JP2021139862A - Liquid chromatography, and control method of liquid chromatography - Google Patents

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

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

In the chromatograph, a technique is known in which a constant-speed gradient program is converted into a constant-pressure gradient program and the liquid feed pump is controlled according to the converted constant-pressure gradient program in order to shorten the measurement time (for example). For example, see Patent Document 1.).

Japanese Unexamined Patent Publication No. 2011-179962

However, it has been found that when the constant pressure gradient program converted from the constant velocity gradient program as described above is used, the measurement content is easily affected by various measurement conditions, so that an appropriate holding time may not be obtained. ..

In view of the above points, it is an object of the present invention to speed up the measurement by the liquid chromatograph and to easily obtain an appropriate holding time.

In order to achieve the above object, the present invention
It ’s a liquid chromatograph,
A pump that sends the eluate while changing the mixing ratio of two or more types of eluent,
A sample injection unit that injects a sample into the eluate sent from the pump,
A separation column to which the eluate into which the sample is injected is supplied by the sample injection section to separate the target component in the sample, and
A detector that analyzes the target component separated by the separation column,
A control unit that controls the measurement operation of the pump, the sample injection unit, the separation column, and the detector, and processes data.
With
The control unit controls the flow rate of the eluent so as to satisfy a predetermined upper limit pressure of the eluent according to the mixing ratio of the eluent and the change timing of the mixing ratio. It is a feature. The flow rate is synonymous with the flow velocity.

As a result, the mixing ratio of the eluent and the change timing of the mixing ratio are appropriately maintained, and the flow velocity of the eluent is controlled according to these, so that high-speed analysis that satisfies a predetermined upper limit pressure is possible. However, the holding time can be easily maintained.

According to the present invention, it is possible to easily maintain an appropriate holding time or degree of separation while speeding up the measurement by the liquid chromatograph. The degree of separation is an index that quantifies the degree of separation between the two peaks and the quality of the two peaks. The term “appropriate retention time” as used herein means that a good degree of separation can be obtained.

It is a schematic block diagram of the system of the liquid chromatograph. It is explanatory drawing which shows the example of the gradient elution program generation process. It is explanatory drawing which shows the example of the gradient elution program.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

(Rough structure of chromatograph)
The liquid chromatograph includes, for example, a plurality of eluents 101 ... As shown in FIG. 1, one or more of them is selected and fed to the autosampler 103 (sample injection section) by the liquid feed pump 102. The sample is to be injected. The target component of the injected sample is separated by a separation column (not shown) installed in a column thermostat 104 for keeping the temperature constant.

The components separated by the separation column are detected by the detector 108 to analyze the target analytical species. In the case of the post-column derivatization method, the components separated by the separation column are mixed with the derivatization reagent 105 fed by the liquid feed pump 106, and when passing through the reactor 107, react to develop color and detect. By being detected by the vessel 108, analysis of analytical species such as amino acids can be performed. For amino acid analysis, for example, ninhydrin is used as the derivatization reagent.

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 has come to control the operation of each unit of the liquid chromatograph and process the detection result. There is. Further, although not particularly limited, for example, a program for analysis operation (gradient elution program of eluent) is generated (functions as a generator), and the operation of each of the above parts is controlled based on the program. It has become. More specifically, for example, the measurement operation control unit 121a sets and controls the hardware of each part of the liquid chromatogram according to the method. The calculation unit 121b is adapted to perform calculation processing of the pressure profile by a theoretical formula (flow velocity, viscosity as a temperature function, system delay volume), retention prediction of a standard sample, closed-loop calculation thereof, and the like. The storage unit 121c is designed to store past pressure profiles, standard sample chromatograms, time programs, and the like. The processing unit 121d is adapted to perform waveform processing of the chromatograms of the acquired standard and unknown samples. Further, various information is displayed on the display unit 121e of the control unit 121.

(Explanation of terms)
The main meanings of the terms used herein will be described. It should be noted that this explanation is mainly for facilitating understanding, and is not necessarily for strict limited interpretation.

"Gradient elution" This is a general operation for eluting a solute while continuously changing the composition of the mobile phase (eluent 101).

"Temperature gradient elution" This is an operation to change the column temperature using a time program. Basically, according to the van't Hoof equation, the holding time changes with the temperature change.

"Flow rate gradient elution" This is an operation to change the flow rate using a time program in the same way. Ideally, when the flow rate changes, the holding capacity does not change, but the holding time does.

"Temperature flow rate gradient elution" This is an operation that comprehensively changes not only the composition of the mobile phase but also the column temperature and flow rate using a time program. However, as the term of the present invention, it is not essential that all three change, and if any one variable changes with time, such as mobile phase composition, temperature, and flow rate, it is used as the term "temperature flow rate gradient elution" in a broad sense. Sometimes used.

(Time program generation example 1)
Hereinafter, an example in which a time program is generated while the operation control of the chromatograph is performed by the control unit 121 will be described with reference to FIG. The following operations are not necessarily all performed by the control unit 121, and the time program may be read from the outside or a part of the time program may be set manually.

(S101) First, a time program for switching the mobile phase (eluent 101) is set in the apparatus according to the separation state of each target analytical species. That is, a so-called general gradient elution program is set. This may be stepwise elution or gradient elution. Further, the time program may also include control of the column temperature according to the separation state of each analysis species of interest so that temperature gradient elution can be performed.

(S102) The measurement operation is executed based on the above time program. In this measurement operation, the standard sample is injected and the column pressure is measured.

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

(S104) Based on the above chromatogram, it is determined whether an appropriate retention time or degree of separation is obtained.

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

Based on the time program adjusted as described above, the processing after (S102) is repeated.

(S106) When the holding time is appropriate in (S104) above, whether the column pressure measured at the time of measurement with the standard sample is appropriate, that is, the column pressure does not exceed the upper limit pressure, and the column pressure does not exceed the upper limit pressure. In addition, it is determined whether or not the pressure is within a predetermined pressure range higher than the predetermined lower limit pressure set for speeding up the measurement. Here, the upper limit pressure of the column pressure is, in reality, 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 or the like. It is preferable to set with. This is to take into consideration that the flow resistance of the column varies not a little. In addition, usually, there may be a time zone in which the flow rate is constant. When it is determined that the column pressure is appropriate, the time program is saved and output, and then the measurement operation for the actual analysis is performed.

(S107) If it is determined in (S106) above that the column pressure is not appropriate, the time program is adjusted so that the flow rate of the eluent is adjusted, and the time program is set in the apparatus. 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 as to approach the upper limit pressure. Here, specifically, the flow velocity can be obtained as follows, for example, where the upper limit pressure when the eluent 101 is sent to the separation column is ΔPmax (Pa).

That is, the calculation unit 121b requests a flow velocity program that shows the flow velocity of the eluent 101, to be exact, the time change of the linear velocity u0 (mm / s). Specifically, based on Kozeny-Carman's equation, ΔP = (η ・ u0 ・ L) / Kv,
It can be obtained as u0 = (ΔPmax · Kv) / (L · η).

here,
Kv (m ^ 2) is a column permit,
L (mm) is the column length,
η (Pa · s) indicates the viscosity of the mixed solution according to the time change of the mixing ratio of the eluent 101. The flow velocity may be determined using the time-varying viscosity based on the temperature program of the separation column (eluent 101). That is, for example, when the gradient elution method is performed using an aqueous solution and an organic solvent as eluents, the viscosity of each liquid is different and the viscosity is temperature-dependent, so that the pressure value changes depending on the mixing ratio and temperature during measurement. The flow velocity u0 is obtained based on the change.

Here, instead of the flow velocity u0, various converted values or equivalent values may be used, such as the flow rate F = Sef · u0 per unit time being used. The same is true for other values in this regard. Here, the above Seff is an effective cross-sectional area through which a liquid can be passed.

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

The actual pressure profile has a certain delay time from the time program. The switching of the mobile phase is the internal volume from the mixing point of the mobile phase to the column, and is called a system delay volume or a duel volume. The flow rate will be switched with a delay of the duel volume.

There is also a delay time associated with switching the column temperature. This is a phenomenon such as a transient response when heat is conducted from a Pertier element to a column, for example. On the other hand, when the flow rate changes, unlike the previous two variables (mobile phase composition and column temperature), the pressure responds almost immediately, so the delay time can be ignored.

(S108) Based on the time program adjusted as described above, the measurement operation is performed while introducing the standard sample and measuring the column pressure in the same manner as in (S102) above.

(S109) Whether or not the column pressure in the above measurement operation is appropriate is determined in the same manner as in the above (S106), and if it is determined that the column pressure is not appropriate, the above (S107) and subsequent steps are repeated to adjust the flow velocity of the eluent. Will be done.

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

(S111) Based on the chromatogram, it is determined whether an appropriate retention time is obtained in the same manner as in (S104). If appropriate, the time program is saved and output, and then measurement operations for actual analysis are performed.

(S112) On the other hand, if the holding time is not appropriate, the time program is adjusted so that the mixing ratio of the eluent (timing of change in the mixing ratio) and the column temperature are adjusted in the same manner as in (S105) above. Is set to, and (S102) and subsequent steps are repeated. That is, if the retention time becomes inappropriate due to the adjustment of the eluent flow rate, the retention time is adjusted again and the measurement operation using the standard sample is repeated so that the retention time can be appropriately maintained while speeding up the measurement. Be made.

(Time program generation example 2)
In addition to the switching of the eluent 101 as described above, a time program may be generated in which the flow rate of the derivatizing reagent 105 used in the case of amino acid analysis is linked.

That is, for example, in the analysis of amino acids, each amino acid separated by the separation column is mixed with the derivatizing reagent 105, reacts and develops color when passing through the reactor 107, and is detected by the detector 108. Is done by. Since the number of ninhydrin molecules in the derivatization reagent is larger than the number of amino acid molecules of the analytical species, even if the number of ninhydrin molecules on the reaction side fluctuates slightly, the number of molecules of Ruehman's purple, which is a reaction product, is in principle. It does not change, and it is considered that the peak height does not change.

However, if the flow velocity or the mixing ratio changes from moment to moment due to the flow velocity program or the mixing ratio program as described above, the absorbance and fluorescence intensity of the mixed solution itself are affected by this, not the reaction product. The detection results of absorbance detection and fluorescence detection fluctuate. This can cause baseline fluctuations.

On the other hand, the absorbance of the mixed solution and the like also vary depending on the amount of the derivatizing reagent 105. For example, if the flow rate of the derivatizing reagent 105 decreases, the absorbance decreases, and if the flow rate of the derivatizing reagent 105 increases, the absorbance increases.

Therefore, by controlling the flow rate of the derivatizing reagent 105 according to at least one of the flow rate of the eluent 101 and the mixing ratio within an acceptable range as the optimum mixing amount of the derivatizing reagent 105, for example, liquid feeding By interlocking the flow rates of the pumps 102 and 106 so that the flow rate ratio becomes constant, the change in the absorbance of the mixed solution can be offset and the baseline of the detector 108 can be stabilized.

(Example of generated time program)
An example of the time program generated as described above will be described with reference to FIG.

The stepwise elution method is used for switching the mobile phases such as B1 and B2 in FIG. 3 by setting them with a time difference of 0.1 min. That is, B1 is 100% up to 20.0 min, and B1 is switched from 20.1 min to 50% and B2 is switched stepwise to 50%.

The flow rate of the liquid feed pump 102 is described as Flow 1, 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 the flow rate is switched to 35.0 min. Here, a flow rate linear gradient elution for 2 minutes is performed from 35.0 min to 37.0 min. * Is added to indicate that it is a gradient linear gradient elution. The stepwise mobile phase composition change from 40.0 to 40.1 min is also reflected in the flow linear gradient elution from 60.0 min to 62.0 min. * Is also attached here.

The temperature on the time program can be switched in steps. The first 0.0 min starts at a flow rate of 0.5 mL / min at 40 ° C. and switches to 30 ° C. at 2.0 min. However, the actual temperature exhibits a temperature profile such as a transient response. Therefore, in order to make the flow rate change follow this temperature change, the flow rate is cut out to 5.0 min, and the flow rate is linearly gradient elution lowered toward 0.45 mL / min of 7.0 min. Therefore, * is also attached here to indicate the gradient. Here, since the viscosity of the eluent increases as the column temperature decreases, it is possible to prevent the pressure from increasing unless the flow rate is reduced. The column temperature of 50 ° C. at 20.0 min is also switched in a stepped manner, but the change in flow rate accompanying this is not particularly set. Such a case may be possible.

In the present invention, it is required to convert the analysis method based on a constant pressure, that is, generally based on a constant pressure reference. This is a so-called method transfer for constant pressure. The index that is generally preserved before and after this conversion, especially in the variable flow velocity range, is the retention capacity rather than the retention time. For example, the volume of the flow velocity, which changes from moment to moment, can be calculated by integrating along the time axis. If the origin of the holding time and the origin of the holding volume are synchronized, the holding volume will change from moment to moment as an integral value.

By the way, the problem here is that before the conversion, the mixing ratio of gradient elution and the switching time of the column temperature were specified by the timetable, but when the flow velocity becomes variable, the time axis is no longer an absolute standard. Therefore, after conversion, instead of the timetable, the mixing ratio, column temperature, or flow velocity must be specified by a program that can be called a volume table based on the holding volume, which is an integral value. Since it is necessary to specify the volume instead of the time, it is desirable to adjust the analysis method with the actual chromatogram in which the standard substance is injected. If you think about it carefully, you can understand both the time profile of the mixed ratio change of the gradient elution method and the time profile of the transient response of the column temperature on the time axis. Therefore, the replacement of the time axis with the volume axis has a great influence on the understanding, and it is also the reason why it is desirable to actually measure the time-responsive phenomenon. However, it should be added that if a time program such as a mixing ratio or a flow velocity can be described by a polynomial function with time as a variable, it can be easily integrated over time.

As mentioned above, the pressure of the mobile phase (eluent) is proportional to the viscosity and also to the flow velocity. The viscosity of the mobile phase depends on the composition of the mobile phase and is also a function of the column temperature. Further, a time change also occurs according to the switching of the mobile phase and the like. Therefore, by controlling the flow velocity in consideration of these factors, high-speed analysis becomes possible while protecting the column against pressure.

Moreover, by confirming the fluctuation of the holding time caused by the adjustment of the flow velocity control and further adjusting the mixing ratio and the column temperature, it becomes possible to perform the analysis with higher accuracy.

101 Eluent 102 Liquid feed pump 103 Auto sampler 104 Column thermostat 105 Derivatization reagent 106 Liquid feed pump 107 Reaction device 108 Detector 121 Control unit 121a Measurement operation control unit 121b Calculation unit 121c Storage unit 121d Processing unit 121e Display unit

Claims (10)

It is a control method of a liquid chromatograph that measures by sending two or more types of eluents.
The process of injecting a standard sample and measuring it, as well as measuring the column pressure,
A step of adjusting the mixing ratio of the eluent and the setting of the change timing of the mixing ratio based on the measurement result of the standard sample, and
The step of adjusting the eluent flow rate setting according to the measured column pressure, and
A method for controlling a liquid chromatograph, which comprises. The method for controlling a liquid chromatograph according to claim 1.
A method for controlling a liquid chromatograph, which comprises adjusting the setting of the flow velocity of the eluent so as to satisfy a predetermined pressure range of the eluate according to the measured column pressure. The method for controlling a liquid chromatograph according to claim 2.
The steps of injecting, measuring, and measuring the column pressure of the standard sample, adjusting the eluent mixing ratio and the setting of the change timing of the mixing ratio, and adjusting the setting of the eluent flow velocity are repeated. A method of controlling a liquid chromatograph, characterized in that it is performed. The method for controlling a liquid chromatograph according to claim 1.
The process of adjusting the column temperature and the setting of the change timing of the column temperature based on the measurement result of the above standard sample, and
A method for controlling a liquid chromatograph, which comprises. It ’s a liquid chromatograph,
A pump that sends the eluate while changing the mixing ratio of two or more types of eluent,
A sample injection unit that injects a sample into the eluate sent from the pump,
A separation column to which the eluate into which the sample is injected is supplied by the sample injection section to separate the target component in the sample, and
A detector that analyzes the target component separated by the separation column,
A control unit that controls the measurement operation of the pump, the sample injection unit, the separation column, and the detector, and processes data.
With
The control unit adjusts the flow velocity of the eluent so as to satisfy a predetermined upper limit pressure of the eluent according to the mixing ratio of the eluent and the change timing of the mixing ratio, and the flow velocity is adjusted. A liquid chromatograph characterized by performing measurement control in which measurement is performed using a standard sample when adjustment is performed. The liquid chromatograph of claim 5.
Further, a column constant temperature device for keeping the temperature of the separation column constant is provided, and the separation column is provided with a constant temperature device.
The control unit further
A liquid chromatograph comprising adjusting the flow velocity of the eluate so as to satisfy a predetermined upper limit pressure of the eluate according to the temperature of the separation column and the timing of change in the temperature of the separation column. The liquid chromatograph according to claim 6.
The liquid chromatograph is characterized in that the control unit adjusts the flow velocity of the eluent at a timing corresponding to the transient response time of the separation column with respect to the timing at which the temperature of the separation column starts to change. The liquid chromatograph according to any one of claims 5 to 7.
Further, a derivatization reagent pump for feeding the derivatization reagent and a reaction device for reacting the derivatization reagent with the sample are provided to analyze amino acids.
The liquid chromatograph is characterized in that the control unit adjusts the flow rate of the derivatizing reagent according to at least one of the flow rate of the eluent and the mixing ratio of the eluent. The liquid chromatograph according to any one of claims 5 to 8, wherein the control unit changes the flow rate of the eluent or the flow rate of the derivatizing reagent in a predetermined time. A characteristic liquid chromatograph. The liquid chromatograph according to any one of claims 5 to 9.
A liquid chromatograph characterized in that it is configured to generate a time program according to the result of the flow velocity adjustment.

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