retention time caused by such flow rate control adjustments and further adjusting the mixing ratio and column temperature, it becomes possible to perform analysis with higher accuracy. [Explanation of Symbols]

[0053] 101 Eluent 102 Liquid transfer pump 103 Autosampler 104 Column Thermostatic Device 105 Derivatization Reagents 106 Liquid transfer pump 107 Reactor 108 detectors 121 Control Unit 121a Measurement Operation Control Unit 121b Arithmetic unit 121c Storage section 121d Processing Unit 121e Display section

Claims

1. A method for controlling a liquid chromatograph that performs measurements by supplying an eluent, The process involves injecting a standard sample and performing measurements, as well as measuring the column pressure. The process involves adjusting the column temperature and the timing of changes in the column temperature based on the measurement results using the above standard sample, The process involves adjusting the flow rate of the eluent according to the measured column pressure, It has, A method for controlling a liquid chromatograph, characterized in that the measurement results using the above-mentioned standard sample are used to determine whether an appropriate retention time or degree of separation has been obtained.

2. A method for controlling a liquid chromatograph according to claim 1, A method for controlling a liquid chromatograph, characterized by adjusting the flow rate of the eluent so as to satisfy a predetermined pressure range of the eluent, according to the column pressure measured above.

3. A method for controlling a liquid chromatograph according to claim 2, A method for controlling a liquid chromatograph, characterized by repeatedly performing the steps of injecting the above-mentioned standard sample, measuring it, measuring the above-mentioned column pressure, adjusting the setting of the column temperature and the timing of the change in column temperature, and adjusting the setting of the above-mentioned flow rate of the eluent.

4. It is a liquid chromatograph, A pump for delivering the eluent, A sample injection unit for injecting the sample into the eluent delivered from the above-mentioned pump, The sample injection unit above supplies the eluent containing the sample, and a separation column separates the target component in the sample. A detector for analyzing the target component separated by the above separation column, A control unit controls the measurement operation of the pump, the sample injection unit, the separation column, and the detector, and processes the data. Equipped with, The control unit is characterized by performing flow rate adjustment to adjust the flow rate of the eluent so as to satisfy a predetermined upper pressure limit of the eluent, according to the temperature of the separation column and the timing of changes in the temperature of the separation column, and measurement control to perform a measurement using a standard sample when the flow rate adjustment is performed, and to determine whether an appropriate retention time or degree of separation has been obtained based on the measurement using the standard sample.

5. A liquid chromatograph according to claim 4, The liquid chromatograph is characterized in that the control unit adjusts the flow rate of the eluent at a timing corresponding to the transient response time of the separation column in relation to the timing of the start of a temperature change in the separation column.

6. A liquid chromatograph according to any one of claims 4 to 5, Furthermore, the system is configured to analyze amino acids by including a derivatization reagent pump for delivering the derivatization reagent and a reaction apparatus for reacting the derivatization reagent with the sample. The control unit is characterized by adjusting the flow rate of the derivatization reagent according to the flow rate of the eluent.

7. A liquid chromatograph according to any one of claims 4 to 6, wherein the control unit is characterized in that it causes a gradient change in the flow rate of the eluent according to claims 4 to 6, or the flow rate of the derivatization reagent according to claim 6, within a predetermined time.

8. A liquid chromatograph according to any one of claims 4 to 7, A liquid chromatograph characterized by being configured to generate a time program corresponding to the results of the above flow rate adjustment.

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