Abstract:
In a liquid chromatograph mass spectrometer, chromatogram data is obtained by carrying out alternately a mass scanning in a positive ion detection mode and a mass scanning in a negative ion detection mode, obtaining the chromatogram data in every positive and negative polarities through summing up the mass spectrum data obtained at the respective mass scannings, and adding the data of both polarities. Since the chromatograms in both polarities are averaged, even if there is a level difference therebetween, a chromatogram in a normal form can be obtained.

Description:
BACKGROUND OF THE INVENTION AND RELATED ART STATEMENT  
         [0001]    The invention relates to a liquid chromatograph mass spectrometer, in particular, an. apparatus suitable for fractionating various components contained in a sample solution by using a liquid chromatograph mass spectrometer.  
           [0002]    Heretofore, there has been known a fraction chromatograph wherein a plurality of components contained in a sample is separated and collected by using a chromatograph device, such as high performance liquid chromatograph (hereinafter referred to as “HPLC”).  
           [0003]    [0003]FIG. 5 is a block diagram for showing an example of a structure of a fraction chromatograph using HPLC. An eluant, i.e. mobile phase, stored in an eluant tank  1  is sucked by a pump  2  and is transferred to flow into a column  4  through a sample introduction portion  3  at a predetermined flow rate. A sample solution injected into the mobile phase at the sample introduction portion  3  is introduced into the column  4  together with the mobile phase, and while passing through the column  4 , its components are separated and eluted. A detector  5  detects sequentially the components eluted from the column  4  and sends detection signals to a signal process portion  6 . All or a part of the eluate passing through the detector  5  is introduced into a fraction collector  8 . The signal process portion  6  prepares a chromatogram based on the detection signals obtained from the detector  5 , and a control portion  7  provides the fraction collector  8  with a control signal for fraction based on a peak appearing on the chromatogram at real time. The fraction collector  8  controls an electromagnetic valve and the like based on the control signal and distributes the eluates to vials corresponding to the respective components.  
           [0004]    By the way, recently, there has been widely used a liquid chromatograph mass spectrometer (hereinafter referred to as “LC/MS”) using a mass spectrometer (hereinafter referred to as “MS”) as a detector of HPLC. In MS, since various components contained in an introduced sample are separated and detected in every mass number, i.e. mass/charge, even if a plurality of components is timewise overlapped, there is an advantage such that these components are separated and subjected to a qualitative analysis and a quantitative analysis.  
           [0005]    In the LC/MS, a mass spectrum can be obtained by carrying out a mass scanning over a set mass region, sequentially detecting strengths of ions separated in every mass number and examining a relationship between the mass number and the strength. Also, a total ion chromatogram (hereinafter referred to simply as “chromatogram”) can be obtained by repeatedly carrying out the mass scannings, integrating the ion strength in every scanning regardless of the mass number and examining a timewise change of the total ion strength. Further, by watching a specific mass number, a mass chromatogram can be obtained by examining a timewise change of the strengths of ions having the mass number in every scanning.  
           [0006]    In case the LC/MS, as described above, is used for the fraction chromatograph, it is necessary to determine a timing of fraction based on the chromatogram data for preparing a chromatogram or mass chromatogram. Normally, the chromatogram data is calculated according to process conditions set beforehand (for example, strengths of ions having a specific mass number are added, strengths of ions in a predetermined mass region are added and the like), from a great number of mass spectrum data obtained by one-time mass scanning. Thus, a chromatogram data can be obtained in every mass scanning. Therefore, for example, in case a spectrometry is carried out while alternatively changing a mass scanning in a positive ion detection mode for detecting positive ions and a mass scanning in a negative ion detection mode for detecting negative ions, a chromatogram data obtained at a certain time point t is a value calculated based on the mass spectrum obtained by the positive ion detection mode, and the subsequent chromatogram datum obtained at t+Δt is a value calculated based on the mass spectrum obtained by the negative ion detection mode.  
           [0007]    Generally, since the levels of the background noises in the positive ion detection mode and the negative ion detection mode are different, the chromatogram prepared based on the mass spectrum obtained in the positive ion detection mode and the chromatogram prepared based on the mass spectrum obtained in the negative ion detection mode are different in levels of the base lines as shown in FIG. 6( a ). Therefore, when the chromatogram data obtained when the positive polarity and the negative polarity are switched over as described above is connected or added in time sequence, the chromatogram curve having sawteeth shapes as shown in FIG. 6( b ) is obtained. Also, in case a component detectable only by the positive ion detection mode and a component detectable only by the negative ion detection mode are mixed, since the respective chromatograms become, for example, as shown in FIG. 7( a ), when the chromatogram data obtained when the positive polarity and the negative polarity are switched over are connected or added in time sequence, the peak waveform becomes sawteeth shapes as shown in FIG. 7( b ).  
           [0008]    In either case, in the control portion  7 , since an accurate starting point and an accurate terminal point of the peak can not be determined by using such chromatogram data, it is impossible to determine the timing of fractionating the respective components, or an erroneous control signal is sent to the fraction collector  8 . In view of the defects as described above, in case the fraction operation is carried out by the conventional LC/MS, since the fraction operation can not be carried out while alternately changing the positive polarity and the negative polarity, it is necessary that the fraction operation in the positive ion detection mode and the fraction operation in the negative ion detection mode are separately carried out. Thus, the fraction operation is not carried out effectively.  
           [0009]    In view of the above problems, the present invention has been made and an object of the present invention is to provide a liquid chromatograph mass spectrometer, wherein even in case a mass spectrometry is carried out while changing the spectrometry conditions, such as alternately changing a positive polarity and a negative polarity, a chromatogram for normally operating a fraction collector can be obtained, so that a proper fraction operation can be done by only one-time spectrometry.  
           [0010]    Further objects and advantages of the invention will be apparent from the following description of the invention.  
         SUMMARY OF THE INVENTION  
         [0011]    In order to solve the above-stated problems, according to the present invention, in a liquid chromatograph mass spectrometer, a sample, components of which are separated in a liquid chromatograph portion in a time-wise direction, i.e. along a passage of time, is introduced into a mass spectrometry portion and a fraction collector, and the fraction collector fractionates and collects the respective components based on the information obtained in the mass spectrometry portion. The liquid chromatograph mass spectrometer includes: a setting device for setting beforehand a plurality of spectrometry conditions when a mass spectrometry is carried out; a spectrometry execution device for executing a cycle of spectrometry by changing the spectrometry condition set by the setting device whenever one-time mass scanning in one cycle is carried out, the periodical spectrometry being repeated sequentially; an operation device for obtaining chromatogram data by adding together a number of mass spectrum data obtained by the one-time mass scanning whenever the cycle of spectrometry is completed and further adding thereto values in the respective mass scannings, or for obtaining the chromatogram data by adding the mass spectrum data with respect to a specific mass number obtained by the respective mass scannings; and a fraction control device for controlling an operation of the fraction collector based on the chromatogram data obtained by the operation device.  
           [0012]    Here, the “spectrometry condition” means a condition which has an effect on the ion generating condition or the ion detecting condition. For example, the spectrometry condition may include a positive ion detection mode for detecting a positive ion and a negative ion detection mode for detecting a negative ion. When the positive ion detection mode and the negative ion detection mode are set by the setting device, the spectrometry execution device carries out alternately one-time mass scanning of the positive ion detection mode and one-time mass scanning of the negative ion detection mode. Since a large number of mass spectrum data with respect to the mass number in a predetermined region can be obtained by the respective mass scannings, the operation device adds the mass spectrum data in each polarity, and further adds thereto the values of the respective polarities to obtain a single chromatogram. More specifically, since the single chromatogram reflects a plurality of mass spectrum data of both polarities, even if the levels on the base lines of the respective chromatograms of the positive and negative ions are different, or a peak is present only in either the positive ion or negative ion chromatogram, they are averaged.  
           [0013]    Incidentally, in case a fraction operation is carried out based on the mass chromatogram, the operation device adds the mass spectrum data with respect to a specific mass number obtained by each mass scanning to obtain the chromatogram data.  
           [0014]    According to the liquid chromatograph mass spectrometer of the present invention, even in case the mass scanning is carried out while changing the spectrometry conditions of the mass spectrometry, there can be obtained a chromatogram wherein the peak waveform can be normally obtained, so that the fractions of the respective components can be properly carried out by the fraction collector. Also, for example, since the fraction operations with respect to the components of both polarities can be carried out by a single spectrometry, it is not necessary to carry out fraction operations for the respective polarities as in the conventional liquid chromatograph mass spectrometer, thus shortening the time required for the fraction operation. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0015]    [0015]FIG. 1 is a block diagram for showing an entire structure of LC/MS of an embodiment according to the present invention;  
         [0016]    [0016]FIG. 2 is a graphic chart for explaining a signal process operation of the present embodiment;  
         [0017]    [0017]FIG. 3 is a flow chart for showing the signal process operation of the present embodiment;  
         [0018]    FIGS.  4 ( a ) and  4 ( b ) are examples of chromatograms obtained in the present embodiment;  
         [0019]    [0019]FIG. 5 is a block diagram for showing a structure of a fraction chromatograph using a general HPLC;  
         [0020]    FIGS.  6 ( a ) and  6 ( b ) are chromatograms for explaining problems in a fraction device using a conventional LC/MS; and  
         [0021]    FIGS.  7 ( a ),  7 ( b ) are other chromatograms for explaining problems in a fraction device using a conventional LC/MS. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0022]    Hereunder, LC/MS of an embodiment of the present invention will be explained with reference to FIG. 1 to FIG. 4( b ).  
         [0023]    [0023]FIG. 1 is a block diagram of an entire LC/MS according to the present embodiment. A sample liquid eluted from a column  4  of an LC is divided into two-flow paths at a predetermined ratio at a flow-path diverging portion  9 , one of which is sent to an MS portion  10  and the other of which is sent to a fraction collector  8 . The MS portion  10  includes a nebulizing or atomizing chamber  11  having a nozzle  12  and a discharge electrode  13 , and a spectrometry chamber  16  having a quadrupole filter  17  and an ion detector  18 . There are provided two intermediate chambers  15  between the nebulizing chamber  11  and the spectrometry chamber  16 . The nebulizing chamber  11  and the first intermediate chamber  15  are connected through a desolvent pipe  14 . The signal detected by the ion detector  18  in the MS portion  10  is inputted into a signal process portion  20 , and, after being subjected to processing as described later at the signal process portion  20 , gives the chromatogram data to a control portion  21 . The control portion  21  controls operations of the respective portions in the MS portion  10 , the fraction collector  8 , and operations of the respective portions of the LC though control signal lines are not shown,.  
         [0024]    Operations of the MS portion  10  are as follows. When the sample solution supplied from the column  4  reaches the nozzle  12 , the sample solution is atomized in the nebulizing chamber  11  as high temperature drops. The dispersed drops collide with gas molecules under the atmospheric pressure, are smashed into further fine drops, and quickly dried, i.e. removal of the solvent, to thereby vaporize the sample molecules. The fine gas particles contact the buffer ions produced by the corona discharge from the discharge electrode  13  to cause a chemical reaction, and ionized. The fine drops containing the generated ions plunge into the desolvent pipe  14  and are further subjected to the desolvent while the fine drops pass through the desolvent pipe  14 . The ions are sent to the spectrometry chamber  16  through the two intermediate chambers  15 , and only objective ions having a specific mass number, i.e. mass/charge, pass through the quadrupole filter  17  disposed in the spectrometry chamber  16  to reach the ion detector  18 . Electric current corresponding to the ion number which has arrived at the ion detector  18  can be taken out therefrom.  
         [0025]    In the MS portion  10 , a positive ion detection mode for detecting the positive ions by generating the positive ions and a negative ion detection mode for detecting the negative ions by generating the negative ions can be switched over in a short time, by changing voltages applied to the respective portions, such as the discharge electrode  13 , and switching the operation of the ion detector  18 .  
         [0026]    Hereunder, operations of the present LC/MS when fraction operations are carried out in both positive and negative polarities alternately will be explained.  
         [0027]    [0027]FIG. 3 is a flow chart for showing operations at the time of the spectrometry in the signal process portion  20  and the control portion  21 , and FIG. 2 is a graphic chart for explaining the operations thereof. An operator inputs various parameters, such as operation conditions of LC, operation conditions of the MS portion  10  and process conditions in the signal process portion  20 , to set therein from the operating portion  22 . These conditions include a mass region at a time of mass scanning, a mass step, a scanning time and so on in the MS portion  10 .  
         [0028]    When the spectrometry starts, first, the control portion  21  sets parameters of the respective portions of the MS portion  10  to be the positive ion detection mode (Step S 1 ), and carries out the mass scanning in a predetermined mass region (Step S 2 ). At the time of the mass scanning, when the voltage applied to the quadrupole filter  17  is controlled, the mass number of the ions having passed through the quadrupole filter  17  and arrived at the ion detector  18  is changed. The signal process portion  20  processes the detection signals which are sequentially changed at the time of the mass scanning, and obtains the mass spectrum data for showing relationships between the mass number and the ion strength (Step S 3 ). The mass spectrum reflects only the positive ion strength as shown in FIG. 2. Among a large number of mass spectrum data, the mass spectrum data is extracted according to the predetermined process conditions, such as mass region, and is added together to obtain chromatogram data A(+) of the positive polarity and stored in a memory (Step S 4 ).  
         [0029]    Then, the control portion  21  sets parameters of the respective portions of the MS portion  10  to become the negative ion detection mode (Step S 5 ), and carries out the mass scanning in a predetermined mass region (Step S 6 ). More specifically, the control portion  21  carries out the mass scanning in the same manner as in the above-explained positive ion detection mode, and the signal process portion  20  processes the detection signals which are sequentially changed at the time of mass scanning to obtain the mass spectrum data showing relationships between the mass number and the ion strength (Step S 7 ). The mass spectrum reflects only the negative ion strength as shown in FIG. 2. Among a large number of mass spectrum data, the mass spectrum data is extracted according to the predetermined process conditions, and is added together to obtain chromatogram data A(−) of the negative polarity (Step S 8 ). Then, when the chromatogram data A(+) and A(−) of the positive and negative polarities are completed, both data is added together to obtain the chromatogram data A and outputted as an analogue value (Step S 9 ). Thereafter, until spectrometries of all components are completed, the above-described processes are repeated by returning from Step S 10  to Step S.  
         [0030]    With the above-described process, as shown in FIG. 2, a chromatogram datum A can be obtained in every two times of the mass scannings (one for the positive polarity and one for the negative polarity). FIGS.  4 ( a ) and  4 ( b ) are chromatograms prepared based on the chromatogram data obtained from the signal process portion  20 . According to the present LC/MS, even in case the chromatograms of the positive and negative polarities are separately prepared and the respective peaks are formed in different positions as shown in FIG. 4( a ), the peaks of both polarities appear on the chromatogram in a normal form as shown in FIG. 4( b ), and the sawteeth shapes as shown in FIG. 7( b ) are not formed.  
         [0031]    When the control portion  21  receives the chromatogram data from the signal process portion  20  at a real time, the control portion  21  detects a starting point of a peak of an objective component to be fractionated and outputs a collection start signal to the fraction collector  8  with a predetermined time delay from the time when the starting point is detected. The time delay is determined by a flow rate of a mobile phase and pipe capacities from the flow path diverging portion  9  to the nozzle  12  of the MS portion  10  and from the flow path diverging portion  9  to an electromagnetic valve of the fraction collector  8 . In the fraction collector  8 , when the objective component arrives at the electromagnetic valve, the electromagnetic valve is opened according to the collecting start signal to start fraction. When a termination point of the peak of the objective component is detected, the control portion  21  sends a collection completion signal to the fraction collector  8  in the same manner. Thus, when the fraction or separation of the objective component is completed, the electromagnetic valve is closed. In case a plurality of components is fractionated, during a period when the electromagnetic valve is closed, a vial bottle is moved by a biaxial arm or the like and an empty vial bottle is set at a fractioning position for the next fraction.  
         [0032]    Incidentally, in case a spectrometry is carried out by using only one polarity without changing the positive polarity and the negative polarity as described above, either the chromatogram datum A(+) or chromatogram datum A(−) in FIG. 3 may be processed as zero.  
         [0033]    While the above embodiment shows the case where the positive polarity and the negative polarity are changed, in addition to this, the same method can be used by changing or shifting the operation conditions of the various mass spectrometries. For example, it is possible to carry out the respective mass spectrometries through change of a mode for cleavage of ions by changing a voltage to be applied to a deflector electrode disposed in the intermediate chamber of the MS portion  10 . Of course, in case the operation conditions include more than three kinds, chromatogram data may be calculated in every mass spectrometries of more than three times corresponding to the operation conditions.  
         [0034]    While the invention has been explained with reference to the specific embodiments of the invention, the explanation is illustrative and the invention is limited only by the appended claims.