CN106290679B - High performance liquid chromatography device and analysis method thereof - Google Patents
High performance liquid chromatography device and analysis method thereof Download PDFInfo
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- CN106290679B CN106290679B CN201510252133.2A CN201510252133A CN106290679B CN 106290679 B CN106290679 B CN 106290679B CN 201510252133 A CN201510252133 A CN 201510252133A CN 106290679 B CN106290679 B CN 106290679B
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Abstract
The invention provides a high performance liquid chromatography device which comprises a constant flow pump, a chromatographic column, a detector and a sensor for measuring the real-time flow of a mobile phase. The constant flow pump is used to provide a mobile phase to carry the components to be tested into the column for separation and then into the detector for detection of each component. The real-time flow measured by the sensor is used for integrating and calculating the peak area of the chromatogram, so that the content of the measured component is calculated based on the peak area. The invention also relates to a high performance liquid chromatography analysis method.
Description
Technical Field
The invention relates to a high performance liquid chromatography device and an analysis method thereof.
Background
At present, when the content of a certain component is quantitatively analyzed by using high performance liquid chromatography, the content is usually calculated according to the peak area of the component in a chromatogram, and the formula of the peak area is usually as follows: a. thei=(Fc)∫htdt of, wherein AiPeak area of chromatographic peak representing measured component, FcExpressed as the mobile phase flow, htExpressed as the real-time peak height at time t. However, in the analysis process of the content of the measured component, the accuracy of the measurement result is not high.
Disclosure of Invention
In view of the above, there is a need for a high performance liquid chromatography apparatus and an analysis method thereof that improve the accuracy of the measurement results of the components to be measured.
A high performance liquid chromatography device comprises a constant flow pump, a chromatographic column and a detector. The constant flow pump is used to provide a mobile phase to carry the components to be tested into the column for separation and then into the detector for detection of each component. The high performance liquid chromatography device also comprises a sensor for measuring the real-time flow of the mobile phase. The real-time flow measured by the sensor is used for integrating and calculating the peak area of the chromatogram, so that the content of the measured component is calculated based on the peak area.
The sensor may be disposed at a front end of the detector, a rear end of the detector, or on the detector.
The detector is provided with a flow cell and the sensor comprises a sensor for measuring or converting a signal for the flow of the mobile phase flowing through the front end of the flow cell, the flow of the mobile phase flowing through the back end of the flow cell.
The signal is the pressure of the pipeline flowing through the front and the back of the flow cell.
The peak area of the chromatogram is calculated to include the form: a. thei=∫htFtIntegral function of dt, where AiPeak area of chromatographic peak representing measured component, FtExpressed as the mobile phase real-time flow at time t, htExpressed as the real-time peak height at time t.
The sensor comprises a flow meter or a pressure sensor.
A high performance liquid chromatography method comprising the steps of:
providing a high performance liquid chromatography device, wherein the high performance liquid chromatography device comprises a chromatographic column, a constant flow pump for providing a mobile phase and a sensor;
injecting a sample containing the component to be detected into the high performance liquid chromatography device, and enabling a mobile phase to carry the sample to flow through the chromatographic column for analysis;
the sensor measures the real-time flow of the mobile phase;
obtaining the real-time peak height of the chromatographic peak of the measured component at the measurement time t;
the real-time peak height at the time t is combined with the real-time flow at the time t to carry out integral calculation on the peak area of the chromatographic peak of the component to be measured;
the content of the component to be measured is calculated based on the peak area.
The peak area of the chromatogram is calculated to include the form: a. thei=∫htFtIntegral function of dt, where AiExpressed as the peak area of the chromatographic peak of the component to be measured, FtExpressed as the mobile phase real-time flow at time t, htExpressed as the real-time peak height at time t.
The sensor comprises a flow meter or a pressure sensor.
The real-time flow measured by the sensor includes the mobile phase flow before passing through the detector, the mobile phase flow after passing through the detector, or the mobile phase flow passing through the detector.
The high performance liquid chromatography device also comprises a detector which is provided with a flow cell, and the real-time flow measured by the sensor comprises the flow of the mobile phase before flowing through the flow cell, the flow of the mobile phase after flowing through the flow cell or a signal which can be converted into the flow of the mobile phase.
The signal is the pressure of the pipeline flowing through the front and the back of the flow cell.
Compared with the prior art, the high performance liquid chromatography device provided by the invention is provided with the sensor for measuring the real-time flow of the detector, and the peak area of the chromatogram is calculated by integrating the real-time flow measured by the sensor. The influence of pipeline flow fluctuation on peak area calculation of a chromatogram is avoided, and the flow precision and performance requirements of the constant flow pump are reduced. The high performance liquid chromatography analysis method improves the accuracy of chromatogram area calculation.
Drawings
FIG. 1 is a system configuration diagram of a first preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 2 is a system configuration diagram of a second preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 3 is a system configuration diagram of a third preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 4 is a system configuration diagram of a fourth preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 5 is a system configuration diagram of a fifth preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 6 is a system configuration diagram of a sixth preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 7 is a system configuration diagram of a seventh preferred embodiment of the high performance liquid chromatography apparatus of the present invention.
FIG. 8 is a graph showing the measurement results of the high performance liquid chromatography apparatus of the present invention using chromatography columns of different numbers of uses.
Description of the main elements
| High performance |
100，200，300，400，500，600，700 |
| Buffer |
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The following detailed description will further illustrate the invention in conjunction with the above-described figures.
Detailed Description
Referring to fig. 1, a high performance liquid chromatography apparatus 100 according to a first embodiment of the present invention is used for measuring the content of a component to be measured, and includes a buffer bottle 1 for storing a mobile phase, a constant flow pump 2, a pulse damper 3, a sample injector 4, a chromatography column 5, a detector 6, a sensor 7, a back pressure adjusting device 8, and an infusion pipeline 9, wherein the infusion pipeline 9 is disposed between the buffer bottle 1, the constant flow pump 2, the pulse damper 3, the sample injector 4, the chromatography column 5, the detector 6, the sensor 7, and the back pressure adjusting device 8.
The constant flow pump 2 is used to provide a constant flow of mobile phase. The pulse damper 3 is capable of monitoring the back end hydraulic system pressure, which generally utilizes the compressibility of the material itself to smooth the line flow. The sample injector 4 is used to inject the components to be measured into the mobile phase, and to load the chromatographic column 5 with the mobile phase. The column is used to separate the component to be measured into a number of individual components, and each individual component flows sequentially out of the column 5 and into the detector 6. The sensor 7 is for example a flow meter or a pressure sensor.
In this embodiment, the sensor 7 is a flow meter, the sensor 7 is disposed between the detector 6 and the back pressure adjusting device 8, and the sensor 7 is used for measuring the flow rate of the mobile phase flowing through the detector 6 in real time.
The detector 6 is provided with a flow cell 61, i.e. the sensor 7 can also measure the flow of the mobile phase before it has passed through the flow cell 61, before it has passed through the flow cell 61 or a signal which can be converted into the flow of the mobile phase in real time. The signal for the mobile phase flow is, for example, the line pressure before and after flowing through the flow cell 61.
It will be appreciated that the sensor 7 may be located at the front end of the detector 6, at the rear end of the detector 6 or on the detector 6.
Referring to fig. 1, when analyzing a component to be measured in the hplc apparatus 100 of the present invention, first, the mobile phase stored in the buffer bottle 1 is injected into the infusion pipeline 9 by the constant flow pump 2. The component to be measured then passes through the sample injector 4 into the mobile phase, which drives the component to be measured into the chromatographic column 5. The detected component is separated into a plurality of single components in the chromatographic column 5, each single component enters the detector 6, and the content of each component is detected and analyzed by the detector 6 in turn. The sensor 7 measures the flow rate of the mobile phase flowing through the detector 6 in real time during the analysis of the component to be measured.
The detector 6 is provided with a flow cell 61, i.e. the sensor 7 can measure the flow of the mobile phase in front of the flow cell 61 or the flow in front of the flow cell 61 in real time.
It will be appreciated that the sensor 7 can also be used to convert the mobile phase flow by measuring in real time the line pressure before and after the flowcell 61.
Referring to FIG. 2, in the second embodiment of the HPLC apparatus 200 of the present invention, the sensor 7 is disposed at the front end of the detector 6, for example, the sensor 7 is disposed between the chromatographic column 5 and the detector 6.
In this embodiment, the sensor 7 is a flow meter, and the sensor 7 is used for measuring the flow rate of the mobile phase flowing through the chromatographic column 5 in real time.
Research finds that when the chromatographic column 5 with a large number of times is used for analyzing the component to be measured, the pressure of the chromatographic column rises due to the fact that impurities in a sample block the chromatographic column, and the flow fluctuation of the chromatographic column is large, so that the accuracy of a measuring result is influenced. In order to improve the accuracy of the measurement result, the flow rate of the mobile phase flowing through the chromatographic column 5 can be measured in real time through the sensor 7, the real-time flow rate is combined with the current real-time peak height, the chromatographic peak area of the target component is calculated in an integrating mode, the content of the measured component is calculated based on the peak area, and therefore the problem that the measured component generates deviation due to the influence of the chromatographic column which is used for a large number of times on the measurement result is solved.
Referring to FIG. 3, in the third embodiment of the HPLC apparatus 300 according to the present invention, the sensor 7 is disposed at the front end of the detector 6, for example, the sensor 7 is disposed between the sample injector 4 and the chromatographic column 5. In this embodiment, the sensor 7 is a flow meter, and the sensor 7 is used for measuring the flow rate of the mobile phase flowing through the detector 6 in real time.
Referring to FIG. 4, in the third embodiment of the HPLC apparatus 400 of the present invention, the sensor 7 is disposed at the front end of the detector 6, for example, the sensor 7 is disposed between the pulse damper 3 and the sample injector 4. In this embodiment, the sensor 7 is a flow meter, and the sensor 7 is used for measuring the flow rate of the mobile phase flowing through the detector 6 in real time.
Referring to fig. 5, in the third embodiment of the hplc apparatus 500 of the present invention, the sensor 7 is disposed at the front end of the detector 6, for example, the sensor 7 is disposed between the chromatographic column 5 and the detector 6. In this embodiment, the sensor 7 is a pressure sensor, and the sensor 7 measures the line pressure before and after flowing through the flow cell 61 in real time.
Referring to fig. 6, in a third embodiment of the hplc apparatus 400 of the present invention, the sensor 7 is disposed on the detector 6. In this embodiment, the sensor 7 is a pressure sensor, the sensor 7 measures the line pressure after the detector 6 in real time, and the sensor 7 measures the line pressure before and after the flow cell 61 in real time.
Referring to FIG. 7, in a third embodiment of the HPLC apparatus 400 according to the present invention, the sensor 7 is disposed between the pulse damper 3 and the sample injector 4. In this embodiment, the sensor 7 is a pressure sensor, and the sensor 7 measures the line pressure before and after flowing through the flow cell 61 in real time.
Referring also to fig. 2-4, similar to fig. 1, the sensor 7 is preferably a flow meter for measuring the flow rate of the mobile phase in real time. Except that the sensors 7 are respectively disposed at different positions to measure the flow rate of the mobile phase in real time.
Referring also to fig. 5-7, similar to fig. 1, the sensor 7 is a pressure sensor for measuring the line pressure. In contrast, the sensors 7 are respectively disposed at different positions to measure the pressures of the respective lines in real time, and then convert the measured pressures of the respective lines into the mobile phase flow rate values.
The high performance liquid chromatography device is provided with a sensor for measuring the real-time flow of the detector, and the peak area of the chromatogram is calculated by integrating the real-time flow measured by the sensor. The influence of pipeline flow fluctuation on peak area calculation of a chromatogram is avoided, and the flow precision and performance requirements of the constant flow pump are reduced.
A high performance liquid chromatography method comprising the steps of:
a high performance liquid chromatography apparatus is provided that includes a constant flow pump 2 for providing a mobile phase, an injector 4, a chromatography column 5, a detector 6, and a sensor 7.
Firstly, the constant flow pump 2 provides a certain flow rate of mobile phase, the component to be measured enters the mobile phase through the sample injector 4 and is loaded into the chromatographic column 5 by the mobile phase, the component to be measured is separated into a plurality of components in the chromatographic column 5, and each component flows out of the chromatographic column in turn and enters the detector 6.
The sensor 7 measures the real-time flow of mobile phase through the column.
The sensor 7 is for example a flow meter or a pressure sensor.
The real-time flow measured by the sensor 7 includes the mobile phase flow before passing through the detector 6, the mobile phase flow after passing through the detector 6, or the mobile phase flow passing through the detector 6.
The detector 6 is provided with a flow cell 61 and the real-time flow measured by the sensor 6 comprises the mobile phase flow before passing through the flow cell 61 or the mobile phase flow after passing through the flow cell 61.
It will be appreciated that the real-time flow measured by the sensor 7 also includes measuring the line pressure before and after the flow cell 61, which can be converted to the mobile phase flow value.
Subsequently, the real-time peak height at the measurement time t of the chromatographic peak of the measured component is obtained.
And integrating the real-time peak height at the time t and the real-time flow at the time t to calculate the peak area of the chromatographic peak of the component to be measured. The peak area of the chromatogram is calculated to include the form: a. thei=∫htFtdt, where i is expressed as the measured component; a. theiA peak area representing a chromatographic peak of the measured component; ft is expressed as the mobile phase real-time flow; ht is expressed as the real-time peak height at time t, where peak height h is expressed as the distance from the highest point of the peak to the bottom of the peak in the chromatogram.
The calculation of the measured component includes the form: m isi= (1/S) AiWherein m isiExpressed as the content of the component to be measured; s is expressed as detector sensitivity.
It will be appreciated that the peak area of the chromatogram is directly proportional to the content of the component being measured.
The applicant finds out through research that the content of the component to be detected is analyzed by the high performance liquid chromatography, and results are deviated due to the use of different chromatographic columns. When the chromatographic column with more times is used for analysis, the pressure of the chromatographic column rises due to the fact that impurities in a sample block the chromatographic column, and the measured real-time flow fluctuates greatly, so that the accuracy of a measuring result is influenced. When a new chromatographic column is used for analysis, the difficulty of constant flow rate provided by the constant flow pump is increased, and the measured real-time flow fluctuation is large, so that the accuracy of the measurement result is influenced.
The high performance liquid chromatography method of the invention measures the flow value of the mobile phase in real time, integrates and calculates the chromatographic peak area of the target component by combining the real-time flow with the real-time peak height at that time, and calculates the content of the measured component based on the peak area. The result shows that the measurement result of the content of the component to be measured is less influenced by the use times of the chromatographic column, so that the accuracy of the measurement result is improved.
The present invention will be specifically described below with reference to examples.
Example comparison of column measurements for different numbers of uses
A glycated hemoglobin standard with a known target value of 5.7% (glycated hemoglobin HbA1c as a percentage of total hemoglobin) was measured using chromatography columns of different numbers of uses. The instrument is a Meyer glycated hemoglobin analyzer and is operated according to the instrument specification.
The method comprises the following steps: in the HPLC analysis method of the present invention, the sensor 7 is a flow meter, and the flow rate (F) of the mobile phase flowing through the chromatographic column at time t is measured in real time by the flow metert) Obtaining a real-time peak height (h) at a measurement time t of a chromatographic peak of the measured componentt) Using an integral function Ai=∫htFtdt calculating the peak area, where AiPeak area of chromatographic peak representing measured component, FtExpressed as the mobile phase real-time flow, htExpressed as the real-time peak height at time t. And calculating the content of the glycosylated hemoglobin according to the peak area.
The method 2 comprises the following steps: in the conventional HPLC analysis method, a constant flow rate (F) of the mobile phase is setc) Obtaining a real-time peak height (h) at a measurement time t of a chromatographic peak of the measured componentt) Using an integral function Ai=(Fc)∫htdt calculating the peak area, where AiPeak area of chromatographic peak representing measured component, FcExpressed as the set flow rate of the mobile phase, htExpressed as the real-time peak height at time t. And calculating the content of the glycosylated hemoglobin according to the peak area.
As shown in the first Table and FIG. 8, the content of glycated hemoglobin measured by method 1 showed little variation in the results, indicating that the measurement results of the sample to be measured are less affected by the number of times the column is used by the HPLC analysis method of the present invention. The result deviation of the content of the glycated hemoglobin measured by the method 2 is large, which indicates that the measurement result of the sample to be measured deviates from the true value more and more along with the increase of the use times of the chromatographic column.
TABLE-methods 1 and 2-measurement results of chromatography columns using different numbers of uses
Note: the chromatographic column 1 is a new column; the chromatography column 2 is a chromatography column used about 500 times; the chromatographic column 3 is a chromatographic column used about 1000 times; the chromatography column 4 is a chromatography column used about 1500 times.
The high performance liquid chromatography method provided by the invention is provided with the sensor to measure the flow value of the mobile phase in real time, and the real-time flow is combined with the current real-time peak height to calculate the chromatographic peak area of the measured component in an integral manner, so that the problem of large peak area calculation deviation of a chromatogram map caused by the influence of pipeline flow fluctuation is solved. By using the high performance liquid chromatography analysis method, the detection result is less influenced by the use times of the chromatographic column, so that the accuracy of the measurement result is improved, the requirement on the performance of the constant flow pump of the instrument can be reduced, and the high performance liquid chromatography analysis method has a better application prospect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and the above embodiments are only used for explaining the claims. The scope of the invention is not limited by the description. Any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present disclosure are included in the scope of the present invention.
Claims (10)
1. A high performance liquid chromatography device, which comprises a constant flow pump, a chromatography column and a detector, wherein the constant flow pump is used for providing a mobile phase to load a component to be detected into the chromatography column for separation, and then the component enters the detector to detect each component, and the high performance liquid chromatography device is characterized in that: the high performance liquid chromatography device also comprises a sensor for measuring the real-time flow of the mobile phase, the real-time flow measured by the sensor is used for integrating and calculating the peak area of a chromatogram, so as to calculate the content of the measured component based on the peak area, and the calculation of the peak area of the chromatogram comprises the following steps: a. thei＝∫htFtIntegral function of dt, where AiPeak area of chromatographic peak representing measured component, FtExpressed as timeReal time flow of mobile phase at t, htExpressed as the real-time peak height at time t.
2. The high performance liquid chromatography apparatus of claim 1, wherein: the sensor may be disposed at a front end of the detector, a rear end of the detector, or on the detector.
3. The high performance liquid chromatography apparatus of claim 2, wherein: the detector is provided with a flow cell and the sensor comprises a sensor for measuring or converting a signal for the flow of the mobile phase flowing through the front end of the flow cell, the flow of the mobile phase flowing through the back end of the flow cell.
4. The high performance liquid chromatography apparatus of claim 3, wherein: the signal is the pressure of the pipeline flowing through the front and the back of the flow cell.
5. The high performance liquid chromatography apparatus of claim 1, wherein: the sensor comprises a flow meter or a pressure sensor.
6. A high performance liquid chromatography method comprising the steps of:
providing a high performance liquid chromatography device, wherein the high performance liquid chromatography device comprises a chromatographic column, a constant flow pump for providing a mobile phase and a sensor;
injecting a sample containing the component to be detected into the high performance liquid chromatography device, and enabling a mobile phase carrying the sample to flow through the chromatographic column;
the sensor measures the real-time flow of the mobile phase;
obtaining the real-time peak height of the chromatographic peak of the measured component at the measurement time t;
the real-time peak height at the time t is combined with the real-time flow at the time t to carry out integration calculation on the peak area of the chromatographic peak of the measured component, and the calculation of the peak area of the chromatographic peak comprises the following forms: a. thei＝∫htFtIntegral function of dt, where AiChromatographic peak expressed as measured componentArea of peak of (1), FtExpressed as the mobile phase real-time flow at time t, htExpressed as the real-time peak height at time t;
the content of the component to be measured is calculated based on the peak area.
7. The high performance liquid chromatography analysis method of claim 6, wherein: the sensor comprises a flow meter or a pressure sensor.
8. The high performance liquid chromatography analysis method of claim 6, wherein: the real-time flow measured by the sensor includes the mobile phase flow before passing through the detector, the mobile phase flow after passing through the detector, or the mobile phase flow passing through the detector.
9. The high performance liquid chromatography analysis method of claim 8, wherein: the high performance liquid chromatography device also comprises a detector which is provided with a flow cell, and the real-time flow measured by the sensor comprises the flow of the mobile phase before flowing through the flow cell, the flow of the mobile phase after flowing through the flow cell or a signal which can be converted into the flow of the mobile phase.
10. The high performance liquid chromatography analysis method of claim 9, wherein: the signal is the pressure of the pipeline flowing through the front and the back of the flow cell.
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