JP2019184330A - Preparative liquid column chromatograph device, method for measuring arrival time in preparative liquid column chromatograph device, and method for preparative by column chromatograph - Google Patents

Abstract

To provide a preparative liquid column chromatograph device which can accurately measure the arrival time from when a detector detected an elution to when the elution arrives the switch of the preparative means and can make the detection by the detector and the time of preparative coincide with each other.SOLUTION: The present invention includes a detector and preparative means, and the switch of the preparative means automatically changes when a set delay time has passed since the detection intensity of the detector exceeds a set threshold value 41.SELECTED DRAWING: Figure 1

Description

The present invention relates to a preparative liquid column chromatograph apparatus, an arrival time measurement method in a preparative liquid column chromatograph apparatus, and a preparative method using a column chromatograph.

The preparative liquid chromatograph is an apparatus for separating each component in a solution in which a plurality of components are mixed according to the principle of chromatograph using the difference in passage speed of different compounds in the column packing material. It is. In recent years, even in such a preparative liquid chromatograph, functions have been advanced and automated, and many attempts have been made to achieve efficient fractionation. Further, not only fractionation but also a liquid chromatograph for fractionation having a qualitative analysis function has been studied.

In such a preparative liquid chromatograph, the eluate after passing through the column is fractionated at regular intervals. In such fraction separation, the present inventors have made an attempt to clarify what the components in each fractionated test tube are by performing qualitative analysis continuously at the same time as the separation. (Patent Document 1 etc.).

In Patent Document 1, by performing qualitative analysis of a sample continuously in preparative liquid column chromatography, it is not necessary to perform qualitative analysis measurement on a preparative fraction test tube for a sample obtained by a fraction collector. Developed a device for obtaining analytical data.

In such a preparative liquid chromatograph, it is necessary to accurately match the obtained qualitative analysis data with the composition inside the test tube actually collected. It was necessary to control with high precision. If this is inaccurate, the qualitative analysis data and the actual sample do not match, so the composition of the preparative fraction test tube does not match.
Until now, there have not been enough attempts to achieve such a match.

In general, when fractionation is performed with a liquid chromatograph, it is important to accurately know the time from when the eluate is detected by the detector until the eluate reaches the changeover switch for fractionation into each fraction. . Then, it is necessary to switch the changeover switch to the sample acquisition side at an accurate timing. If this timing is inaccurate, the peak indicated by the chromatogram does not coincide with the preparative fraction, and the target component for the preparative purpose cannot be accurately recovered. That is, the qualitative analysis data continuously measured and the composition of the fraction test tube cannot be matched accurately.

Patent Documents 2 and 3 disclose means for determining such a sorting start time. However, the method of Patent Document 2 calculates the delay time by theoretical calculation based on the liquid flow rate and the like. However, in reality, there may be individual differences between devices, pump performance may decrease over time, and the outflow rate may change slightly depending on the temperature and pressure during measurement. It is difficult to match the theoretical calculation value with the actual arrival time with high accuracy.
Since the method of Patent Document 3 performs LC analysis for each fraction collected, it is not a simple method, and it takes time and cost.

Furthermore, as described above, in order to accurately know the components contained in each fraction test tube by integrating and averaging the qualitative analysis data measured continuously, the sample acquisition time by the sorting means, the detector It is necessary to accurately know the relationship with the detection time according to (C). Even if the deviation here is very slight, if the deviation occurs between the acquisition time of the fraction test tube and the acquisition time of the qualitative analysis data, the above-mentioned purpose cannot be suitably achieved. End up. Sufficient studies have not been made to reduce the deviation at a level that can cope with the improvement of such problems.

JP, 2006-3954, A Japanese Patent No. 3268820 International Publication 2010-021008

In order to solve the above-mentioned problem, the present application is an arrival time from when the eluate is detected by the detector (C) until reaching the changeover switch of the sorting means (B) in the preparative liquid column chromatograph. It is an object of the present invention to provide a preparative liquid column chromatograph capable of accurately measuring the detection time and accurately matching the detection by the detector (C) with the preparative time.

The present invention is a preparative liquid column chromatograph apparatus provided with a detector (C) and a preparative means (B),
A sorting liquid characterized in that the selector switch of the sorting means (B) is automatically switched after the set delay time has elapsed from the time when the detection intensity in the detector (C) exceeds a set threshold value. It is a column chromatograph apparatus.

The present invention includes a fractionation means (B) having a changeover switch for switching a flow path through which the eluate from the column flows, an eluate detector (C), and an eluate analysis means (D). In a preparative liquid column chromatograph,
The fractionation is characterized by comprising in-liquid mark generating means (X) for measuring the arrival time from when the sample is detected by the detector (C) until reaching the selector switch of the sorting means (B). This is a liquid column chromatograph apparatus.

The in-liquid mark generating means (X) preferably generates bubbles in the eluate.
The in-liquid mark generating means (X) has a shape that can be mounted on the column mounting portion, and when measuring the arrival time, the column (A) is replaced with the in-liquid mark generating means (X). It is preferable that the measurement is performed.

The sorting means (B) preferably includes reading means (Z) for reading the position of the in-liquid mark generated by the in-liquid mark generating means (X) in the pipe in the vicinity of the changeover switch.

The preparative liquid column chromatograph apparatus of the present invention further includes a display means (E) for displaying the analysis result, a calculation means (F) for controlling the display means (E) and a data storage means (G), and data. Data storage means (G) to store,
The data storage means (G) stores the continuous measurement results of the detector (C), the continuous measurement results of the analysis means (D), and the fraction acquisition times of the sorting means (B). Is preferred.
The display means (E) preferably displays the result of integrating or averaging the continuous measurement results of the analysis means (D) according to the fraction acquisition times of the fractionation means (B).

The present invention, in the above-described preparative liquid column chromatograph apparatus,
The state in which the in-liquid mark generating means (X) is present in the eluate flow path,
The eluate is allowed to flow out, the changeover switch of the sorting means (B) is switched at the initially set switching time, and at the same time, the liquid feeding pump is stopped,
The arrival time measurement method in the preparative liquid column chromatograph is characterized in that the error in the arrival time is measured by confirming the position of the in-liquid mark in the pipe at that time.

The present invention is a chromatography method using the preparative liquid column chromatograph apparatus described above,
Step (1) of measuring the arrival time in the preparative liquid column chromatograph apparatus by the above method,
Using the arrival time measured in step (1), the switching time (i) of the changeover switch of the sorting means (B), the relationship (ii) between the measurement time and the measurement result of the analysis means (D), and And (2) correcting at least one of the effluent flow rates (iii)
In addition, the chromatography is performed by the step (3) of performing the chromatography while continuously performing the qualitative analysis by the analysis means (D),
It is also a fractionation method using a column chromatograph characterized in that the qualitative analysis result obtained in step (3) is processed by reflecting the correction result in step (2).

According to the present invention, it is possible to accurately measure the arrival time from when the eluate passes through the detector (C) until it reaches the changeover switch in the sorting means (B), whereby the sorting means (B ) Can be accurately controlled. Thereby, the qualitative analysis data by the qualitative analysis means can be made to coincide with the qualitative analysis data of the test tube components collected by the fraction collector with high accuracy.

For this reason, it is not necessary to perform a qualitative analysis on the test tube components again, and the experimental efficiency can be greatly improved.

It is a schematic diagram which shows an example of the liquid column chromatograph apparatus for fractionation of this invention. It is a schematic diagram which shows an example of the liquid column chromatograph apparatus for fractionation of this invention. It is a schematic diagram which shows an example of the liquid column chromatograph apparatus for fractionation of this invention. It is a schematic diagram which shows the operation state of the changeover switch of a sorting means (B). It is a schematic diagram which shows the state of the selector switch at the time of arrival time measurement. It is a schematic diagram which shows the state in which the reading means (Z) exists in the piping vicinity of the selector switch vicinity. It is a figure which shows the state which displayed the qualitative analysis data in an example of the display in the image display apparatus of the liquid chromatograph apparatus for fractionation of this invention. It is a figure which shows that fractionation is performed suitably when the arrival time by this invention is measured. It is a schematic diagram which shows an example of the in-liquid mark generation | occurrence | production means (X) used by this invention. It is a schematic diagram which shows an example of the in-liquid mark generation | occurrence | production means (X) used by this invention. It is a figure for demonstrating an example of the processing method of the measurement data by the analysis means (D) at the time of measuring the arrival time in this invention. It is a figure for demonstrating an example of the processing method of the measurement data by the analysis means (D) at the time of measuring the arrival time in this invention. In the preparative liquid column chromatograph apparatus of the present invention, the measurement of the detector when measuring the arrival time from when the sample is detected by the detector (C) until it reaches the selector switch of the preparative means (B) It is a figure which shows a result.

Hereinafter, the present invention will be described in detail.
The subject of the present invention is a preparative column chromatograph apparatus also called medium pressure column chromatography. That is, it is not a chromatographic apparatus for analysis, but an apparatus for separating and acquiring a sample in which a plurality of types of compounds are mixed.

According to the first aspect of the present invention, when the sample is separated, the changeover switch in the detector (B) is switched at the most appropriate timing, whereby precise separation can be performed.

The column chromatograph for fractionation according to the first aspect of the present invention is such that the detector (C) automatically switches the selector switch of the fractionating means (B) when the delay time elapses from the time when the peak exceeding the set threshold is detected. Switching is performed. An example of the first aspect of the present invention is shown in FIG.

In the preparative column chromatograph, a dead volume of about 1 to 3 ml is generally present between the detector (C) such as a UV cell and the switching nozzle of the preparative means (B). .

Thus, for example, if the dead volume is 2 ml and the flow rate is 20 ml / min,
There is a delay time (Td) of Td = (2/20) × 60 = 6 seconds.
In the present specification, the delay time means “the arrival time from the detection by the detector (C) until reaching the changeover switch of the sorting means (B)”, and hereinafter used in this sense. To do.
Such a delay time becomes shorter as the flow rate becomes larger, and becomes longer as the flow rate becomes smaller. If the dead volume is 2ml, the flow rate is 5ml / min.
There is a long delay time of Td = (2/5) × 60 = 24 seconds. Then, taking this delay time into account, the changeover switch in the sorting means (B) is switched to perform sample acquisition.

Thus, a dead volume of about 1 to 3 ml is generally present between the detector (C) and the switching nozzle of the sorting means (B). For this reason, it is preferable to switch the changeover switch in consideration of the time until the sample actually reaches the changeover switch of the sorting means (B) after the sample is detected by the detector (C).

In the first aspect of the present invention, this point is examined, the threshold value is set in the detector (C), and a detection value exceeding the threshold value is detected. Is to switch. Thereby, when the sample detected by the detector (C) reaches the switching nozzle of the sorting means (B), switching can be performed accurately.

The threshold value can be appropriately set according to the sample, and can be set as a detection value that can confirm that the target sample compound has started to elute. What can be specifically used as the detector (C) will be described in detail below.

The preparative chromatography apparatus of the present invention is preferably one that performs various controls by a computer. Therefore, it is preferable that the above-described changeover of the changeover switch is also performed by a computer. That is, after starting the separation by chromatograph, after detecting that the detection value of the detector (C) exceeds the threshold value, the selector switch of the sorting means (B) is switched based on the delay time described above. Preferably, the power time is automatically calculated, and the changeover switch is automatically switched based on the calculated switching time.

With the configuration as described above, in order to optimize the switching time in the preparative chromatograph, it is necessary to accurately measure the delay time. That is, if the set value of the delay time is incorrect, the above-described method of the first aspect of the present invention cannot be optimally performed.

The present inventors have also studied this point and have completed a method for accurately measuring the delay time in a preparative chromatograph. This is the second aspect of the present invention.

As described above, a dead volume of about 1 to 3 ml generally exists between the detector (C) and the switching nozzle of the sorting means (B). Then, the delay time is calculated from the dead volume and the flow rate.

On the other hand, since the inner diameter of the flow path around the switching nozzle is about 2 mm, when the flow rate is 20 ml / min, the distance that the sample moves in the flow path per second is
(20/60) ÷ (0.1 × 0.1 × π) = 10.6 (cm)
It becomes the length of.
That is, measuring the distance of 10 cm forward and backward around the switching nozzle of the fraction collector is equivalent to measuring the flow rate deviation of less than ± 1 second.
In other words, for the in-liquid mark generated by the in-liquid mark generating means (X), a delay time of 0.1 seconds can be measured by measuring in centimeter units that can be easily seen by a human in the vicinity of the switching nozzle. It becomes.

Furthermore, when the flow rate is 20 ml / min, the volume of the solvent corresponding to a deviation of 0.1 second is
(20/60) × 0.1 = 0.03 ml
Thus, this amount is sufficiently small with respect to the capacity of the test tube of the fraction collector (several tens of ml), so that the delay time can be accurately corrected.

From the above, as described in detail below, the mark is generated by the in-liquid mark generating means (X), and this is observed in the vicinity of the switching nozzle of the fraction collector, so that the delay time can be corrected with high accuracy. The inventors have found that this can be done. If the control of these devices is performed in increments of 0.1 to 0.01 seconds, it is only necessary to input the delay time acquired based on the position of the visually observed mark, and under accurate time management. Sample acquisition can be performed.

Further, depending on the configuration of the apparatus, other complicated control may be performed, so that the switching time of the selector switch of the sorting means (B) may be controlled only in increments of 1 second. Even in such a case, the analysis results by the detector (C) and the analysis means (D) can be used effectively.
For example, for the switching time set in increments of 1 second, the analysis means (D) is set to perform 10 measurements per second (see FIG. 11). Although the switching timing is in 1 second increments, the measured values of the detector (C) and the analyzing means (D) are obtained in 0.1 second increments. Taking into account the difference Tc between the measured delay time and the measured delay time, the measured data may be adopted while being shifted by Tc. Then, the contents of the test tube can be analyzed with accuracy within 0.1 seconds.

Further, even when the switching time is controlled in increments of 1 second and the measurement of the analysis means (D) is performed at intervals of about 1 second and the same control interval, plotting each measurement data, FIG. As described above, the analysis value corrected in consideration of the difference Tc between the switching time and the accurately measured delay time is selected, so that the measurement result used for the analysis can be obtained with an error substantially within one second. You can also choose.

In addition, such an aspect of the present invention can be grasped as a strict measurement method of “flow rate”.
In other words, the “delay time” described above is corrected by the delay time itself, but the reason why such a “delay time” essentially occurs is that the flow rate of the eluate is not strictly controlled. This is due to that.
Therefore, highly accurate correction of the delay time by the pump flow rate control value can also be performed. Correction may be performed by converting the delay time read by the above-described method into a pump flow rate error and thereby changing the control value of the pump flow rate.

Further, correction can be performed by incorporating a flow meter in the flow path after pumping and automatically controlling the pump flow rate to a set value so that the delay time does not have to be changed.
In such correction, the set value can be corrected automatically by controlling the pump flow rate so that the delay time does not need to be changed.

When performing such correction,
Td (seconds) = [(dead volume (ml)) / (pump flow rate (ml / min))] × 60
The correction can be performed based on the relational expression.
As a result, precise preparative column chromatography can be performed by adjusting settings including adjusting the pump flow rate.

By the above operation, by visually observing a value of about millimeter to centimeter around the nozzle, the “delay time” or “dead volume” can be accurately measured, and the sample acquisition time can be precisely controlled, The present inventors have found that the time can be exactly matched with the measurement result of the continuous qualitative analysis by the analysis means (D), thereby completing the present invention.

The present invention is a method for performing a precise measurement of “delay time” required for performing such a preparative column chromatography. In the present invention, it is described as “measurement of arrival time”, but “measurement of dead volume” described above is also included in this “measurement of arrival time”.
Because if the “dead volume (ie, the pipe volume from the detector (C) to the changeover switch)” can be measured accurately, the “delay time” can be easily calculated from the pump flow rate. This is because they can be regarded as the same.

Hereinafter, the preparative chromatography apparatus for measuring the arrival time according to the present invention will be described in detail.
As described above, according to the present invention, the in-liquid mark generating means for accurately measuring the arrival time from when the sample is detected by the detector (C) until it reaches the changeover switch of the sorting means (B). X).
As a result, the analysis result by the analysis means (D) and the composition of the fraction test tube sorted by the sorting means (B) can be matched with high accuracy.
For this reason, about the fraction test tube obtained after fractionation, it becomes possible to know the composition with high accuracy without performing qualitative analysis again. As a result, the labor of the experiment worker can be greatly reduced, which greatly contributes to the efficiency of the experiment.

To some extent, the time from when the sample is theoretically detected by the detector (C) until it reaches the changeover switch of the sorting means (B) is estimated by the above-described Patent Documents 2 and 3 or other known methods. can do.
In practice, however, individual differences between devices, changes in pump performance due to long-term use, flow fluctuations due to use conditions, and the like occur. In order to correct such a slight deviation from the theoretical value, the time from when the sample is detected by the detector (C) until it reaches the changeover switch of the sorting means (B) is accurately measured periodically. Measurement and correction are required.

The present invention is characterized in that the in-liquid mark generating means (X) is used to accurately measure the arrival time, and the in-liquid mark generated thereby is used. Then, when the changeover time of the changeover switch set in the apparatus reaches, the liquid supply is stopped, and in this state, the position of the mark in the liquid is confirmed to accurately measure the arrival time.

This point will be described more specifically with reference to the schematic diagrams of FIGS.
4 and 5 are schematic views showing the changeover switch provided in the sorting means (B). FIG. 4 shows the liquid flow during the preparative column chromatography operation using the preparative means (B).
In preparative liquid column chromatography, the pipe is filled with the effluent at the time when the preparative operation is started. When separation starts and the effluent begins to flow by the pump, the liquid is sequentially pushed out of the pipe. For this reason, since the component which begins to flow out first is the component which existed in piping at the time of a separation start, it cannot contain the sample in it.

For this reason, the first eluate is discharged from the outlet of FIG. 4a. Then, when sample elution starts, the changeover switch is switched to the fraction collector side as shown in FIG. After that, the test tubes for acquisition are changed at regular intervals, the composition is confirmed for each test tube, and the experiment proceeds to the next stage.
For this reason, if the timing of switching from the discharge port to the fraction collector is deviated, the above-described precise sorting cannot be performed. Furthermore, accurate qualitative analysis data on the composition of each fraction cannot be obtained.

Such switching of the changeover switch is generally performed by the control means of the sorting means (B), specifically, it is controlled by a computer, and is automatically switched when the set changeover time is reached. is there.

FIG. 4 shows a schematic diagram when the arrival time is measured using a preparative liquid column chromatograph equipped with the in-liquid mark generating means (X) of the present invention.
FIG. 4 shows a state when liquid feeding is stopped during the time when the liquid mark generating means (X) is provided in the liquid feeding line to generate the liquid mark and the switching is performed from the discharge port side to the fraction collector.
At this time, if the switching time and the arrival time completely coincide with each other, the in-liquid mark exists at a position where the three tubes intersect. However, if the switching time and the arrival time are slightly deviated, the in-liquid mark is present at a position in the middle of the liquid feeding pipe. This state is shown as FIGS. 4a and b. In FIG. 4a, the in-liquid mark has advanced toward the discharge port because the switching time is too late, and in FIG. 4b, the in-liquid mark has not yet reached the changeover switch because the switching time is too early.

By such a test, the difference between the switching time and the arrival time set by the apparatus can be calculated from the position of the mark in liquid during liquid feeding from the switching unit. This value can be obtained as the volume shown in FIG. 5 based on the position of the mark in the liquid. This volume may be used as it is, or this volume may be converted into the movement time of the liquid.

FIG. 6 is a schematic diagram showing a pipe in the vicinity of the changeover switch including a reading means (Z) for reading the position of the in-liquid mark generated by the in-liquid mark generating means (X). . It is preferable that the reading means (Z) is provided in this way because the deviation shown in FIG. 5 can be obtained immediately.
The unit of this scale is not particularly limited, and if it can be calculated in the form of length, volume, time, etc., it can be a value suitable for use in the present invention by performing conversion described in detail below. it can.

Such precise measurement of the arrival time has a particularly favorable effect, particularly when continuous qualitative analysis is performed simultaneously with the separation operation. Furthermore, when performing continuous qualitative analysis and integrating or averaging the qualitative analysis data obtained in this way, the most favorable effect is achieved. That is, when integrating and averaging in order to know the composition of the sample in the fraction test tube, it is necessary to accurately determine the time range in which integration and averaging should be performed. This range can be accurately determined by measuring the correct arrival time by the method of the present invention.
As a result, the time range when integrating or averaging the results of continuous qualitative analysis completely matches the actually acquired acquisition range. As a result, highly accurate analysis results can be obtained, and work efficiency in chemical experiments can be greatly improved.

The in-liquid mark generated by the in-liquid mark generating means (X) can be detected when the in-liquid mark passes the detector (C), and when the liquid feeding is stopped at the switching time. There is no particular limitation as long as the location of the mark in the liquid can be confirmed. Such confirmation of the position of the mark in the liquid is preferably performed by visual observation, but the position may be confirmed by a sensor or the like.

Specific examples include air bubbles and coloring. When bubbles are intentionally generated in the liquid, the properties of the bubbles and the effluent are significantly different, so that the passage of bubbles is clearly detected when passing through the detector (C). When the switch is switched and the flow of the liquid is stopped, the deviation can be measured based on the position where the mark generated by the in-liquid mark generating means (X) is stopped, and the correction can be performed thereby. it can.

The means for generating bubbles as the in-liquid mark generating means (X) is not particularly limited, but may be configured as shown in FIG.
That is, in the six-way valve 31, the syringe 32 is connected to one side and further has a loop. Air is introduced into the syringe 32 and introduced into the system.
In this state, when the six-way valve is connected in the direction of the flow path flowing from the pump to the detector (C), the component packed in the loop flows.

Alternatively, as shown in FIG. 10, a method using a column-shaped container can also be used. That is, as a state where air is put in a container having a flow path, this may be used by being installed in the flow path instead of the column.

The bubble generating means shown in FIG. 10 can form a silica region containing a normally used solvent with the cavity region for storing the bubbles on the lower side, and in this state, it is set in the channel and the bubble can be put into the channel. it can. (In this case, the space between the pump and the switching nozzle is filled with a solvent, and then the pump is temporarily stopped and the bubble generating means is attached.)

If air bubbles are used as the mark in the liquid, bubbles will be present at the tip of the eluate when liquid feeding is stopped at the switching time, so where the tip of the solution is present in the pipe, and the solution is in the pipe It is possible to easily determine how far outflow has occurred.

When coloring is performed as the in-liquid mark generating means (X), it can be basically performed by the same method using the apparatus shown in FIGS.
That is, in the submerged mark generating means (X) using the six-way valve of FIG. 9, a similar test can be performed by injecting a colored liquid instead of air injected from the syringe.

Coloring can also be performed by the column-shaped submerged mark generating means (X) shown in FIG. In this case, for example, contrary to the case of generating bubbles, if the colored liquid is accumulated in the silica region and then installed in the flow path filled with the solvent, the correction value of the delay time can be determined at the tip of the colored liquid. it can.

Further, as shown in FIG. 10, the colored liquid is accumulated in the upper region 34 with the silica region facing up, and the bubbles are accumulated in the lower region 35, and then, in the same manner as in the above-described two examples, it is attached in the flow path filled with the solvent. For example, the correction value of the delay time can be confirmed in a form that can be easily detected by both humans and the detector (C) due to the effects of both the bubbles and the colored liquid.

Further, in the detection, the concentration of the colored liquid is preferably such that a sharp rise is caused in the detector (C).
In other words, if the color density is dilute or the color is originally light, it will rise slowly in a short period of time, and an accurate delay time may not be measured. A diagram showing such a rising state is shown in FIG. That is, since the analysis value by the detector (C) increases rapidly, it is difficult to cause an error in the time for determining that the sample has passed the detector. Further, the error can be minimized by setting the threshold value for detecting the rise of the detector low.

The deviation observed from the pipe is basically measured as “length”. To convert this into time, for example, the following method can be employed.

The difference between the switching time set by the device and the actual arrival time can be calculated by the following equations (1) and (2).
The volume of the solution moved from the changeover switch: y (ml) is the tube inner diameter: R (cm) and the displacement scale amount: x (cm).
y = [(πR ² ) / 4] × x (1)
If the flow rate of the solution in the tube is Fp (ml / min), TD = (60 × y) / Fp (2) using the above equation (1).
(TD: deviation time)

Such a calculation may be performed manually, or may be performed by a program that automatically calculates by inputting each of the parameters described above.
Therefore, the following data analysis (for example, integration or average of the measurement results of the analysis means (D)) is performed by setting the switching time accurately or considering the difference between the precisely measured arrival time and the switching time. Etc.).

Further, in the preparative liquid chromatograph apparatus of the present invention, in order to control the apparatus, the display means (F) for displaying the analysis result, the calculation control means (G) for controlling the display means, and the data for storing the data It is preferable to have a storage means (H). Further, the sorting means (B) of the present invention has a changeover switch, and this changeover switch is automatically switched after a predetermined time has elapsed.
Regarding the control of the changeover switches in (F) to (H) and (B), a personal computer storing a program capable of performing calculation, control, data storage, display, etc. for achieving a predetermined purpose Etc. Although not described in detail in the following description, basically, control using a personal computer or the like is performed.

The essence of the technology of the present invention is as described above. Next, the preparative liquid chromatograph apparatus of the present invention will be described from the viewpoint of a more specific apparatus configuration.
The schematic diagram which shows more specifically the preparative liquid chromatograph apparatus of the present invention is shown in FIGS. This will be described in detail below with reference to schematic diagrams.

The preparative liquid chromatograph apparatus of the present invention separates components by a column, but the main part of the present invention relates to the separation and analysis of the eluate after separation. Therefore, any commonly used column can be used.

In the present invention, when measuring the arrival time using the in-liquid mark generating means (X), it is not necessary to use a column. Therefore, in the schematic diagram shown in FIG. 2, the portion indicated as the column or in-liquid mark generating means (X) 1 is provided with a column during the separation operation, and in the case of measuring the arrival time, the in-liquid mark generating means It is preferable that the mode is switched as in (X).

More specifically, for example, a method in which a cartridge type column is installed by being fitted into the column mounting position can be mentioned. Then, the submerged mark generating means (X) is also of the same size and shape, and can be switched to the mode for measuring the arrival time by fitting it in the column.

The installation of the column and the in-liquid mark generating means (X) in the apparatus is not limited to the above-described method. For example, as shown in FIG. Another method may be used such as separately providing a liquid feed line for the mark generating means (X) and switching the liquid flow path.

In the present invention, the sorting means (B) is provided with a change-over switch that can be freely opened and closed. As this change-over switch, a known switch can be used and can be changed by a computer. That is, when the time instructed by the computer arrives, it automatically switches and the acquisition of the sample is started by the fraction collector.

The control of the selector switch of the sorting means (B) is preferably based on detection of a sample by the detector (C) described in detail below.
That is, the detector (C) described in detail below is used to confirm the presence or absence of dissolved components in the eluate, and only the eluate containing dissolved components is obtained as a fraction, and discarded if no dissolved components are present. It may be. A known system can be used as such a system.

In the present invention, the eluate that has passed through the column (A) is fractionated by the fractionating means (B), and is collected in a test tube as a single or a plurality of fractions such as fractions 1, 2, 3,. The At that time, fraction 1 is a fraction from 0 to 10 seconds from the start of outflow, fraction 2 is a fraction from 1 to 2 minutes from the start of outflow, etc. Fraction 2 is fraction data associated with an outflow amount such as 5 to 10 ml of the outflow start, and is stored in the data storage means (H).

At the time of the sorting operation using the column, the fraction test tube is obtained in consideration of the measured corrected volume (or corrected time) described above.
For example, if the set time set as the switch switching time is 0.1 seconds earlier than the actual sample elution start time, sample acquisition was started from (set time−0.1 seconds). , Qualitative analysis result data processing may be performed. (Same for late)

An example of such fraction data is shown in FIG. 73 in FIG. 7 indicates what time zone component each fraction test tube has acquired.

The liquid chromatograph apparatus of this invention is equipped with a detector (C). That is, the detector (C) is used to measure whether or not a dissolved component is present in the eluate, thereby performing chromatography while confirming the elution position of the dissolved component. As the detector (C), those widely used in various chromatographic apparatuses can be used, and the detector (C) is selected from the group consisting of ultraviolet absorption, differential refractive index, evaporation scattered light, mass spectrometry, and circular dichroism. Can be used.

The function as the detector (C) may be performed by the eluate analysis means (D) described in detail below, or separately from the eluate analysis means (D), the detector (C) It may be provided. In addition, since the analysis means (D) described in detail below is a means for performing a qualitative analysis, the analysis means (D) needs to be an analysis that can clarify what kind of compound the target compound is. The detector only needs to be able to confirm the presence or absence of the eluted component, and therefore, the analysis may be performed in a form suitable for such a purpose.

For example, when a mass spectrometer is used as both a detector (C) and an analysis means (D), the total ion mode is used to check only the presence or absence of eluted components in the liquid. It may be used as data of (C), and data as a chart may be used as data of the analysis means (D).

The measurement result performed by the detector (C) is also sent to the data storage means (H) and stored. The data stored here is called an elution curve and shows the relationship between time and the presence or absence of an eluate. FIG. 6 is an example of information displayed on the display means (F). The elution curve measured by the detector (C) can be shown as an elution curve with time on the horizontal axis as 72 in FIG.

As shown in FIG. 6, when the relationship between the elution curve obtained by the detector (C) and the acquisition time of each fraction test tube by the sorting means (B) is displayed in parallel, the fraction test tube is The component corresponding to the elution peak is preferable in terms of easy understanding.

The preparative liquid chromatograph apparatus of the present invention is to perform the display means (F) while displaying the detection result by the detector (C) and the fraction data by the preparative means (B) in an overlapping manner. preferable.

In the preparative liquid chromatograph apparatus of the present invention, the analysis means (D) includes at least one qualitative analysis means for continuously qualitatively analyzing the eluate. That is, the effluent is continuously subjected to qualitative analysis, and the obtained data is stored in the data storage means (H).

The analysis means (D) is not particularly limited as long as it can qualitatively analyze the compound to be separated and confirm that the target compound is separated. For example, ultraviolet absorption, mass spectrometry And nuclear magnetic resonance (NMR). Two or more of these may be provided. The qualitative analysis needs to be performed to such an extent that it can be confirmed that the target is the target and that the target is separated with the purity desired by the observer. Among these, it is particularly preferable to use mass spectrometry.

For example, when UV absorption is performed, the absorbance at a single wavelength is not measured, but the UV absorption at a wide range of wavelengths is measured. By doing so, it is possible to easily determine whether or not the target compound is present at a high concentration in a predetermined fraction by confirming the absorption pattern.

Moreover, if it is a mass spectrum, it will be necessary to create the chart which measured the molecular weight. As a result, the target substance can be identified and the presence of impurities can be confirmed in the same manner as the ultraviolet absorption described above.

Furthermore, in recent years, NMR analysis techniques have improved, and attempts have been made to connect NMR as detection means in various chromatographs. These known methods may be applied to the present invention, and NMR measurement may be continuously performed on the separated eluate.

The analysis by the analysis means (D) is performed on the outflow line in the case of the analysis means that does not cause decomposition by analysis, such as ultraviolet absorption, and the effluent after the analysis is directly in the fraction. In the case of an analysis method in which the sample is decomposed by analysis, such as mass spectrometry, a part of the effluent may be introduced into the measurement line and continuously measured.

The analytical devices constituting these analytical means (D) are each commercially available, and these are combined with a preparative liquid chromatograph and integrated as a whole by providing a computer storing a control program. Can be used.

Since the preparative liquid chromatograph apparatus of the present invention has the above-described configuration, the observer obtained the analysis means (D) while referring to the data of the detector (C) and the fraction data. Specify the location where qualitative analysis data is required. In this way, the qualitative analysis data designated by the observer is displayed on the display means (F).

By specifying the position in this way, the observer can easily obtain the desired qualitative analysis data, obtain the analysis data at the time of fractionation by the preparative liquid chromatograph device, and Can be done accurately.

The qualitative analysis data is preferably measured continuously at regular intervals. Therefore, the spectrum is measured as a spectrum that gradually changes in accordance with the composition change of the eluate. And in the measuring apparatus of this invention, it is preferable to calculate the integral value or average value corresponding to a specific fraction, and to make this into fraction data.

That is, as described above, in the present invention, the elution time corresponding to the components in the test tube in each fraction can be acquired as extremely accurate data with strict correction. it can.
For this reason, it is necessary to know very accurately whether the qualitative analysis data corresponding to the sample in the test tube of the fraction can be obtained by integrating or averaging the range of the qualitative analysis data measured continuously. Can do. For this reason, the qualitative analysis data of the sample in the test tube can be automatically acquired without performing the qualitative analysis of the sample in the test tube again.

The instruction by the observer is not particularly limited. For example, if a specific point of the measurement result by the detector is designated, the analysis result by the analysis means (D) at that time can be displayed on the screen. Accordingly, analysis data on the contents can be obtained without performing a qualitative analysis on the contents of the fraction test tube.

Such a state is shown in FIG. In FIG. 7, when an operator designates a specific fraction 73, an integral value or an average value in a range designated thereby is displayed as qualitative analysis data 74. In 74, the spectrum peak of B displayed in 75, the B 'derived ion spectrum peak B', and the molecular ion spectrum peak of C are displayed. Thus, the operator can obtain the analysis result of the fraction test tube as the automatically obtained qualitative analysis data without performing the qualitative analysis of the fraction test tube.

The present invention is also a method for measuring arrival time measurement in a preparative liquid column chromatograph using the preparative liquid column chromatograph provided with the in-liquid mark generating means (X) as described above. Such a method is as described above, and the above-described effects can be obtained by performing liquid column chromatography after performing such arrival time measurement in a preparative liquid column chromatograph apparatus. it can.

The present invention further relates to a chromatography method using the above-described preparative liquid column chromatograph apparatus, and the step (1) of measuring the arrival time in the preparative liquid column chromatograph apparatus by the above-described method,
Using the arrival time measured in step (1), the switching time (i) of the changeover switch of the sorting means (B), the relationship (ii) between the measurement time and the measurement result of the analysis means (D), and And (2) correcting at least one of the effluent flow rates (iii)
In addition, the chromatography is performed by the step (3) of performing the chromatography while continuously performing the qualitative analysis by the analysis means (D),
It is also a fractionation method using a column chromatograph characterized in that the qualitative analysis result obtained in step (3) is processed by reflecting the correction result in step (2).

Each step of such a column chromatographic fractionation method is as described above. Therefore, fractionation by column chromatography can be performed according to such a method. Moreover, any of the steps (1), (2) and (3) described above may be performed first. That is, after performing the correction in the steps (1) and (2), the step (3) may be performed, or after performing the step (3), the correction in the steps (1) and (2) is performed. Also good.

By such a chromatographic method, the contents of the test tube and the spectrum can be compared more accurately, and the experimental work can be made much more efficient.

Hereinafter, the present invention will be described based on examples.
(Test method)
This experiment has the outline shown in FIGS. 1 and 2, and as shown in FIG. 9, the in-liquid mark generating means (X) (bubble generating means) is installed between the column and the detector (C). The equipment used was used. Furthermore, a scale is provided in the vicinity of the changeover switch so that numerical values can be easily read visually.
At the start of the experiment, the arrival time from when the sample was detected by the detector (C) until it reached the selector switch of the sorting means (B) was a theoretical value calculated from the volume and flow rate in the pipe.
In this state, air was introduced into the loop using the six-way valve shown in FIG. 9, and then air was pushed out to form bubbles in the liquid, which was then fed by a pump. In this arrival time measurement test, only the solvent is fed.
Then, when the switching time of the initial setting reached, the switch was switched and the pump was stopped at the same time to stop the liquid feeding.
The deviation was measured by reading the bubble position at that time, and as a result, TD = 16 seconds was calculated.

After confirming this point, a separation operation by preparative liquid chromatography was performed on a sample in which two kinds of compounds were mixed. In this test, the test is performed based on the premise that it is necessary to obtain pure compound 2.
In such fractionation, TD = 0, 8, 16, 24, and 32 seconds are set, and the changeover switch of the fractionation means (B) is switched at this time, so that the fraction is collected at regular intervals from the start of compound 1 elution. A sample was obtained. And the test tube (test tube 12 in the description of a figure) from which it was judged by analysis of the elution curve by a detector that compound 2 was acquired with high purity was acquired, and mass spectrometry was performed about this test tube 12. .

As a result, when fractionation was performed with TD = 0 and 8, a sample in which compounds 1 and 2 were mixed in the fraction was obtained. On the other hand, when correction is performed based on TD = 16 seconds calculated by the above-described method, only the target compound 2 is present in the test tube 12 predicted from the measurement result of the detector, A sample without Compound 1 was obtained.
On the other hand, when TD = 24 seconds and 32 seconds, a sample containing only compound 2 is obtained, but the peak intensity of the compound is weaker than that when TD = 16. For this reason, it is estimated that the acquisition time is too late and the sample after the peak position is acquired.

In view of the above experimental results, the content of the test tube is measured by accurately measuring the arrival time from the detection by the detector (C) by the method of the present invention until the switch of the sorting means (B) is reached. It is clear that the object and spectrum can be compared more accurately.

With the preparative liquid chromatograph device of the present invention, it is possible to start the separation of the solution accurately and to perform the qualitative analysis together. Thereby, each fraction and the data obtained by qualitative analysis can be made to respond | correspond correctly, and the qualitative analysis data which correctly reflected the composition obtained in each fraction can be obtained.

1 column or submerged mark generating means 2 fraction collector 3 detector 4 qualitative analyzing means 5 controller 11 eluent 12 injector 13 pump 14 liquid sensor 15 drain pipe 17 submerged mark generating means 31 six-way valve 32 syringe 33 switching valve 41 threshold 71 Qualitative analysis data acquisition range selected from 71 chromatograms 72 Chromatogram 73 Qualitative analysis data 75, 76 Chromatogram 77-81 Mass spectrum 82 Average spectrum displayed on fraction display means corresponding to qualitative analysis data acquisition range

Claims (9)

A preparative liquid column chromatograph apparatus comprising a detector (C) and a preparative means (B),
For the sorting, the changeover switch of the sorting means (B) is automatically switched after the set delay time has elapsed from the time when the detection intensity in the detector (C) exceeds the set threshold. Liquid column chromatograph. A preparative liquid comprising a sorting means (B) having a changeover switch for switching the flow path through which the eluate flows from the column, an eluent detector (C), and an eluent analyzing means (D) In column chromatograph equipment,
The fractionation is characterized by comprising in-liquid mark generating means (X) for measuring the arrival time from when the sample is detected by the detector (C) until reaching the selector switch of the sorting means (B). Liquid column chromatography equipment. The preparative liquid column chromatograph apparatus according to claim 2, wherein the in-liquid mark generating means (X) generates bubbles in the eluate. The in-liquid mark generating means (X) has a shape that can be mounted on the column mounting portion. When measuring the arrival time, the column (A) is replaced with the in-liquid mark generating means (X). The preparative liquid column chromatograph apparatus according to claim 2 or 3, wherein measurement is performed. The sorting means (B) comprises a reading means (Z) for reading the position of the in-liquid mark generated by the in-liquid mark generating means (X) in the pipe near the changeover switch. Alternatively, the preparative liquid column chromatograph apparatus according to 4. Furthermore, the display unit (E) for displaying the analysis result, the calculation unit (F) for controlling the display unit (E) and the data storage unit (G), and the data storage unit (G) for storing the data are provided.
The data storage means (G) stores the continuous measurement results of the detector (C), the continuous measurement results of the analysis means (D), and the fraction acquisition times of the sorting means (B). Item 6. A preparative liquid column chromatograph according to Item 2, 3, 4 or 5. The display means (E) displays results obtained by integrating or averaging the continuous measurement results of the analysis means (D) according to the fraction acquisition times of the fractionation means (B). The preparative liquid column chromatograph according to 4 or 5. In the preparative liquid column chromatograph of claim 2, 3 or 4,
The state in which the in-liquid mark generating means (X) is present in the eluate flow path,
The eluate is allowed to flow out, the changeover switch of the sorting means (B) is switched at the initially set switching time, and at the same time, the liquid feeding pump is stopped,
An arrival time measurement method in a preparative liquid column chromatograph, wherein an error in the arrival time is measured by confirming a position of the in-liquid mark in the pipe at that time. A chromatography method using the preparative liquid column chromatograph according to claim 2, 3, 4, 5, 6 or 7,
A step (1) of measuring an arrival time in a preparative liquid column chromatograph by the method according to claim 8;
Using the arrival time measured in step (1), the switching time (i) of the changeover switch of the sorting means (B), the relationship (ii) between the measurement time and the measurement result of the analysis means (D), and And (2) correcting at least one of the effluent flow rates (iii)
In addition, the chromatography is performed by the step (3) of performing the chromatography while continuously performing the qualitative analysis by the analysis means (D),
A fractionation method using a column chromatograph, wherein the qualitative analysis result obtained in step (3) is processed by reflecting the correction result in step (2).

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