JP4082304B2 - Preparative liquid chromatograph - Google Patents

Description

  The present invention relates to a preparative liquid chromatograph apparatus that separates components in a sample solution using a liquid chromatograph.

  Conventionally, for example, as described in Patent Document 1, a plurality of components contained in a sample are separated and collected using a chromatographic apparatus including a high performance liquid chromatograph (hereinafter referred to as “HPLC”). A so-called preparative chromatograph apparatus is known.

Japanese Patent Laid-Open No. 5-302918 ([0003], FIG. 3)

  FIG. 1 is an example of the configuration of a preparative chromatograph using HPLC. The eluent (mobile phase) stored in the eluent tank 11 is sucked by the 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 sequentially detects components eluted from the column 14 and sends a detection signal to the signal processing unit 16. All or part of the eluate that has passed through the detector 15 is introduced into the fraction collector 18. The signal processing unit 16 creates a chromatogram based on the detection signal obtained from the detector 15, and the control unit 17 gives the chromatogram data to the fraction collector 18. The fraction collector 18 controls a solenoid valve or the like based on the peak appearing in the chromatogram data, and fractionates the eluate into different vials for each component.

  In recent years, liquid chromatograph mass spectrometers (hereinafter referred to as “LC / MS”) using mass spectrometers (hereinafter referred to as “MS”) have been widely used as HPLC detectors. . In MS, various components contained in the introduced sample can be detected separately for each mass number (mass / charge), so even if multiple components overlap in time, they are separated. Qualitative analysis and quantitative analysis. In LC / MS, the mass spectrum can be obtained by mass-scanning the set mass range, detecting the ion intensity separated for each mass number, and examining the relationship with the mass number. In addition, mass scans are repeated, and the ion intensity is accumulated regardless of the mass number for each scan to obtain a total ion chromatogram by examining the temporal change in the total ion intensity, or focusing on a specific mass number A mass chromatogram can be obtained by examining the time change of the ion intensity of the mass number for each scanning. Based on the data of the total ion chromatogram and mass chromatogram obtained using MS as a detector in this way, the fraction collector performs the control as described above, and the eluate can be collected.

  As described above, when the fraction collector is controlled using measurement data obtained from MS or other detectors, the following two problems arise due to the measurement data.

The first point is caused by the fact that the control time interval of the fraction collector does not match the time interval of the measurement data.
The chromatogram data is transmitted from the detector to the fraction collector at a time interval according to the measurement time interval. This time interval varies depending on the measurement conditions. For example, when MS is used as a detector, the time required for one mass scan differs depending on whether the MS scans the entire mass range or focuses on a specific number of masses. The time interval is different. Further, when mass scanning is performed by paying attention to only a specific plurality of mass numbers, the measurement time interval differs depending on the number of types. Furthermore, when adding mass spectra from a plurality of mass scans in order to increase measurement accuracy, the measurement time interval differs depending on the number of additions.

  On the other hand, in the fraction collector, control for fractionation is performed at predetermined time intervals. Therefore, normally, the control time interval of the fraction collector and the time interval of the chromatogram data do not coincide. As a result, the operation of the fraction collector may become unstable.

  The second point is caused by spike noise. Spike noise in MS is caused by the ionized sample and neutral charge particles entering the background. In other detectors, noise similar to spike noise may occur due to a power source or the like. Hereinafter, these noises are collectively referred to as “spike noise”. Such spike noise usually enters only for a moment, and only one point in the chromatogram is larger than the other data. When such spike noise of only a moment is sent to the fraction collector as a sorting control signal, the fraction collector may operate erroneously and execute control for sorting.

  The problem to be solved by the present invention is to stably operate the fraction collector regardless of the difference between the time interval of the measured chromatogram data and the time interval for controlling the fraction collector and the presence of spike noise. It is to provide a preparative liquid chromatograph apparatus capable of

The first aspect of the preparative liquid chromatograph apparatus according to the present invention, which has been made to solve the above-mentioned problems, introduces a sample that has been subjected to component separation in the time direction by the liquid chromatograph section into the analysis section and the fraction collector. In a preparative liquid chromatograph that fractionates and collects each component with a fraction collector based on analysis information by the analysis unit,
a) a chromatogram data determining means for determining chromatogram data at each time based on the analysis information;
b) sequentially acquiring the chromatogram data, interpolating the chromatogram data at a predetermined time interval equal to or an integral multiple of the control time interval of the fraction collector to create control data, and Control data creation means for transmitting to the fraction collector at predetermined time intervals ;
It is characterized by providing.

  The chromatogram data determining means determines chromatogram data based on the analysis information obtained in the analysis unit. Here, MS can be used for an analysis part, for example. In that case, chromatogram data is determined based on the mass spectrum obtained in MS. The determined chromatogram data may be, for example, the total ion chromatogram data or the mass chromatogram data. The time interval at which chromatogram data is obtained varies depending on the analysis conditions such as the range of mass scanning in MS and the number of integrations. Even when an analyzer other than MS is used, the time interval of chromatogram data may differ depending on the analysis conditions and instrument settings.

  The control data creation means interpolates the chromatogram data at predetermined time intervals based on the chromatogram data obtained at the time intervals (hereinafter referred to as “measurement time intervals”) depending on the analysis conditions and the like. Create interpolation data. For interpolation, typically, interpolation based on a linear function (linear interpolation) can be used. Further, interpolation may be performed using a higher-order function or a power function higher than a quadratic function. The predetermined time interval is usually a time interval when control is performed in the fraction collector (hereinafter referred to as “control time interval”). If data can be obtained at the control time interval, for example, the predetermined time interval may be an integer multiple of the control time interval.

  The control data creating means transmits the data thus obtained by interpolation to the fraction collector as control data. The fraction collector controls sorting based on this control data. Since the control data can be obtained at control time intervals (or an integral multiple thereof) by the operation of the control data creating means, the fraction collector can operate stably.

In addition, the second aspect of the preparative liquid chromatograph apparatus according to the present invention introduces a sample whose components have been separated in the time direction by the liquid chromatograph section into the analysis section and the fraction collector, and provides analysis information by the analysis section. In a preparative liquid chromatograph that fractionates and collects each component based on a fraction collector,
c) Chromatogram data determining means for determining chromatogram data at each time based on the analysis information;
d) sequentially acquiring the chromatogram data, correcting the intermediate value among the three points before and after the time of interest as chromatogram data at the time of interest, and obtaining the obtained data from the fraction collector Control data creation means for transmitting to the fraction collector at a predetermined time interval equal to or an integral multiple of the control time interval ;
It is characterized by providing.

  The chromatogram data determining means is the same as that in the first aspect. The control data creation means receives the chromatogram data from the chromatogram data determination means, and calculates an intermediate value among the values of the chromatogram data at three points before and after the time of interest (therefore, the number of data is three). The corrected chromatogram data at the time of interest is used. This is control data. The three points of data can be, for example, data obtained at a time closest to the time of interest and data before and after the data, or data obtained immediately before the time of interest and data before and after the data. When data with a large value at only one point is present in the data of three points due to spike noise, the data cannot be the “intermediate value”, so such spike noise is used in the control data. Is eliminated. The control data creating means transmits the control data thus obtained to the fraction collector. The fraction collector does not malfunction due to the effects of spike noise.

  Furthermore, it is desirable that the preparative liquid chromatograph apparatus of the present invention has a configuration in which the features of the above two aspects are combined. That is, in the preparative liquid chromatograph apparatus according to the first aspect, the control data creation means further calculates an intermediate value among three values before and after the time of interest based on the data obtained by the interpolation. Correction is performed as chromatogram data at the time of interest, and the obtained data is transmitted to the fraction collector. Alternatively, in the preparative liquid chromatograph apparatus according to the second aspect, the control data creation means further interpolates the data at predetermined time intervals based on the data obtained by the correction, and obtains the obtained data. May be transmitted to the fraction collector. In any case, since data that is not affected by spike noise is transmitted to the fraction collector at control time intervals, the fraction collector can operate stably without malfunctioning.

  In the preparative liquid chromatograph of the first aspect of the present invention, the obtained chromatogram data is interpolated at a predetermined time interval, so that the control is performed at every control time interval of the fraction collector regardless of the analysis conditions. Therefore, the operation of the fraction collector can be stabilized.

  In the preparative liquid chromatograph apparatus according to the second aspect of the present invention, spike noise can be eliminated by using an intermediate value among the data values of the three points before and after as chromatogram data at the time of interest. it can. Therefore, malfunction of the fraction collector due to spike noise can be prevented.

  Further, by combining the features of these two aspects, interpolating chromatogram data, and further setting the intermediate value of the three points before and after the time of interest as the chromatogram data at the time of interest, the above 2 Two effects can be obtained together. Needless to say, the same effect can be obtained by interpolating at intermediate time values among the three points before and after the time of interest as chromatogram data at the time of interest and interpolating at predetermined time intervals based on the data.

  As an example of the preparative liquid chromatograph apparatus of the present invention, a configuration of an example of a preparative liquid chromatograph mass spectrometer is shown in FIG. Configuration and process until the sample is injected into the mobile phase sucked from the eluent tank 11 by the pump 12 in the sample introduction unit 13 and the sample solution is separated in the time direction and eluted while passing through the column 14. Is the same as described above. The sample solution eluted from the column 14 is branched into two flow paths at a predetermined diversion ratio in the flow path branching section 19, and one is sent to the MS section 20 and the other is sent to the fraction collector 18.

  The MS unit 20 performs mass spectrometry by ionizing the introduced sample by a predetermined method. FIG. 2 shows an example of the MS unit 20. The sample solution is sprayed from the nozzle 22 to the atomization chamber 21, and the sample in the sample solution is ionized by corona discharge from the discharge electrode 23. While the fine droplets containing ions pass through the desolvation tube 24, the solvent evaporates, and the ions are sent to the analysis chamber 26 through the intermediate chamber 25. In the analysis chamber 26, only ions having a specific mass number pass through the quadrupole filter 27 and are detected by the detector 28. Note that, here, an example of the configuration of the MS unit 20 is shown, and the present invention can be similarly applied even when an MS unit having another configuration is used.

  The chromatogram data determination unit 31 first obtains a mass spectrum at a certain time based on a signal detected by the detector 28 of the MS unit 20. This mass spectrum may be obtained by adding the detection signals obtained not only by one mass scan but also by a plurality of mass scans for each mass number. In order to perform such addition, for example, a DSP (Digital Signal Processor) for integrating digital signals obtained by A / D conversion of signals from the detector can be used. Next, chromatogram data at that time is determined from the obtained mass spectrum by a predetermined method. The chromatogram data calculated here includes, for example, total ion chromatogram data obtained by adding up all mass data of the mass spectrum, mass ion chromatogram data using a value at a specific mass number of the mass spectrum, and the like. .

  The control data creation unit 32 creates data used for controlling the fraction collector 18 based on the chromatogram data, and transmits the data to the fraction collector 18. Details thereof will be described later.

  Based on the control data, the fraction collector 18 performs the following control in order to separate the target component from the sample solution branched at the flow path branching portion 19 and sent to the fraction collector 18. First, the peak start point is detected from the control data based on a predetermined reference, and when a predetermined delay time has elapsed from the time of the start point, the electromagnetic valve is opened and the sample is dispensed into a vial. The delay time here is provided for the sample branched at the flow path branching portion 19 because it is slower to reach the fraction collector 18 than when it is introduced into the MS portion 20 to obtain chromatogram data. It varies depending on the flow rate of the mobile phase and the capacity of the piping. This delay time is determined in advance by measurement. When the fraction collector 18 detects the peak end point from the control data based on a predetermined reference, the fraction collector 18 closes the solenoid valve after the predetermined delay time elapses and ends the sorting. When fractionating a plurality of components, the vial is moved by a biaxial arm or the like every time the separation of one component is completed, and an empty vial is set at the sorting position.

  The chromatogram data determination unit 31, the control data creation unit 32 and the MS unit 20 are controlled by the control unit 33. In addition, a storage unit 34 for temporarily storing data for creating control data is provided. Setting conditions and the like can be input from the input unit 35 by the measurer.

Hereinafter, data processing in the chromatogram data determination unit 31 and the control data creation unit 32 will be described.
First, an overview of the processing performed in each of these units will be described by taking as an example the case of obtaining mass chromatogram data from measurement in the MS unit 20 for a plurality of different (j types) mass numbers. A signal corresponding to each of the j types of mass numbers is output from the detector 28 by mass scanning in the MS unit 20 and input to the chromatogram data determining unit 31. The chromatogram data determination unit 31 is provided with a DSP, and a signal from the detector 28 is A / D converted and introduced into this DSP. In this DSP, a signal of one mass number is acquired from a signal of one mass scan, and this is repeated a predetermined number of times (k times) to calculate mass chromatogram data at that mass number. The DSP sequentially performs this operation for j types of mass numbers, and calculates mass chromatogram data for each mass number. Of the obtained mass chromatogram data, those having a predetermined mass number may be used for the subsequent processing, or the chromatogram data of j types of mass numbers are binarized, and the logical sum or logical product is obtained. The subsequent processing may be performed by using them. In any case, the time interval ΔT for obtaining the mass chromatogram data is ΔT = S × j × k, where S is the sampling time interval for the A / D conversion.

On the other hand, the fraction collector 18 controls fractionation at a predetermined control time interval Δt based on chromatogram data. In the fraction collector 18 used in this embodiment, the control time interval Δt is 0.2 seconds. Although it is desirable that the control time interval Δt and the measurement time interval ΔT of chromatogram data coincide with each other, actually, as shown in FIG. FIG. 3A shows a case where Δt <ΔT. The chromatogram data A (t) indicated by a circle in the figure is obtained at t = T ₁ , t = T ₂ = T ₁ + ΔT,. Is performed at a time interval Δt shorter than ΔT at time t = t ₁ , t ₂ (= t ₁ + Δt), t ₃ ,. With direct A (t) to control the fraction collector 18, the value of the chromatogram data at control time t ₃ immediately before t = T ₂ is A (T _1), in the control time t ₄ immediately thereafter The value of the chromatogram data is A (T ₂ ). Therefore, in this example, the chromatogram data changes from A (T ₁ ) to A (T ₂ ) over a long time ΔT, but in this example it changes at a shorter time Δt. Since the fraction collector 18 misidentifies as described above, the control in the fraction collector 18 becomes unstable. FIG. 3B shows a case where Δt> ΔT. In this case, the difference in chromatogram data resulting from the difference between the measurement time interval Δt of the chromatogram data and the control time interval ΔT is not as great as in the above case, but it still makes the control in the fraction collector 18 unstable. Cause. In order to avoid such a problem, the apparatus of this embodiment interpolates chromatogram data A (t) to create data A ′ (t) for each time Δt.

  Further, as shown in FIG. 3 (c), spike noise may occur in which one point of the chromatogram data marked with a circle becomes large. In the apparatus of the present embodiment, processing for removing such spike noise is also performed, and the obtained data A ″ (t) is transmitted to the fraction collector 18 as control data. The fraction collector 18 performs fractionation based on the control data.

Hereinafter, details of processing in the chromatogram data determination unit 31 and the control data creation unit 32 will be described with reference to the flowchart of FIG. 4. First, measurement in the MS unit 20 is started by a predetermined operation by the measurer. The time at which chromatogram data A (t) is determined by the chromatogram data determination unit 31 is T _m (m = 1, 2,...), And the data after correction by the control data creation unit 32 is obtained. T _n (n = 1, 2,...) First, in step S1, the control unit 33 sets m = 1 and n = 0 as initial conditions. In fact, since there is no data at n = 0, the control unit 33 sets A ′ (t ₀ ) = 0 as an initial condition. Similarly, the control unit 33 sets A (T ₋₁ ) = 0 and A (T ₀ ) = 0. These initial values are stored in the storage unit 34.

The MS unit 20 performs mass scanning on the introduced sample by a predetermined method (step S2). Chromatogram data determination unit 31 creates a mass spectrum from the detection signal of the detector 28, to create the data of the chromatogram A (T _m) at time t = T _m from the mass spectrum. (Step S3). The obtained data A (T _m ) is sent to the control data creation unit 32 and stored in the storage unit 34. Next, in step S4, whether or not the control unit 33 is past the time for performing interpolation of the data of the chromatogram, i.e., whether the time Δt from the time t = t _n, which was most recently interpolation has elapsed Determine. Accordingly, the determination formula in step S4 is T _m > t _{n + 1} = t _n + Δt. If this judgment formula is satisfied, the process proceeds to step S5.If not, steps S2 to S3 are repeatedly executed until the judgment formula is satisfied, and chromatogram data A (T _{m + 1} ), A (T _{m + 2} ),. Decide on ..

In step S 5, the control data creation unit 32 acquires the previous chromatogram data A (T _m−1 ) from the storage unit 33, and from this data A (T _m−1 ) and the chromatogram data determination unit 31. Based on the sent data A (T _m ), an interpolation value A ′ (t _{n + 1} ) of chromatogram data at t = t _{n + 1} is obtained by linear interpolation. Since the slope of the straight line connecting the two values A (T _m ) and A (T _m-1 ) is {A (T _m ) -A (T _m-1 )} / ΔT, A ′ (t _{n +1} )
A '(t _{n + 1} ) = {A (T _m ) -A (T _m-1 )} (t _{n + 1} -T _m-1 ) / ΔT + A (T _m-1 )… (1)
Is required. A ′ (t _{n + 1} ) is stored in the storage unit 34.

The control data creation unit 32 further acquires A ′ (t _n ) and A ′ (t _n−1 ) that have already been obtained (or set in step S1) from the storage unit 34, and these three data Among the values of A '(t _{n + 1} ), A' (t _n ), A '(t _n-1 ), the second value is the corrected value of chromatogram data at time t = t _n A ″ (t _n ) is determined (step S6). This correction eliminates the influence of spike noise because the data is not used even if a large value resulting from spike noise is included in the three values. A ″ (t _n ) thus obtained becomes control data for the fraction collector 18. The control data creation unit 32 transmits the control data A ″ (t _n ) to the fraction collector 18 (step S7). The fraction collector 18 controls sorting as described above based on the transmitted control data A ″ (t _n ).

The control unit 33 adds 1 to the value of n in step S8, and determines whether or not the time t _{n + 1} has passed the measurement end time t _max in step S9. If t _{n + 1} has passed t _max , the measurement in the MS unit is terminated, otherwise the process proceeds to step S10. In step S10, the control unit 33 determines whether or not T _m > t _{n + 1} for _n changed in step S8. If T _m > t _{n + 1} , the processing from steps S5 to S9 is repeated. The condition of this step S10 corresponds to the case where there are a plurality of times for performing the control between the times T _m-1 and T _m, and the above iterative process is performed at those times A ′ (t) and A ″. This is for calculating all of (t). If T _m <t _{n + 1} , that is, if all data to be interpolated is calculated between times T _m−1 and T _m , 1 is added to the value of m in step S11, and the process returns to step S2.

  In this way, S2 to S11 are repeatedly executed, and the correction value A ″ (t) of the chromatogram data is sequentially obtained. This A ″ (t) is obtained at the same time interval as the control time interval Δt in the fraction collector 18, and further, the influence of spike noise is eliminated, so that the fraction collector 18 can be operated stably. .

The schematic block diagram which shows an example of the conventional preparative liquid chromatograph mass spectrometer. The block diagram which shows one Example of the liquid chromatograph mass spectrometer which concerns on this invention. The graph for demonstrating control time interval (DELTA) t, measurement time interval (DELTA) T of chromatogram data, and spike noise. The flowchart explaining the data processing of the chromatogram in this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Eluent tank 12 ... Pump 13 ... Sample introduction part 14 ... Column 15 ... Detector 16 ... Signal processing part 17 ... Control part 18 ... Fraction collector 19 ... Flow path branching part 20 ... MS part 21 ... Atomization chamber 22 ... Nozzle 23 ... Discharge electrode 24 ... Desolvation tube 25 ... Intermediate chamber 26 ... Analysis chamber 27 ... Quadrupole filter 28 ... Detector 31 ... Chromatogram data decision unit 32 ... Control data creation unit 33 ... Control unit 34 ... Storage unit 35 ... Input section

Claims (4)

In a preparative liquid chromatograph that introduces a sample separated in the time direction in the liquid chromatograph part into the analysis part and the fraction collector, and fractionates and collects each component based on the analysis information by the analysis part,
a) a chromatogram data determining means for determining chromatogram data at each time based on the analysis information;
b) sequentially acquiring the chromatogram data, interpolating the chromatogram data at a predetermined time interval equal to or an integral multiple of the control time interval of the fraction collector to create control data, and Control data creation means for transmitting to the fraction collector at predetermined time intervals ;
A preparative liquid chromatograph apparatus comprising:   The control data creation means further performs correction based on the data obtained by the interpolation to make the intermediate value of the three values before and after the time of interest as chromatogram data at the time of interest. The preparative liquid chromatograph according to claim 1, wherein the collected data is transmitted to a fraction collector. In a preparative liquid chromatograph that introduces a sample separated in the time direction in the liquid chromatograph part into the analysis part and the fraction collector, and fractionates and collects each component based on the analysis information by the analysis part,
a) a chromatogram data determining means for determining chromatogram data at each time based on the analysis information;
b) sequentially acquiring the chromatogram data, correcting the intermediate value of the three values before and after the time of interest as chromatogram data at the time of interest, and obtaining the obtained data from the fraction collector Control data creation means for transmitting to the fraction collector at a predetermined time interval equal to or an integral multiple of the control time interval ;
A preparative liquid chromatograph apparatus comprising:   4. The control data creation means further interpolates the data at predetermined time intervals based on the data obtained by the correction, and transmits the obtained data to a fraction collector. The preparative liquid chromatograph apparatus described.

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