JP4861107B2 - Chromatograph apparatus and analysis method - Google Patents

Description

  The present invention relates to a chromatographic apparatus and an analysis method for analyzing a sample by chromatography.

  In an analysis using a chromatographic apparatus, optimization of separation conditions can be cited as a method for increasing the analysis throughput. However, since the reduction in the resolution of the detection peak due to the reduction in the separation time causes a reduction in the analysis accuracy, it is difficult to significantly reduce the separation time.

  As a method for shortening the analysis time without changing the separation conditions, there are methods for shortening the time required for the sample injection preparation operation between analyzes and the time required for controlling the analyzer (for example, see Patent Document 1, Patent Document 2, etc.). As is known, the contribution to shortening the analysis time is small, and it is difficult to drastically shorten.

  Also known is a method of shortening the analysis time by processing a plurality of samples in parallel (see, for example, Patent Document 3 and Patent Document 4), but the optimal value of the sample injection interval at the time of multiple sample injection is analyzed. A method for calculating according to the detection condition of the sample has not been known so far.

JP 2005-257575 A JP 2001-153853 A Japanese Patent No. 2834224 JP 2005-127814 A

  An object of the present invention is to provide a chromatographic apparatus and method capable of shortening the time required for analyzing a plurality of samples without changing the separation conditions and by a simple method without reducing the analysis accuracy. That is.

The present invention detects a sample injection part for injecting an analysis sample, a separation column for separating the analysis sample injected by the sample injection part for each component, and a component of the analysis sample separated by the separation column A detector, an input unit for inputting the number of analysis samples, and the sample injection unit, the separation column, the detector, and the input unit so as to analyze the number of analysis samples specified by the input unit. In a chromatographic apparatus including a liquid chromatograph including a control unit that controls each unit and a recording unit that records a chromatogram obtained by analysis, a detection time range from the start to the end of detection of the component is stored. Storage means; processing means for calculating an injection interval for injecting the plurality of analysis samples into the sample injection portion based on a detection time range stored in the storage means; and the injection By applying a septum and having a control means for controlling each unit so that the analysis of multiple analytes is performed.

According to the present invention, it is possible to greatly shorten the time without analysis lowering the analysis accuracy.

  The sample injection interval can be obtained by a simple method of calculating based on the qualitative condition of the detection target component in the sample and the detection time range of each component.

  Before describing the embodiments of the present invention, the outline of the present invention will be briefly described.

  In the analysis of the sample using the chromatograph apparatus, the sample injected into the sample injection unit is separated into each component by the separation column, and then detected in order from the component reaching the detector.

  The detection component is qualitatively determined based on the time (detection time) required from the sample injection to the detector and quantified based on the peak area value and the like.

  In the analysis using a mass spectrometer, qualitative analysis is performed based on the mass-to-charge ratio m / z inherent in the components. Therefore, it is possible to qualify and quantify a plurality of components simultaneously as long as the components have different mass-to-charge ratios.

  Conventionally, when analyzing a plurality of samples using a chromatographic apparatus, the first sample is injected, and after the detection of all detection target components in the sample is completed, the method moves to the analysis of the next sample Is common.

  At this time, the time required for analyzing a plurality of samples is calculated by the product of the time required for analyzing one sample and the number of samples, and the time required for analysis becomes longer in proportion to the number of samples.

In the present invention, without changing the separation conditions and, without also lowering the analysis accuracy, it is possible to analyze in parallel a plurality samples, significantly reducing the time required for the analysis of multiple samples It becomes possible.

  Examples will be described below.

Figure 1 shows the structure of the analytical instrument system for a liquid chromatograph / mass spectrometer (LC / MS) using the present invention.

  This analytical instrument apparatus includes a unit of an eluent pump 10 for supplying an eluent, a unit of an autosampler 20 for injecting an analysis sample, a unit of a cleaning pump 30 for supplying a cleaning liquid to the autosampler, and a cleaning liquid channel switching valve. The analysis unit includes 31 units, a column oven 40 unit for keeping the column temperature constant, and a mass spectrometer 50 unit.

  Furthermore, the analytical instrument device includes a control unit 60 that controls the entire analytical instrument device, a recording unit 70 that records analysis data, an analysis unit 80 that analyzes analysis data, a CRT 90, a printer 100, a mouse 110, and a keyboard 120. Have.

  The autosampler 20 includes a washing port, an injection port, a sample rack, a nozzle, a sample loop, a syringe, an injection valve, and a syringe valve.

  The sample rack of the autosampler 20 includes a place for storing a microplate into which an analysis sample has been dispensed (details of the autosampler are not shown). Moreover, the piping which connects between each said unit is shown as the continuous line, and the wiring which communicates between each unit is shown with the dotted line.

  The eluent sent from the eluent pump 10 sequentially flows through the injection valve of the autosampler 20 to the column oven 40 and the mass spectrometer 50.

  By switching the injection valve, the eluent flows through the flow path returning from the injection valve to the injection valve via the sample loop, nozzle, and injection port.

  At the time of sample injection, the sample is sucked with a nozzle and then the injection valve is switched to introduce the sample into the analysis channel. The cleaning liquid flows to the cleaning port of the autosampler and is used for cleaning the nozzle.

  FIG. 2 shows the overall flow of the analysis procedure.

  First, before starting the continuous analysis of a plurality of samples, a standard sample containing the detection target components in the sample is analyzed, and analysis conditions for detection in a state where all the detection target components in the sample are separated are determined. .

  Analytical conditions for liquid chromatographs include eluent types, flow rate studies, column selection, etc., and analytical conditions for mass spectrometers include mass-to-charge ratios of detection target components in the sample, detection time (sample For example, the time required for the component to reach the detector after injection) and the detection time range.

  The detection time range of each component is set based on each time of the peak start point and end point (time taken from the start to end of component detection) of each component in the mass spectrometer. When the examination of the analysis conditions is completed, the examination result is registered in the control unit 60. The control unit 60 controls each unit according to the registered analysis conditions and analyzes the sample.

  FIG. 3 shows an example of setting values of detection conditions for the detection target component.

  Here, the mass-to-charge ratio, the detection time, and the detection time range of the detection target component are set together with the component name of the detection target component in the sample or a symbol specifying the component.

  Next, the sample injection interval when continuously injecting a plurality of samples is calculated based on the mass-to-charge ratio of the detection target component and the detection time range. This sample injection interval means a waiting time from when a sample is injected until the next sample is injected.

  The sample injection interval is calculated based on the qualitative condition of the detection target component and the detection time range. When a mass spectrometer is used as the detector, the mass-to-charge ratio of the detection target component is used as the qualitative condition, and when the DAD detector is used, the absorption wavelength band of the detection target component is used as the qualitative condition.

In either case, if detection peaks of components with the same qualitative conditions overlap, accurate quantification becomes difficult and causes a decrease in quantification accuracy. the condition of calculating the fee Note input interval.

  In particular, among the calculated sample injection intervals, when the waiting time is set to be the shortest, the time required for analyzing a plurality of samples is the shortest. The calculation conditions for the sample injection interval will be described later.

  After obtaining the sample injection interval, the number of analysis samples is input to the control unit 60 and analysis is started. When the analysis is started, the nozzle of the autosampler 20 sucks a specified amount of the analysis sample from the vial of the analysis sample installed in the sample rack and injects it into the injection port.

  The sample is introduced into the analysis channel by switching the injection valve simultaneously with the injection. At the same time, a start signal is sent from the autosampler 20 to the mass spectrometer 50 to start data acquisition and analysis.

  The time when the analysis sample is injected (sample injection time) is registered in the analysis unit 80 in a state associated with the analysis sample. Further, based on the pre-registered component detection time and sample injection time, the time at which each component reaches the detector (scheduled detection time) is calculated, and the result is registered in the analysis unit 80.

  After the first sample injection, after the time corresponding to the sample injection interval calculated in advance has elapsed, the next sample is injected and the sample injection time is recorded in the recording unit 70.

  Thereafter, the same processing is performed until the analysis of the preset number of analysis samples is completed.

  An analysis sample is injected, components in the sample are separated by a separation column, and components reaching the mass spectrometer 50 are sequentially detected.

  The detection time and mass-to-charge ratio of the component actually detected are compared with the detection time and mass-to-charge ratio of the detection target component of each analysis sample registered in the analysis unit 80, and the detected component is identified and analyzed. Is recorded in the recording unit 70.

  Next, conditions for calculating the sample injection interval will be described.

  Here, a case where a mass spectrometer is used as a detector will be described.

  As already described, in order to analyze a plurality of samples with high accuracy, it is necessary that the detection time ranges of components that are difficult to separate and analyze with a detector do not overlap among the detection target components in the sample. .

  Therefore, the following is a calculation condition of the sample injection interval.

    (1) It is longer than the detection time range of any detection target component contained in the sample.

(2) Components that are difficult to detect separately from each other in the sample (the mass-to-charge ratio is the same or
Is within the specified approximate value), if there are multiple detection times of those components
After setting the multi-component detection time range (multi-component detection time range spanning multiple components from the start to the end of component detection) that includes the entire range, the condition (1) is applied.

At this time, it is also possible to any detection time range plurality Ingredient set by the user to set a sample injection interval so as not to overlap.

  For example, when there is a component that suppresses ionization of other components, the sample injection interval is set so that the detection time range of the inhibitory component and the target component does not overlap, thereby avoiding a decrease in the analysis accuracy of the target component it can.

  In this case, in (2) above, a detection time range including all of the detection time ranges of the inhibition component and the target component may be set, and the condition (1) may be applied.

  The above process creates a program that automatically calculates the sample injection interval based on the detection conditions of the detection target component in the sample, and sets the sample injection interval to be calculated at the same time as setting the detection condition. It is also possible to save the trouble of calculating the injection interval.

The range user selects multiple component detection time, the plurality of components detection time range is memorize in the memory means of the memory unit.

  Specific analysis values are shown below.

  This example shows the results of analyzing a saccharide 4-component mixed sample (fructose, glucose, sucrose, raffinose) in which the contained components are known and the concentration of each component is unknown.

  Each sample is analyzed by adding a fixed amount of each component isotope as an internal standard. The standard sample is analyzed before the analysis is started, and based on the result, the mass-to-charge ratio, the detection time, and the detection time range of the detection target component in this example are determined as follows.

Fructose (m / z: 179) Detection time: 3.7 minutes
Detection time range: (3.4 minutes-4.0 minutes)
Glucose (m / z: 179) Detection time: 4.5 minutes
Detection time range: (4.1 minutes -4.9 minutes)
Sucrose (m / z: 341) Detection time: 6.1 minutes
Detection time range: (5.5 minutes-6.7 minutes)
Raffinose (m / z: 503) Detection time: 10.2 minutes
Detection time range: (9.2 minutes -11.2 minutes)
Here, the time between sample injections is t, the number of sample injections is n (n = 1, 2,..., N), and the scheduled detection time T of each component is expressed as follows.

Expected detection time for fructose: T _n (fructose) = t (n−1) +3.7
Glucose detection scheduled time: T _n (glucose) = t (n−1) +4.5
Scheduled detection time of sucrose: T _n (sucrose) = t (n−1) +6.1
Raffinose scheduled detection time: T _n (Raffinose) = t (n−1) +10.2
The detection time range of each component can be expressed as follows.

Detection time range of fructose (m / z = 179) Start point: t (n-1) +3.4
End point: t (n-1) +4.0
Detection time range of glucose (m / z = 179) Start point: t (n-1) +4.1
End point: t (n-1) +4.9
Detection time range of sucrose (m / z = 341) Start point: t (n-1) +5.5
End point: t (n-1) +6.7
Detection time range of raffinose (m / z = 503) Start point: t (n-1) +9.2
End point: t (n-1) +11.2
FIG. 4 shows the relationship between the mass-to-charge ratio of each component and the detection time range at this time.

  FIG. 4 shows chromatograms of fructose, glucose (each m / z = 179), sucrose (m / z = 341) and raffinose (m / z = 503), with the horizontal axis representing time and the vertical axis representing the intensity of each peak. Is shown.

  The detection time range of each component is the range indicated by shading in the figure. Here, since fructose and glucose have the same mass-to-charge ratio, a detection time range including each detection time range of fructose and glucose is set from (2) of the sample injection interval calculation condition at the time of multiple sample injection.

  As a result, the detection time range of m / z = 179 is from the start point of the fructose detection time range to the end point of the glucose detection time range, and is set as follows.

Detection time range of m / z = 179 Start point: t (n-1) +3.4
End point: t (n-1) +4.9
The minimum value of the sample injection interval is calculated based on the above setting conditions. As a result, since the maximum detection time range is 2.0 minutes of raffinose (m / z = 503), the minimum value t _min of the sample injection interval is 2.0. The result is registered in the control unit 60, and the analysis unit is controlled to inject the sample every time.

  The above processing simplifies the process of setting analysis conditions before the start of analysis by creating a program that automatically sets the mass-to-charge ratio and detection time range of peaks detected in the analysis results of standard samples. You can also

  Next, various conditions for continuously analyzing a plurality of samples are set, and continuous analysis is started. In this example, the analysis was performed by setting the number of analysis samples to 10, the sample injection amount of 10 ul, and the time between sample injections of 2.0 minutes. FIG. 5 shows chromatograms at m / z = 179 and m / z = 503 (the horizontal axis is time). As determined from the above calculation conditions, when a sample is injected with a sample injection interval of 2 minutes, a plurality of samples can be analyzed without overlapping the detection time ranges of the detection target components.

  When analysis is started and an analysis sample is injected, the mass spectrometer 50 starts data acquisition and analysis. After 2 minutes have passed since the first sample was injected, the next analysis sample was injected, and thereafter, the same processing was repeated for a total of 10 analyzes.

At this time, when a 0-minute time injected with analytical sample of first time _(I 1 = 0), the sample injection time _{I n is, I n = 0,2,4,6,8, ··· 2} (n -1)..., 18 (n is the number of sample injections, n = 1, 2,..., 10).

  Further, the scheduled detection time of each component is calculated as follows and registered in the control unit 60 and the analysis unit 70.

Detection time of fructose (m / z = 179): T = 3.7, 5.7,.
2 (n-1) +3.7, ...,
21.7
Detection time of glucose (m / z = 179): T = 4.4, 6.4,.
2 (n-1) +4.4, ...,
22.4
Detection time of sucrose (m / z = 341): T = 6.1, 8.1,.
2 (n-1) +6.1, ...,
24.1
Scheduled detection time of raffinose (m / z = 503): T = 10.2,
12.2, ...
2 (n-1) +10.2,
..., 28.2
(Where n is the number of sample injections: n = 1, 2,..., 10)
By using the above relational expression, the component detected by the mass spectrometer can be easily assigned to the original analysis sample. For example, when a substance with m / z = 179 is detected at time T = 13.7, it is found from the mass-to-charge ratio of the detected substance that the substance is fructose or glucose.

  Next, it is possible to determine that the detected substance is fructose by comparing the detection time of the detected substance with the scheduled detection times of fructose and glucose.

  Moreover, since the calculation formula for the scheduled detection time of fructose is obtained as I = 10 and n = 6, the detected fructose has a sample injection time of 10 minutes and is included in the sixth injected analysis sample. It is determined that it is a component that was present.

  As a result, the analysis result of the detected component is recorded in the recording unit 70 as the quantitative result of the fructose of the analysis sample injected sixth. The same processing is performed for the other components, and each analysis sample and the detection result of each component are all associated and recorded in the recording unit 70.

  In addition, the operation can be simplified by creating a program that automatically performs the above processing in the control unit 60, the analysis unit 80, and the recording unit 70.

  Here, it is also possible to perform analysis using a detector that performs qualitative and quantitative measurement based on the physical quantity unique to the target component, such as a diode array detector (DAD), instead of a mass spectrometer. It is clear from the content of the invention.

1 is a schematic configuration diagram of an LC / MS analysis instrument apparatus according to an embodiment of the present invention. The work process figure of the analysis in the LC / MS analytical instrument apparatus concerning the Example of this invention. The figure which shows the example of the setting value of the detection conditions in the LC / MS analyzer apparatus concerning the Example of this invention. The figure which concerns on the Example of this invention and shows the chromatogram obtained on detection conditions. The figure which concerns on the Example of this invention and shows the chromatogram obtained when a sample is inject | poured.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Eluent pump, 20 ... Autosampler, 30 ... Washing pump, 40 ... Column oven, 50 ... Mass spectrometer, 60 ... Control part, 70 ... Recording part, 80 ... Analysis part, 90 ... CRT, 100 ... Printer, 110 ... mouse, 120 ... keyboard.

Claims (1)

A sample injection part for injecting an analysis sample;
A separation column for separating the analysis sample injected by the sample injection unit into components;
A detector for detecting a component of the analysis sample separated by the separation column;
An input unit for inputting the number of the analysis samples;
A control unit that controls each unit such as the sample injection unit, the separation column, the detector, and the input unit so as to perform analysis of the number of analysis samples specified by the input unit;
In a chromatograph apparatus including a liquid chromatograph provided with a recording unit for recording a chromatogram obtained by analysis,
Storage means for storing a detection time range from the start to the end of detection of the component;
Processing means for calculating an injection interval for injecting a plurality of the analysis samples into the sample injection unit based on the detection time range stored in the storage means;
Wherein the injection interval by applying have a control means for controlling each unit so that the analysis of multiple analytes is performed,
Using a mass spectrometer as the detector,
The mass-to-charge ratio of the component analyzed and detected by the mass spectrometer and the detection time range are stored in the storage means,
In the detection time range, the processing means sets a time interval equal to or greater than a maximum detection time range width as the injection interval,
A user-selected multi-component detection time range that extends over any of a plurality of components selected by the user from the detection target components included in the analysis sample is newly set, and the user-selected multi-component detection time range and the detection time A chromatograph apparatus characterized in that a longest detection time range from the range is the injection time interval .

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