JP5195686B2 - Liquid chromatograph - Google Patents

Description

  The present invention relates to a liquid chromatograph apparatus (hereinafter abbreviated as LC), and in particular, a two-dimensional analysis of components separated by a first-stage (first-dimension) LC by a second-stage (second-dimension) LC. The type of LC.

  In LC, various components contained in a sample can be separated and detected in a time direction by a column. However, when the sample to be measured contains a very large number of components or a plurality of components with similar properties, one type of column or one type of column Under analytical conditions, each component may not be sufficiently separated. In order to analyze such a sample, conventionally, an LC called a two-dimensional (multi-dimensional) type LC has been used (see Patent Document 1). In the two-dimensional LC, the eluate fractionated in the first dimension LC is introduced into the second dimension LC set with different separation conditions from the first dimension LC and separated in the first dimension LC. A plurality of components that could not be separated can be detected separately.

  Among such two-dimensional LCs, one using a preparative LC as the first-dimensional LC is known. In the following description, a two-dimensional LC using a preparative LC in the first dimension is simply referred to as a two-dimensional LC. In this type of two-dimensional LC, first, the eluate in the interval (the period from the peak start point to the end point) in which the target component is detected is fractionated into a container such as a vial by the first dimension preparative LC. To do. Next, the eluate is collected from the vial containing the eluate containing the target component, introduced into the second-dimensional LC, and LC analysis is performed. Patent Document 2 discloses an apparatus that uses a liquid handler to introduce the eluate separated by the first-dimensional LC into the second-dimensional LC. In the liquid handler, the probe moves to the sorting position by the first-dimensional LC and inhales the separated eluate, and then the probe moves to the injection port of the second-dimensional LC and sucks first. The sample is injected by discharging the eluted solution.

  In order to perform the analysis of the second-dimensional LC with the two-dimensional LC, it is necessary to designate the sample injection amount as the analysis condition. As described above, when performing fractionation control such that the eluate in the section where the target component is detected is dispensed into a vial, the amount of eluate collected in the vial varies depending on the size of the peak, etc. Absent. Therefore, if the analysis by the second-dimensional LC is performed after specifying a predetermined sample injection amount in advance, the amount of the eluate collected by the first-dimensional preparative LC is considerably larger than the injection amount. Conversely, there may be cases where it is insufficient. In the former case, the separated eluate remains, and the sample cannot be used efficiently. On the other hand, in the latter case, an insufficient amount of air is introduced into the flow path, which not only causes noise on the detection data, but air may accumulate in the flow path and may not escape. It interferes with the analysis.

  In order to avoid such problems, it is necessary to determine the sample injection amount of the second-dimensional LC in accordance with the volume of the eluate collected by the first-dimensional preparative LC. However, in order to do so, an analyst must confirm the results of LC separation in the first dimension (for example, the start time and end time of the fractionation) and calculate the volume of the actually separated eluate by calculation. did not become. Therefore, it is not only very troublesome for the analyst, but there is a problem that it takes time from the end of the first-dimensional LC analysis to the start of the second-dimensional LC analysis, resulting in a decrease in throughput.

JP 2003-254955 A JP 2004-355241 A

  The present invention has been made in view of the above-mentioned problems, and the object of the present invention is appropriate for the second-dimensional LC regardless of the amount of the eluate separated by the first-dimensional preparative LC. An object of the present invention is to provide a two-dimensional LC capable of introducing an amount of sample (eluate).

The present invention, which has been made to solve the above-mentioned problems, separates components in a sample by the first-dimensional liquid chromatographic analysis, dispenses the sample solution containing the components into different containers, and dispenses them into the containers. A two-dimensional liquid chromatograph apparatus for introducing the sample liquid into a second-dimensional liquid chromatograph and executing analysis;
a) Liquid for calculating the amount of sample liquid dispensed in one or more containers based on the information on the start time and end time of the separation obtained during the separation in the first-dimensional liquid chromatographic analysis A quantity calculating means;
b) analysis condition setting means for setting the injection amount of each sample liquid in the second-dimensional liquid chromatographic analysis using the liquid volume of the sample liquid dispensed in the one or more containers;
It is characterized by having.

  Usually, in the first-dimensional LC (preparative LC), the start and end timing of the fractionation are determined based on the signal obtained by the detector, that is, the chromatogram signal. For example, by comparing the chromatogram signal with a predetermined threshold or comparing the amount of change in the chromatogram signal with time (gradient of the chromatogram curve) with the threshold, the peak start point and end point of the target component And the sample liquid obtained in the section from the peak start point to the end point is dispensed into one container. The liquid amount calculation means obtains information on the start time and end time at the time of such preparative control, and information on the flow rate of the mobile phase of the first-dimensional LC (this is the analysis condition of the first-dimensional LC) And the volume of the sample solution dispensed into the container is calculated from this information. When the sample liquid is dispensed into a plurality of containers, the volume of the sample liquid is obtained for each container.

  The analysis condition setting unit sets the injection amount of the sample liquid with reference to the information on the liquid amount calculated by the liquid amount calculation unit when setting the analysis condition in the second-dimensional LC. For example, when analyzing a sample solution dispensed in a certain container with the second-dimensional LC, the same amount as the dispensed sample solution may be set as the injection amount of the sample solution. In other words, the larger the amount of sample liquid dispensed by the first dimension LC, the more the amount injected into the second dimension LC, and the greater the amount of sample liquid dispensed by the first dimension LC. The smaller the amount, the smaller the amount injected into the second-dimensional LC.

  However, considering, for example, the loss of the sample solution accompanying the suction / discharge of the sample solution, the volatilization of the sample solution, etc., the entire amount of the sample solution contained in the container is not always available. Therefore, in the liquid chromatograph apparatus according to one aspect of the present invention, the analysis condition setting means may obtain the injection amount by multiplying the fractionation volume by a predetermined coefficient less than 1. This coefficient may be appropriately determined within a range of, for example, about 0.95 to 0.98.

  According to the liquid chromatograph apparatus according to the present invention, even when the amount of the sample liquid dispensed into the container in the first-order preparative LC is not constant, the sample is always appropriate, that is, not excessive or insufficient. The liquid can be injected into the second dimension LC. Accordingly, the sample can be effectively used and unwanted air can be prevented from being injected into the flow path during sample injection. In addition, it is not necessary for the analyst himself to calculate the fractionation volume, and the fractionation volume is obtained almost simultaneously when the fractionation in the first dimension LC is completed. Throughput is also improved.

1 is a schematic configuration diagram of a two-dimensional LC according to an embodiment of the present invention. The flowchart of preparative LC operation | movement of the 1st dimension in the 2-dimensional type | mold LC of a present Example. The flowchart of the analysis schedule automatic preparation of the 2nd dimension using the analysis result of the 1st dimension in the 2D type LC of a present Example. The schematic diagram which shows the arrangement | sequence of the vial processed in 2D type | mold LC of a present Example. The schematic diagram for demonstrating the characteristic operation | movement in the two-dimensional type | mold LC of a present Example.

  Hereinafter, a two-dimensional LC according to an embodiment of the present invention will be described with reference to the accompanying drawings.

  FIG. 1 is a schematic configuration diagram of a two-dimensional LC according to the present embodiment. This two-dimensional LC includes a six-way valve 3 that can be switched between two states of a solid line and a dotted line in FIG. The a-port of the 6-way valve 3 is connected to the first liquid feed pump 2 for sucking and feeding the mobile phase from the first mobile phase container 1, and the c-port is used to suck and feed the mobile phase from the second mobile phase container 8. A second liquid feed pump 9 for feeding is connected. An autosampler 4 including an injector 5 for injecting a sample solution into the flow path is provided in the flow path between the b port and the e port of the 6-way valve 3. Further, a first detector 7 is connected to the f port of the 6-way valve 3 via the first column 6, and a second detector 11 is connected to the d port via the second column 10. The first detector 7 may be of any type as long as it does not consume a sample solution, but an ultraviolet-visible spectroscopic detector or the like is often used. On the other hand, since the second detector 11 may be a detector that consumes the sample solution, a mass spectrometer can be used in addition to an ultraviolet-visible spectroscopic detector.

  A fraction collector 12 is connected to the outlet side of the first detector 7. The fraction collector 12 includes a sorting unit 121 including a sorting valve 122, a sorting nozzle 123, and the like, a sorting control unit 125, and an A / D converter 126. Further, in order to control the operation of each unit in an integrated manner and to perform data processing, a control / processing unit 20 embodied by a personal computer (PC) is provided. The control / processing unit 20 includes an operation unit such as a keyboard. 21 and the display part 22 are connected.

  In the fraction collector 12, the sorting control unit 125 has a function of controlling the operation of the sorting unit 121 based on the detection signal (chromatogram signal) obtained by the first detector 7. Can also be incorporated in the control / processing unit 20. In the present embodiment, the sorting control unit 125 also has a function of calculating the sorting volume as will be described later, but it is obvious that this can also be incorporated in the control / processing unit 20.

  In FIG. 1, an autosampler 4 having a function of selecting a designated vial from a plurality of samples prepared in each vial, aspirating the sample liquid in the vial, and applying it to the injector 5; The fractionation part 121 of the fraction collector 12 having the function of injecting the eluate (sample solution) into one vial selected from the prepared vials is described separately. It can be realized with a liquid handler.

  A characteristic analysis operation of the two-dimensional LC according to the present embodiment will be described with reference to FIGS. FIG. 2 is a flow chart of preparative LC operation in the first dimension, FIG. 3 is a flow chart of automatic creation of an analysis schedule of the second dimension using the analysis result of the first dimension, and FIG. 4 is a flowchart of a vial handled by the above liquid handler, for example. FIG. 5 is a schematic diagram for explaining a characteristic operation.

  Before the analysis is executed, the analyst inputs and sets a schedule for the first-dimensional LC analysis using the operation unit 21. FIG. 5A is an example of the first-dimensional analysis schedule. In this example, 10 μL of the sample solution in the vial number “1” prepared in the rack of the autosampler 4 is injected, and LC analysis is performed under the conditions described in the analysis condition file named “Method 1”. The target component is dispensed into the sorting containers “101” to “103”. As shown in FIG. 4, the sorting containers are arranged in the same rack as the vial in which the sample solution is prepared, a part of which is used for the first-dimensional LC analysis, and the other one-dimensional. It is a container for collecting a sample liquid in the LC analysis of the eye and a sample used for the second-dimensional LC analysis.

  The analysis condition file includes sorting conditions such as peak detection conditions in addition to separation conditions such as the liquid flow rate of the mobile phase. In general, the peak detection method includes a method of determining that a peak is detected when the chromatogram signal level exceeds a specified threshold level LEVEL, and a peak detection when the upward slope of the chromatogram curve exceeds a predetermined value. There is a method for determining that the peak has appeared, and determining that the peak has ended when the downward slope of the curve has exceeded a predetermined value and has become equal to or less than the predetermined value. Either one method or both methods The peak detection is performed by using together. Here, in order to simplify the description, it is assumed that peak detection is performed by simple determination of the signal level with respect to the threshold LEVEL, but the peak detection method is not limited to this.

  When the first-dimensional LC analysis is started, the first liquid feeding pump 2 sucks the mobile phase from the first mobile phase container 1 under the control of the control / processing unit 20, and the first liquid phase pump 1 through the injector 5 The mobile phase is fed to the column 6 at a substantially constant flow rate. The autosampler 4 prepares a prescribed amount of sample solution according to the analysis schedule (in the case of FIG. 5A, only 10 μL of the sample solution in the vial with the vial number “1” is prepared), and the injector 5 at a predetermined timing. Injects the sample solution into the mobile phase. The injected sample solution is introduced into the first column 6 along the flow of the mobile phase, and various components in the sample solution are separated in the process of passing through the first column 6. However, a contaminant component having properties similar to the target component overlaps the target component. An eluate (sample liquid) containing a component eluted from the first column 6 with a time shift is introduced into the first detector 7, and the first detector 7 has a detection signal having an intensity corresponding to the concentration of the introduced component. Is output. The sample liquid that has passed through the first detector 7 is introduced into the fraction collector 12 as it is.

  In the fraction collector 12, the fractionation control unit 125 switches the fractionation valve 122 so that the introduced sample liquid flows into the waste liquid flow path (the valve on the side toward the fractionation nozzle 123 is turned off). A detection signal (chromatogram signal) from the first detector 7 is converted into a digital value by the A / D converter 126 and input to the sorting control unit 125. As the analysis starts, the preparative control unit 125 reads the chromatogram signal level at predetermined sampling time intervals (step S1), and compares the signal level with the threshold value LEVEL to determine whether the peak start point is reached. Is determined (step S2). When it is desired to fractionate only the target component having a known retention time, the retention time is included in the sorting conditions, and only the peak that appears in the vicinity of the retention time may be detected.

  If it is determined in step S2 that it is not the peak start point, the sorting control unit 125 determines whether or not it is the peak end point (step S5). The peak end point is also determined by comparing the chromatogram signal level with the threshold LEVEL. If it is determined that it is neither the peak start point nor the end point, it waits for a predetermined sampling time (step S10) and determines whether or not the analysis is complete (step S11). And return.

  When the sample liquid containing the target component starts to reach the first detector 7, the chromatogram signal level rises, and it is determined in step S2 that it is the peak start point. Then, the sorting control unit 125 switches the sorting valve 122 (turns on the valve on the sorting nozzle 123 side), and injects the sample solution into the designated vial (step S3). That is, the sample solution is injected into the sorting container numbered “101” for the first peak of the target component. Further, the sorting control unit 125 stores the sorting start time in the internal memory (step S4), and proceeds to the above-described step S10.

  As described above, after waiting for the sampling time after being determined to be the peak start point, the process returns to step S1, and steps S1 → S2 → S5 → S10 → S11 are repeated until the peak end point is reached. When the target component is not present in the sample liquid introduced into the first detector 7, it is determined that the peak end point is reached in step S5, and the sorting control unit 125 sorts the sample liquid so that it flows to the waste liquid channel side. The valve 122 is switched (step S6). Thereby, the dispensing of the sample solution into the first vial is completed, and the dispensing control unit 125 stores the sorting end time in the internal memory (step S7). Thereafter, the fractionation control unit 125 calculates a time T [min] required for the fractionation by subtracting the fractionation start time from the stored fractionation end time, and at this time T, the mobile phase by the first liquid feeding pump 2 is calculated. Is multiplied by the delivery flow rate P [mL / min] to calculate the amount of the sample solution just collected, that is, the fractionation volume for the fractionation container (vial) "101" (step S8). Then, after notifying the control / processing unit 20 of the sorting information including the vial number, the start / end time of the sorting, and the sorting volume (step S9), the process proceeds to step S10.

  Each time the peak of the target component appears in the chromatogram, the processes of steps S3 and S4 are executed at the peak start point, and the processes of steps S6 to S9 are executed at the peak end point, and each of the vials numbered "101" to "103" A sample solution corresponding to the peak is collected. That is, as shown in FIG. 4, the sample solution containing the component derived from the sample solution in the vial having the number “1” is dispensed into three vials having the numbers “101” to “103”. In addition, sorting information is notified to the control / processing unit 20 for each peak. The control / processing unit 20 stores the notified sorting information in a storage device such as an HDD.

  When the analyst instructs the creation of the sorting report at the operation unit 21 at the stage where the first-dimensional LC analysis is completed (or at an arbitrary time thereafter), the control / processing unit 20 stores the data stored in the storage device. Based on the collection information, a sorting report having a format as shown in FIG. 5B is created and displayed on the screen of the display unit 22. That is, in this sorting report, the actual amount of sample liquid (sorting volume) collected in each sorting container is shown. This allows the analyst to know the sorting volume accurately without having to calculate it.

  Subsequently, the control / processing unit 20 automatically creates a second-dimensional analysis schedule according to the flowchart shown in FIG. That is, first, the counter value i is set to 1 (step S21), and then it is determined whether or not all the first-dimensional analysis results have been referred to (step S22). If there is an analysis result that has not yet been referred to, the process proceeds to step S23, and the i-th vial number and the corresponding preparative volume in the first-dimensional LC analysis result are displayed as the vial number and sample in the second-dimensional analysis schedule. Set to injection volume. Then, the counter value i is incremented (step S24), and the process returns to step S22. By repeating the above process until it is determined as Yes in step S22, the numerical values (number of the sorting container and the sorting volume) in the sorting report shown in FIG. 5B are shown in FIG. 5C. It is set in each column (vial number and injection amount) in the second-dimensional analysis schedule as shown.

  The analyst calculates the amount of the sample liquid collected in the sorting container from the start time and end time of the sorting in the first dimension LC analysis, and based on the calculation result, the sample injection of the second dimension LC analysis is performed. Even if the amount is not determined, an appropriate injection amount corresponding to the amount of the sample solution collected in the first-dimensional LC analysis, that is, an appropriate injection amount is automatically set in the second-dimensional LC analysis. . Accordingly, it is possible to prevent air from being injected into the mobile phase without leaving the collected sample solution, and conversely, there is not enough sample solution to be injected. In addition, such setting is automatically performed, so that the burden on the analyst is reduced, and the problem of excess or shortage of the injection amount due to the calculation error or setting error of the analyst is eliminated.

  In the example shown in FIG. 5 (c), the entire amount of the sample solution dispensed into the sorting container, that is, the vial is set to be injected into the second-dimensional LC. Considering the decrease in the amount of liquid due to adhering or volatilizing, it is safer to keep the sample injection amount slightly smaller than the fractionation volume (aeration can be avoided). Therefore, the safety factor K is determined in advance as, for example, 0.98, 0.95, and the value obtained by multiplying the preparative volume by this safety factor K is set as the injection amount in the second-dimensional LC analysis. It is preferable. For example, when K = 0.98, the injection amounts shown in FIG. 5C are 1.47, 1.61, 1.74 [mL] from the top.

When the second-dimensional LC analysis is started after the second-dimensional analysis schedule is set as described above, the six-way valve 3 is indicated by a dotted line in FIG. 1 under the control of the control / processing unit 20. When switched to the connected state, the second liquid feeding pump 9 sucks the mobile phase from the second mobile phase container 8 and feeds the mobile phase to the second column 10 through the injector 5 at a substantially constant flow rate. The autosampler 4 prepares a specified amount of sample solution according to the analysis schedule (in the case of FIG. 5C, first prepare 1.5 mL of the sample solution in the vial with the vial number “101”) at a predetermined timing. The injector 5 injects a sample solution into the mobile phase. The injected sample solution is introduced into the second column 10 along the flow of the mobile phase, and various components in the sample solution are separated in the process of passing through the second column 10.

  An eluate (sample liquid) containing a component eluted from the second column 10 with a time shift is introduced into the second detector 11, and the second detector 11 has a detection signal having an intensity corresponding to the concentration of the introduced component. Is output. The control / processing unit 20 receives this detection signal, creates a chromatogram, and performs qualitative analysis and quantitative analysis based on the peak appearing in the chromatogram. When the second-dimensional LC analysis for the sample solution in the vial with the vial number “101” is completed, the second-dimensional LC analysis for the sample solution in the vials with the vial numbers “102” and “103” is subsequently executed. The

  The above-described embodiment is an example of the present invention, and it is a matter of course that modifications, corrections, and additions may be appropriately made within the scope of the present invention, and included in the scope of the claims of the present application.

DESCRIPTION OF SYMBOLS 1 ... 1st mobile phase container 2 ... 1st liquid feeding pump 3 ... 6 way valve 4 ... Autosampler 5 ... Injector 6 ... 1st column 7 ... 1st detector 8 ... 2nd mobile phase container 9 ... 2nd liquid feeding Pump 10 ... Second column 11 ... Second detector 12 ... Fraction collector 121 ... Sorting unit 122 ... Sorting valve 123 ... Sorting nozzle 125 ... Sorting control unit 126 ... A / D converter 20 ... Control / processing unit 21 ... Operation unit 22 ... Display unit

Claims (2)

The components in the sample are separated by the first-dimensional liquid chromatographic analysis, the sample solution containing the components is dispensed into different containers, and the sample solution dispensed in the containers is introduced into the second-dimensional liquid chromatograph. A two-dimensional liquid chromatograph that performs analysis.
a) Liquid for calculating the amount of sample liquid dispensed in one or more containers based on the information on the start time and end time of the separation obtained during the separation in the first-dimensional liquid chromatographic analysis A quantity calculating means;
b) analysis condition setting means for setting the injection amount of each sample liquid in the second-dimensional liquid chromatographic analysis using the liquid volume of the sample liquid dispensed in the one or more containers;
A liquid chromatograph apparatus comprising: The liquid chromatograph apparatus according to claim 1,
The liquid chromatograph apparatus characterized in that the analysis condition setting means obtains an injection amount by multiplying a preparative volume by a predetermined coefficient less than 1.

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