JP2007183173A - Preparative chromatograph device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparative chromatograph device capable of specifying more appropriate positions as a preparative start point and a termination point. <P>SOLUTION: The preparative chromatograph device has a function of calculating a time zone (preparative operable region) capable of setting the start point or termination point of the preparative isolation by a fraction collector at the present time based on the time (delay time) required by arrival of a sample component having passed a detector at the fraction collector. The preparative operable region 31 is superimposed and displayed on chromatogram of a sample during analysis on a screen of a monitor, and an operator is allowed to specify any time in the preparative operable region 31 as the preparative start point and termination point. When automatic preparative isolation is performed, the peak start/terminal points in the preparative isolation operable region 31 can be updated to more desirable positions or disabled based on the latest chromatogram. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a preparative chromatograph apparatus.

  2. Description of the Related Art So-called preparative chromatograph apparatuses that separate and collect a plurality of components contained in a sample using a chromatograph apparatus such as a high-performance liquid chromatograph have been known (for example, Patent Document 1). See).

  Such a preparative chromatograph apparatus includes a separation unit having a liquid feed pump and a column, a detector provided in the subsequent stage, a fraction collector, and a control unit for controlling them. After the sample components separated and eluted are detected by passing through a detector such as an ultraviolet-visible spectrophotometer, they are introduced into a fraction collector in the subsequent stage and separated into different vials for each component.

  Here, as a method of controlling the sorting operation by the fraction collector, a method called manual sorting in which an operator determines the timing of sorting and an automatic sorting in which the apparatus side automatically determines the timing of sorting is used. There is.

  In the above manual fractionation, the operator observes the chromatogram of the sample under analysis displayed on the monitor almost in real time, and performs the key operation etc. when it is judged appropriate from the shape of the chromatogram. Instruct the start or end of At this time, in the conventional preparative chromatograph apparatus, when receiving an instruction to start or end the fractionation from the operator, the fraction collector immediately starts or ends the fractionation, or is detected by the detector. Taking into account the time required for the components to reach the fraction collector (this is called the “delay time”), the sample components detected by the detector at the time of receiving the instruction are collected at the timing when they reach the fraction collector. The sorting is performed after adding the time correction so as to be performed.

  On the other hand, in the case of automatic sorting, the control unit embodied by a microcomputer provided in the fraction collector, a personal computer equipped with predetermined software, or a dedicated control device (system controller) detects the sample being analyzed. When a signal is received from the detector and the intensity change (temporal increase / decrease) of the detection signal is calculated and the value exceeds or falls below a preset value (this is called the “Slope value”) Are determined as the peak start and end points. For example, when the slope value is 200 μV / sec, first, the point in time when the slope of the change in signal intensity of the detector exceeds 200 μV / sec is recognized as the peak start point. Thereafter, the slope of the signal intensity change becomes a negative value near the peak top, and further becomes a value lower than −200 μV / sec. Then, the point in time when the slope of the change in signal intensity becomes larger than −200 μV / sec is recognized as the peak end point.

JP 2000-214151 A ([0002], [0003], FIG. 1)

  In the case of manual fractionation, in the conventional fractionation chromatograph apparatus, only the part currently detected by the detector can be designated as the start point or end point of the fractionation. Therefore, in the apparatus that performs the control in consideration of the delay time as described above, in the portion that has been detected by the detector in the past and has not reached the fraction collector (hereinafter referred to as “sorting operation possible region”). Even when it is desired to start or end sorting, such an area cannot be designated as a sorting start point or an end point. Therefore, for example, even if there is a valley between peaks in the sortable region, and the operator wants to start sorting from the valley portion, such sorting cannot be performed.

  In the case of the above-described conventional automatic sorting, the control unit calculates the intensity change of the detector signal at a fixed short time interval, and determines the peak start point / end point from the calculated value. For example, as shown in FIG. 7 (a), when the signal intensity fluctuates up and down in the vicinity of the peak top of the chromatogram, it is erroneously determined as the peak end point, and sorting is not performed until the correct peak end point, As shown in FIG. 7B, when the signal intensity is saturated, there is a problem that the start / end of sorting is repeated.

  Then, the problem which this invention tends to solve is providing the preparative chromatograph apparatus which can designate a more suitable position as a fractionation start point or an end point.

The first aspect of the preparative chromatograph apparatus according to the present invention, which has been made to solve the above problems, introduces a sample separated in the time direction by a chromatograph into a detector and a fraction collector, In a preparative chromatograph apparatus for fractionating and collecting each component in a sample with the fraction collector based on detection information by a detector,
a) a chromatogram generating means for generating a chromatogram based on a detection signal from the detector;
b) a delay time setting means for setting the time required for the sample component passing through the detector to reach the fraction collector;
c) Sorting operation possible region calculating means for calculating a time region in which the start point or end point of the sorting by the fraction collector can be set at the present time in consideration of the delay time;
d) display means for superimposing the preparative operation possible region on the chromatogram and displaying on the screen;
e) a sorting point designating means for allowing an operator to designate an arbitrary time as a sorting start point or an end point in the sorting operation enabled area;
f) a fractionation control means for controlling the operation of the fraction collector based on the designation in the fractionation point designation means;
It is characterized by having.

In addition, the second aspect of the preparative chromatograph apparatus according to the present invention, which has been made to solve the above-mentioned problems, introduces a sample whose components have been separated in the time direction by a chromatograph into a detector and a fraction collector. In the preparative chromatograph apparatus for fractionating and collecting each component in the sample with the fraction collector based on the detection information by the detector,
a) a chromatogram generating means for generating a chromatogram based on a detection signal from the detector;
b) a delay time setting means for setting the time required for the sample component passing through the detector to reach the fraction collector;
c) a preparative operation possible region calculating means for calculating a time region in which the start point or the end point of the fraction collection by the fraction collector can be set at the present time in consideration of the delay time;
d) peak detection means for detecting a peak start point or an end point existing on the chromatogram based on the chromatogram obtained from the chromatogram generation means;
e) a fractionation control means for operating the fraction collector at a timing when the peak start point or end point detected on the latest chromatogram deviates from the fractionation operable region;
It is characterized by having.

  Here, the delay time setting means may allow the operator to input the delay time directly, or based on the pipe capacity from the detector to the fraction collector, the liquid feed speed of the liquid feed pump, etc. The delay time may be calculated automatically.

  According to the preparative chromatograph apparatus according to the first aspect of the present invention having the above-described configuration, not only the portion currently detected by the detector but also a wider time range (sorting operation) considering the delay time. The sorting start point and end point can be specified over the possible area. Therefore, the operator determines the peak start point / end point based on the shape of the entire chromatogram obtained up to the present while visually recognizing the chromatogram of the sample under analysis that is constantly updated on the monitor screen. This makes it possible to specify a more accurate sorting timing.

  Further, according to the preparative chromatograph apparatus according to the second aspect of the present invention, the preparative timing is not always determined based on the change in the detector intensity signal at a very short time interval as in the prior art. Waveform analysis is performed based on the latest chromatogram data, and optimal points are detected as sorting start and end points in the region where sorting is possible. For this reason, since waveform analysis can be performed in the presence of data points around to some extent, it is possible to determine a more appropriate position as a peak start point / end point and perform highly accurate sorting.

  Hereinafter, the best mode for carrying out the present invention will be described with reference to examples.

  FIG. 1 is a block diagram showing a main configuration of a preparative liquid chromatograph apparatus (hereinafter referred to as “preparative LC”) according to the present embodiment. The eluent (mobile phase) stored in the eluent tank 11 is sucked by the liquid feed pump 12 and flows to the column 14 through the sample introduction unit 13 at a constant flow rate. The sample solution injected into the mobile phase in the sample introduction unit 13 rides on the mobile phase, is introduced into the column 14, and is separated and eluted in the time direction while passing through the column 14. The detector 15, which is an ultraviolet-visible spectrophotometer, sequentially detects components eluting from the column 14 and sends a detection signal to the signal processing unit 21. All or part of the eluate that has passed through the detector 15 is introduced into the fraction collector 16.

  The signal processing unit 21 creates a chromatogram based on the detection signal obtained from the detector 15, and the waveform analysis unit 22 determines the peak start point on the chromatogram and the chromatogram based on a predetermined waveform analysis parameter at the time of automatic sorting. The end point is detected. The fractionation time determination unit 23 starts fractionation by the fraction collector 16 based on the peak start point / end point detected by the waveform analysis unit 22 or the peak start point / end point information designated by the operator. Alternatively, the end time is determined and a control signal is sent to the sorting control unit 17. The sorting control unit 17 opens and closes the electromagnetic valve (sorting valve) of the fraction collector 16 according to the control signal, and sorts the eluate into different vials for each component.

  The signal processing unit 21, the waveform analysis unit 22, the sorting time determination unit 23, the storage unit 24, and the sorting operation available area derivation unit 25 described later are a personal computer (in the figure) on which a predetermined processing program is installed. (Hereinafter abbreviated as “PC”) 20. Further, the personal computer 20 is provided with an input unit 26 having a mouse and a keyboard for inputting an operator's instruction and a display unit 27 having a monitor for displaying a chromatogram and the like.

  Hereinafter, a procedure for performing manual sorting using the sorting LC of the present embodiment will be described with reference to FIGS. FIG. 2 is a diagram showing an example of a screen display at the time of manual sorting in the sorting LC of the present embodiment, and FIG. 3 is a flowchart showing a manual sorting procedure. First, the operator uses the input unit 26 to set chromatograph analysis conditions such as column temperature and pump flow rate, and sets a delay time and stores it in the storage unit 24 (step S1). Here, the delay time is obtained, for example, by a calculation formula of “delay time = pipe volume between the detector and the fraction collector (ml) / liquid feed speed of the liquid feed pump (ml / min)”. In addition to such a method that the operator directly inputs a numerical value by operating the input unit 26, the delay time is automatically calculated on the apparatus side based on the above formula. May be stored.

  Next, when the operator instructs the start of sorting by a predetermined operation, chromatographic analysis of the target sample is started (step S2). The sample components temporally separated according to the holding time when passing through the column 14 are sequentially detected by the detector 15, and detection data obtained by sampling the detection signals at a predetermined time interval is sent to the signal processing unit 21. Is done. The signal processing unit 21 generates a chromatogram indicating a change in signal intensity over time based on the detected data (step S3), and the chromatogram is displayed on the monitor of the display unit 27. In addition, in the sorting operation possible region deriving unit 25, based on the delay time stored in the storage unit 24, a time region in which the start point or end point of sorting by the fraction collector 16 can be set at the present time, that is, a detector. 15 (hereinafter referred to as “sorting operation possible region”) corresponding to the component that has passed through 15 and has not yet reached the fraction collector 16 (step S4), and is displayed on the monitor of the display unit 27. It is displayed superimposed on the chromatogram of the sample under analysis (step S5).

  Since the chromatogram of the sample under analysis is updated every time a new detection signal is obtained in the signal processing unit 21 or every time a predetermined number of new detection signals are obtained, the chromatogram shown in FIG. In this way, a chromatogram in which the curve extends with the passage of time is drawn, and as the chromatogram extends, the preparative operation possible region 31 also moves sequentially in the time axis direction (FIG. 2 (b)).

  Subsequently, the operator designates a position that is considered to be appropriate as a sorting start point in the sorting operation possible region 31 by a predetermined operation while observing the waveform of the chromatogram of the sample being analyzed displayed on the monitor. (Step S6). The position designated as the sorting start point is indicated by an arrow as shown in FIG. As a method for specifying the sorting start point and end point, for example, when specifying the sorting start point, the “S” key is used on the keyboard, and when specifying the sorting end point, the keyboard is used. There is a method to specify the sorting start point and the sorting end point by combining the key operation and mouse operation, such as clicking any point on the chromatogram while pressing the “E” key. Conceivable.

  The fractionation time determination unit 23 recognizes the position on the chromatogram designated by the operator, and the time when the component corresponding to the position on the chromatogram reaches the fraction collector 16 based on the delay time (the fractionation start) Time) T is determined (step S7). Then, the fractionation time determination unit 23 sends a command to the fractionation control unit 17 so that the fraction collector 16 starts or ends the fractionation at time T, and at time T, the fractionation control unit 17 causes the fraction collector 16 to By opening the sorting valve, sampling of sample components is started (step S8). As a method of sending a command to the sorting control unit 17 so that sorting is started at time T, a method in which a time program is stored in the sorting control unit 17 in advance so that the fraction collector 16 operates at time T is used. Alternatively, the data processing apparatus may directly send a command for starting the sorting to the sorting control unit 17 at time T.

  Thereafter, as shown in FIG. 2 (c), when the operator designates an appropriate position on the chromatogram as the sorting end point in the sortable region 31 (step S9), the chromatogram is similarly obtained as described above. The time (sorting end time) T ′ at which the component corresponding to the position on the gram reaches the fraction collector 16 is determined by the sorting time determination unit 23 (step S10). The valve is closed and the sorting operation is completed (step S11). The above steps S6 to S11 are repeated as necessary until the chromatographic analysis is completed, and the desired sample components are separated into different vials.

  As shown in FIG. 2 (d), the region within the peak sandwiched between the sorting start point and the sorting end point (sorting region 32) is shown on the chromatogram. Is shaded. Thereby, the operator can visually recognize the area actually sorted by the fraction collector 16 easily.

  Next, a procedure for performing automatic sorting using the sorting LC of the present embodiment will be described with reference to FIGS. FIG. 4 is a diagram showing an example of a screen display at the time of automatic sorting in the sorting LC of this embodiment, and FIG. 5 shows the start / end of sorting from detection of peak start / end points in automatic sorting. It is a flowchart which shows the procedure until.

  First, the operator sets analysis conditions and delay time in the same manner as described above, and also sets parameters (waveform analysis parameters) related to chromatogram peak recognition and stores them in the storage unit. As the waveform analysis parameters, the number of data points (Width) averaged during data smoothing, the slope (Slope) of the detection signal, the minimum peak area value (Min. Area), and the like are conceivable.

  When the operator performs a predetermined operation to instruct the start of analysis, a chromatogram is sequentially created based on the detection signal from the detector 15 as in the case of the manual fractionation, and the fractionation operable region Is calculated, and both are superimposed and displayed on the monitor.

  The waveform analysis unit 22 performs waveform analysis every time new chromatogram data is obtained from the signal processing unit 21 or whenever a predetermined number of new chromatogram data is obtained (step S21), and is stored in the storage unit. Based on the waveform analysis parameters, the peak start point or end point on the chromatogram is detected (FIG. 4A). If the detected peak start point or end point is located within the operable region, the information is temporarily stored in the storage unit (step S22). In addition, when the detected peak start point is located before the fractionation operation possible region 31 (that is, the part that has already reached the fraction collector 16), the fractionation starts immediately at that point or A command is sent to the sorting control unit 17 so as to be terminated.

  Thereafter, when the point recognized as the peak start point or end point in the preparative operation possible region 31 soon reaches a predetermined threshold value that deviates from the preparative operation possible region 31 (step S23, FIG. 4 (b)), At the time, the peak start point or end point is determined as the sorting start point or end point by the fraction collector 16. Then, the time (the fractionation start time T or the fractionation end time T ′) at which the component corresponding to the fractionation start point or the end point reaches the fraction collector 16 is determined based on the delay time. Calculated (step S26), a command is sent to the sorting control unit 17 so that sorting is started at the time T or T '. As a method of sending a command to the sorting control unit 17 so that sorting is started at time T or T ′, the time program is controlled in advance so that the fraction collector 16 operates at time T or T ′. The data may be stored in the unit 17 or the data processing apparatus may directly send a command for starting the sorting to the sorting control unit 17 at time T or T ′. The area (sorting area 32) where the sorting has been executed as described above is displayed in a shaded manner as described above (FIG. 4D).

  In the preparative LC of the present embodiment, as described above, each time the chromatogram data is updated by the signal processing unit 21 or every time a predetermined number of new chromatogram data is obtained, the waveform analysis is performed to start the peak start point. Alternatively, the end point is detected, and the sorting operation is always executed based on the analysis result of the latest chromatogram data. Therefore, for the peak start point or end point that was once recognized as the peak start / end point on the chromatogram but is still in the preparative operation possible region 31 (however, the point existing within the above threshold is excluded) If it is determined by subsequent update of the chromatogram data that the point is not suitable as a sorting start point / end point, or if a point more suitable as a sorting start point / end point is detected, the result Based on the above, the peak start point or end point information stored in the storage unit 24 is updated (steps S24 → S25 → S22). For example, when the updated chromatogram data point includes a point having a signal intensity smaller than the detected peak end point, the point is newly stored in the storage unit 24 as the peak end point. In addition, if the peak area value of the chromatogram cut by the line (baseline) connecting the detected peak start point and end point is smaller than a predetermined value (Min.Area), The peak is determined as noise, and the information of the peak start point / end point is deleted from the storage unit 24.

  Thus, according to the preparative LC of the present embodiment, the preparative operation possible region is calculated based on the delay time, and the peak detection / end point in the preparative operable region is calculated based on the latest chromatogram data. Can be updated to a more preferable position or invalidated, so that it is possible to perform sorting with higher accuracy than in the past.

  In addition to the above, the preparative chromatograph device of the present invention may be capable of performing preparative control combining manual preparative and automatic preparative. For example, if the operator wants to start sorting at a certain position within the sortable region even during automatic sorting, the operator By using the input unit 26 to specify an appropriate position on the chromatogram, the operator can sort the desired area.

  Further, during the automatic sorting, a sorting prohibition button 40 as shown in FIG. 6 is displayed on the monitor screen, and the fraction collector 16 can be sorted as shown in FIG. 6A. When the operator determines that the point recognized as the peak start point / end point in the region 31 is inappropriate in view of the shape of the chromatogram, by clicking the sorting prohibition button 40, The peak start point / end point included in the preparative operation possible region 31 may be invalidated (FIG. 6B).

  The best mode for carrying out the present invention has been described above using the embodiments. However, the present invention is not limited to the above embodiments, and various modifications are allowed within the scope of the present invention. It is. For example, in the above embodiment, a UV detector is used as a detector. However, the type of detector is not limited to this. For example, a mass spectrometer (MS) is used as a preparative chromatograph of the present invention. It can also be used as a detector. In this case, as shown in FIG. 8, a splitter 18 is provided on the pipe to branch the component eluted from the column 14 in two directions, one being introduced into the detector (MS) 19 and the other being introduced into the fraction collector 16. Like that. In such a case, the length of the pipe or the like is adjusted so that an appropriate time difference (that is, a delay time) is generated between the arrival of the sample component at the detector and the arrival at the fraction collector.

The block diagram which shows the principal part structure of preparative LC which is one Example of this invention. The figure which shows the screen display at the time of the manual fractionation in the fractionation LC of the Example. The flowchart which shows the procedure of the manual fractionation in the fractionation LC of the Example. The figure which shows the screen display at the time of automatic fractionation in the fractionation LC of the Example. The flowchart which shows the procedure of the automatic fractionation in the preparative LC of the Example. The figure explaining another example of the fractionation control in the fractionation chromatograph apparatus of this invention. The figure explaining the problem at the time of performing automatic fractionation in the conventional fractionation chromatograph apparatus. The block diagram which shows another structural example of the preparative chromatograph apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Eluent tank 12 ... Liquid feed pump 13 ... Sample introduction part 14 ... Column 15, 19 ... Detector 16 ... Fraction collector 17 ... Sorting control part 18 ... Splitter 20 ... Personal computer 21 ... Signal processing part 22 ... Waveform analysis Unit 23 ... Sorting time determination unit 24 ... Storage unit 25 ... Sorting operation possible area derivation unit 26 ... Input unit 27 ... Display unit 31 ... Sorting operation possible area 40 ... Sorting prohibit button

Claims (2)

A preparative chromatograph device that introduces a sample separated in the time direction by a chromatograph into a detector and a fraction collector, and fractionates and collects each component in the sample by the fraction collector based on detection information by the detector In
a) a chromatogram generating means for generating a chromatogram based on a detection signal from the detector;
b) a delay time setting means for setting the time required for the sample component passing through the detector to reach the fraction collector;
c) Sorting operation possible region calculating means for calculating a time region in which the start point or end point of the sorting by the fraction collector can be set at the present time in consideration of the delay time;
d) display means for superimposing the preparative operation possible region on the chromatogram and displaying on the screen;
e) a sorting point designating means for allowing an operator to designate an arbitrary time as a sorting start point or an end point in the sorting operation enabled area;
f) a fractionation control means for controlling the operation of the fraction collector based on the designation in the fractionation point designation means;
A preparative chromatograph apparatus comprising: A preparative chromatograph device that introduces a sample separated in the time direction by a chromatograph into a detector and a fraction collector, and fractionates and collects each component in the sample by the fraction collector based on detection information by the detector In
a) a chromatogram generating means for generating a chromatogram based on a detection signal from the detector;
b) a delay time setting means for setting the time required for the sample component passing through the detector to reach the fraction collector;
c) Sorting operation possible region calculating means for calculating a time region in which the start point or end point of the sorting by the fraction collector can be set at the present time in consideration of the delay time;
d) peak detection means for detecting a peak start point or an end point existing on the chromatogram based on the chromatogram obtained from the chromatogram generation means;
e) a fractionation control means for operating the fraction collector at a timing when the peak start point or end point detected on the latest chromatogram deviates from the fractionation operable region;
A preparative chromatograph apparatus comprising:

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