Liquid chromatography, and control method of liquid chromatography

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 liquid chromatographs using gradient elution. [Background technology]

[0002] In chromatography, a technique is known in which a constant-speed gradient program is converted into a constant-pressure gradient program in order to shorten measurement time, and a liquid delivery 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] JP 2011-179962 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, it was found that when a constant pressure gradient program converted from a constant rate gradient program as described above is used, the measurement content is easily affected by various measurement conditions, and therefore an appropriate retention time may not be obtained.

[0005] In view of the above, an object of the present invention is to increase the speed of measurement by liquid chromatography while easily obtaining an appropriate retention time. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides A method for controlling a liquid chromatograph that delivers an eluent and performs measurements, comprising the steps of: A step of injecting a standard sample and performing a measurement while measuring the column pressure; adjusting the settings of the column temperature and the timing of changing the column temperature based on the measurement results of the standard sample; adjusting the eluent flow rate setting in response to the measured column pressure; having The measurement result using the standard sample is characterized in that it is a judgment result as to whether an appropriate retention time or degree of resolution has been obtained.

[0007] Also, A liquid chromatograph comprising: A pump for delivering an eluent; a sample injection unit that injects a sample into the eluent pumped by the pump; a separation column to which an eluent containing a sample is supplied by the sample injection section and which separates a target component in the sample; A detector for analyzing the target component separated by the separation column; a control unit that controls the measurement operations of the pump, the sample injection unit, the separation column, and the detector, and processes 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 limit pressure of the eluent in accordance with the temperature of the separation column and the timing of change in the temperature of the separation column, and measurement control to perform measurement using a standard sample when the flow rate adjustment is performed and determine whether an appropriate retention time or resolution is obtained by the measurement using the standard sample.

[0008] This means: Column temperature Since the timing of the change in pressure is appropriately maintained and the flow rate of the eluent is controlled accordingly, it is possible to easily maintain the retention time appropriately while enabling high-speed analysis that satisfies a predetermined upper limit pressure. Effect of the Invention

[0009] According to the present invention, it is possible to easily maintain an appropriate retention time or resolution while increasing the speed of measurement by liquid chromatography. Resolution is an index that quantifies the degree of separation of two peaks, whether good or bad. In this specification, the expression "appropriate retention time" refers to obtaining a good resolution. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of a liquid chromatograph system. [Diagram 2] FIG. 1 is an explanatory diagram showing an example of a gradient elution program generation process. [Diagram 3] FIG. 2 is an explanatory diagram showing an example of a gradient elution program. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

[0012] (Schematic configuration of chromatograph) 1, the liquid chromatograph is equipped with a plurality of eluents 101, one or more of which are selected and sent to an autosampler 103 (sample injection section) by a liquid delivery pump 102, where the sample is injected. The injected sample is separated into target components in a separation column (not shown) installed in a column thermostat 104 for keeping the temperature constant.

[0013] The components separated in the separation column are detected by a detector 108, thereby analyzing the target analytes. In the case of the post-column derivatization method, the components separated in the separation column are mixed with a derivatization reagent 105 delivered by a liquid delivery pump 106, react and develop color as they pass through a reaction device 107, and are detected by a detector 108, thereby analyzing the analytes such as amino acids. 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 operation of each unit of the liquid chromatograph and process the detection results. In addition, although not particularly limited, for example, it generates (functions as a generation device) a program for analysis operation (gradient elution program of eluent) and controls the operation of each unit based on this. More specifically, for example, the measurement operation control unit 121a sets and controls each hardware unit of the liquid chromatogram according to a method. The calculation unit 121b performs calculation processing of the pressure profile according to a theoretical formula (flow rate, viscosity as a function of temperature, system delay volume), prediction of retention of the standard sample, closed loop calculation thereof, etc. The storage unit 121c stores the past pressure profile, the standard sample chromatogram, and the time program, etc. The processing unit 121d performs waveform processing of the chromatograms of the acquired standard and unknown samples. Moreover, the display unit 121e of the control unit 121 is adapted to display various types of information.

[0015] (Terminology explanation) The main meanings of terms used in this specification will be explained. Note that this explanation is mainly for the purpose of facilitating understanding, and is not necessarily intended to strictly limit interpretation.

[0016] "Gradient elution" is a general operation in which a 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, the retention time changes with temperature according to the van't Hoof equation.

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

[0019] "Temperature-flow gradient elution" is an operation in which not only the composition of the mobile phase but also the column temperature and flow rate are changed comprehensively using a time program. However, as a term of the present invention, it is not essential that all three are changed, and the term "temperature-flow gradient elution" in the broad sense may be used if any one of the variables, the mobile phase composition, the temperature, or the flow rate, changes over time.

[0020] (Time program generation example 1) An example of generating a time program while the control unit 121 controls the operation of the chromatograph will be described below with reference to Fig. 2. Note that the following operations are not necessarily all performed by the control unit 121, and the time program may be read from an external source or part of it may be set manually.

[0021] (S101) First, a time program for switching the mobile phase (eluent 101) is set in the device according to the separation state of each target analyte. That is, a so-called general gradient elution program is set. This may be a program for performing stepwise elution or gradient elution. This time program may also 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 time program, a measurement operation is performed, in which a standard sample is injected and the column pressure is measured.

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

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

[0025] (S105) If the retention time is not appropriate, the time program is adjusted and set in the device so that the mixing ratio of the eluent (the timing of changing 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 above processing from (S102) onwards is repeated.

[0027] (S106) If the retention time is appropriate in (S104), 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 higher than a predetermined lower limit pressure set to speed up the measurement. In reality, the upper limit pressure of the column pressure is preferably set with a certain margin, such as 90% or 80%, with respect to the actual upper limit pressure of the column installed in the column thermostat 104 or the pump 102. This is to take into consideration the fact that the flow resistance of the column varies to some extent. In addition, usually, there may be a time period with a constant flow rate. If the column pressure is determined to be appropriate, the time program is stored or output, and the measurement operation for the actual analysis is performed thereafter.

[0028] (S107) If the column pressure is determined to be inappropriate in (S106) above, the time program is adjusted so that the flow rate of the eluent is adjusted and set in the device. That is, for example, when the pressure is lower than the lower limit pressure, the measurement time can be shortened by increasing the flow rate of the eluent so that it approaches the upper limit pressure. Here, specifically, for example, the flow rate can be calculated as follows, assuming that the upper limit pressure when the eluent 101 is sent to the separation column is ΔPmax (Pa).

[0029] That is, the calculation unit 121b determines a flow rate program that indicates the time change of the flow rate of the eluent 101, specifically, the linear velocity u0 (mm / s). Specifically, based on the Kozeny Carman equation, ΔP=(η·u0·L) / Kv, It can be calculated as u0 = (ΔPmax Kv) / (L η).

[0030] Where: Kv(m^2) is column permeability, L (mm) is the column length, η (Pa s) indicates the viscosity of the mixture according to the change over time in the mixture ratio of the eluent 101. The flow rate may be calculated using the viscosity that changes over time based on the temperature program of the separation column (eluent 101). That is, for example, when gradient elution is performed using an aqueous solution and an organic solvent as the eluent, the viscosities of the liquids are different and are temperature dependent, so that the flow rate u0 is calculated based on the change in pressure value due to the mixture ratio and temperature during measurement.

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

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

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

[0034] There is also a delay time associated with switching the column temperature. This is a phenomenon similar to a transient response, such as when heat is conducted from a Peltier element to a column. On the other hand, when changing the flow rate, the pressure responds almost immediately, unlike the previous two variables (mobile phase composition and column temperature), so the delay time can usually 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 in the same manner as in (S102) above.

[0036] (S109) It is determined whether the column pressure in the measurement operation is appropriate in the same manner as in (S106) above. If it is determined to be inappropriate, (S107) and subsequent steps are repeated to adjust the flow rate of the eluent.

[0037] (S110) If it is determined in the above (S109) that the column pressure is appropriate, a chromatogram is generated based on the standard sample injected in the measurement operation in (S108).

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

[0039] (S112) On the other hand, if the retention time is not appropriate, the time program is adjusted and set in the device so that the mixture ratio of the eluent (timing of change in the mixture ratio) and the column temperature are adjusted in the same manner as in (S105) above, and (S102) and subsequent steps are repeated. In other words, if the retention time becomes inappropriate due to the adjustment of the flow rate of the eluent, it is readjusted and the measurement operation using the standard sample is repeated so that the retention time is kept appropriate while the measurement speed is increased.

[0040] (Time program generation example 2) In addition to switching the eluent 101 as described above, a time program may be generated that links the flow rate of the derivatization reagent 105 used in the analysis of amino acids.

[0041] That is, for example, analysis of amino acids is performed by mixing each amino acid separated in a separation column with a derivatization reagent 105, reacting and coloring as it passes through a reaction device 107, and detecting the color with a detector 108. Since the number of ninhydrin molecules in the derivatization reagent is in excess of the number of amino acid molecules to be analyzed, even if the number of ninhydrin molecules on the reacting side varies slightly, the number of molecules of Ruhemann's purple, which is the reaction product, does not change in principle, and it is considered that the peak height does not change.

[0042] However, when the flow rate or mixture ratio changes from moment to moment due to the flow rate program or mixture ratio program as described above, the absorbance and fluorescence intensity of the mixture itself, rather than that of the reaction product, are affected accordingly, causing fluctuations in the results of absorbance detection or fluorescence detection, which can cause baseline fluctuations.

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

[0044] Therefore, by controlling the flow rate of the derivatization reagent 105 according to at least one of the flow rate of the eluent 101 and the mixing ratio within an allowable range as the optimal mixing amount of the derivatization reagent 105, more specifically, by interlocking the flow rates of the liquid delivery pumps 102 and 106 so that the flow rate ratio is constant, it is possible to offset the change in absorbance of the mixed solution and stabilize the baseline of the detector 108.

[0045] (Example of a generated time program) An example of a time program generated as described above will be described with reference to FIG.

[0046] In Figure 3, the mobile phases B1, B2, etc. are switched with a time difference of 0.1 min to use the stepwise elution method. That is, B1 is 100% up to 20.0 min, and from 20.1 min, B1 is switched to 50% and B2 is switched to 50% in a stepwise manner.

[0047] The flow rate of the liquid delivery pump 102 is indicated as Flow1, but in consideration of the system delay volume, even if the mobile phase composition is switched to 20.0 min, the flow rate is delayed and switched to 35.0 min. Here, a two-minute linear flow rate gradient elution is performed from 35.0 min to 37.0 min. An * is added to indicate that this is a gradient linear gradient elution. The stepwise mobile phase composition change from 40.0 to 40.1 min is also reflected in the linear flow rate gradient elution from 60.0 min to 62.0 min. An * is also added here.

[0048] The temperature in the time program is switched in a stepwise manner. It starts at 40°C at 0.5mL / min at 0.0min, and switches to 30°C at 2.0min. However, the actual temperature shows a temperature profile like a transient response. Therefore, in order to change the flow rate to follow this temperature change, it is cut out at 5.0min, and the flow rate is reduced in a linear gradient elution manner to 0.45mL / min at 7.0min. Therefore, the gradient is also indicated here with an *. Here, the viscosity of the eluent increases as the column temperature decreases, so if the flow rate is not reduced, the pressure will not increase. The column temperature at 20.0min is also switched in a stepwise manner to 50°C, but there is no particular change in the flow rate associated with this. This may be the case.

[0049] In the present invention, it is required to convert the analytical method based on a constant pressure, i.e., on a roughly constant pressure basis. This is the so-called method transfer for making the method constant pressure. Before and after this conversion, the index that is roughly preserved, especially in the variable flow rate range, is the retention volume, not the retention time. For example, the volume can be calculated by integrating the flow rate, which changes from moment to moment, along the time axis. If the origin of the retention time and the origin of the retention volume are synchronized, the retention volume also changes from moment to moment as an integral value.

[0050] Now, the issue here is that before the conversion, the mixing ratio of gradient elution and the switching time of column temperature were specified by a time table, but when the flow rate becomes variable, the time axis is no longer an absolute standard. Therefore, after the conversion, instead of a time table, the mixing ratio, column temperature, and flow rate must be specified by a program that can be called a volume table based on the retention volume, which is an integrated value. There are circumstances in which it is necessary to specify by volume rather than time, so it is desirable to adjust the analysis method using an actual chromatogram in which a standard substance is injected. If you think about it carefully, the time profile of the change in the mixing ratio of gradient elution and the time profile of the transient response of the column temperature can both be understood with the time axis. Therefore, the replacement of the time axis with the volume axis has a large impact on understanding, and is also the reason why it is desirable to actually measure time-responsive phenomena. However, it should be added that if the time program of the mixing ratio, flow rate, etc. can be described by a polynomial function with time as a variable, it can be easily integrated along time.

[0051] As described above, the pressure of the mobile phase (eluent) is proportional to the viscosity and also to the flow rate. The viscosity of the mobile phase depends on the composition of the mobile phase and is also a function of the column temperature. Furthermore, the viscosity also changes over time depending on switching of the mobile phase. Thus, by controlling the flow rate while taking these factors into consideration, high-speed analysis can be performed while protecting the column against pressure.

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

[0053] 101 Eluent 102 Liquid delivery pump 103 Autosampler 104 Column Thermostat 105 Derivatization Reagents 106 Liquid delivery pump 107 Reactor 108 Detector 121 Control Unit 121a Measurement operation control section 121b Arithmetic unit 121c Storage section 121d Processing section 121e Display section

Claims

1. A method for controlling a liquid chromatograph that delivers two or more types of eluent and performs measurements, comprising the steps of: A step of injecting a standard sample and performing a measurement while measuring the column pressure; adjusting the mixing ratio of the eluent and the timing of changing the mixing ratio based on the measurement result of the standard sample; adjusting the eluent flow rate setting in response to the measured column pressure; A method for controlling a liquid chromatograph, comprising:

2. 2. A method for controlling a liquid chromatograph according to claim 1, comprising: A method for controlling a liquid chromatograph, comprising adjusting a flow rate setting of the eluent in response to the measured column pressure so as to satisfy a predetermined pressure range of the eluent.

3. 3. A method for controlling a liquid chromatograph according to claim 2, comprising the steps of: A method for controlling a liquid chromatograph, comprising the steps of: injecting and measuring the standard sample, and measuring the column pressure; adjusting the settings of the mixing ratio of the eluent and the timing of change in the mixing ratio; and adjusting the settings of the flow rate of the eluent.

4. 2. A method for controlling a liquid chromatograph according to claim 1, comprising: adjusting the settings of the column temperature and the timing of changing the column temperature based on the measurement results of the standard sample; A method for controlling a liquid chromatograph, comprising:

5. A liquid chromatograph comprising: a pump that delivers an eluent while changing a mixing ratio of two or more kinds of eluent; a sample injection unit that injects a sample into the eluent pumped by the pump; a separation column to which an eluent containing a sample is supplied by the sample injection section and which separates a target component in the sample; A detector for analyzing the target component separated by the separation column; a control unit that controls the measurement operations of the pump, the sample injection unit, the separation column, and the detector, and processes data; Equipped with the control unit performs flow rate adjustment to adjust the flow rate of the eluent so as to satisfy a predetermined upper limit pressure of the eluent in accordance with a mixing ratio of the eluent and a timing of change in the mixing ratio, and performs measurement control to perform measurement using a standard sample when the flow rate adjustment is performed.

6. 6. The liquid chromatograph of claim 5, Further, a column thermostat for keeping the temperature of the separation column constant is provided, The control unit further A liquid chromatograph comprising: a flow rate of the eluent being adjusted in accordance with a temperature of the separation column and a timing of change in the temperature of the separation column so as to satisfy a predetermined upper limit pressure of the eluent.

7. 7. The liquid chromatograph of claim 6, The liquid chromatograph according to claim 1, wherein the control unit adjusts a flow rate of the eluent at a timing corresponding to a transient response time of the separation column relative to a timing at which a change in temperature of the separation column starts.

8. The liquid chromatograph according to any one of claims 5 to 7, The apparatus further includes a derivatization reagent pump for delivering a derivatization reagent, and a reaction device for reacting the derivatization reagent with a sample, thereby being configured to analyze amino acids; The liquid chromatograph, wherein the control unit adjusts a flow rate of the derivatization reagent in accordance with at least one of a flow rate of the eluent and a mixing ratio of the eluent.

9. 9. The liquid chromatograph according to claim 5, wherein the control unit changes a flow rate of the eluent or a flow rate of the derivatization reagent with a gradient within a predetermined time.

10. The liquid chromatograph according to any one of claims 5 to 9, A liquid chromatograph configured to generate a time program according to a result of the flow rate adjustment.

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