CN112763637A - Variable wavelength data acquisition and processing method of liquid chromatograph ultraviolet detector - Google Patents
Variable wavelength data acquisition and processing method of liquid chromatograph ultraviolet detector Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 16
- 238000003672 processing method Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000005070 sampling Methods 0.000 claims description 19
- 230000005540 biological transmission Effects 0.000 claims description 10
- 238000001228 spectrum Methods 0.000 claims description 8
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 abstract description 12
- 238000004458 analytical method Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 6
- 230000035772 mutation Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000218176 Corydalis Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
Abstract
The invention relates to a variable wavelength data acquisition and processing method of an ultraviolet detector of a liquid chromatograph, in particular to a data acquisition and processing method which can realize an optimized variable wavelength function and can carry out combined control operation detection with a gradient elution program. It comprises 4 steps. The data acquisition and processing method can effectively improve the baseline stability of the detector under specific conditions (variable wavelength), and improve the detection efficiency; and seamless connection of spectrograms of different wavelength bands can be realized. By updating the subsequent wavelength pre-acquisition part in the wavelength changing process and sequentially connecting the subsequent wavelength pre-acquisition part with the data of the previous wavelength, the distortion and base line mutation caused by real-time acquisition and display of the data are avoided, the base line consistency and the data rationality of the data in each wavelength acquisition time period are ensured, the spectrogram data is collected in a unified base line and a coordinate range to facilitate processing and analysis, and the combined application of the variable wavelength and gradient elution is expanded and realized.
Description
Technical Field
The invention relates to a variable wavelength data acquisition and processing method of an ultraviolet detector of a liquid chromatograph, in particular to a data acquisition and processing method which can realize an optimized variable wavelength function and can carry out combined control operation detection with a gradient elution program, and belongs to the technical field of liquid chromatogram detection.
Background
The ultraviolet-visible spectrophotometry is a method for determining the absorbance of a substance within the wavelength range of 190-800 nm, and is used for identification, impurity inspection and quantitative determination. The absorption spectrum of the measured substance is obtained by measuring the absorbance of the substance at different wavelengths and drawing a relation graph of the absorbance and the wavelength. The liquid phase ultraviolet-visible detector (UV-VIS) is an extended application, and can realize dual-wavelength detection. Two common methods are mainly adopted, one method adopts two sets of independent light splitting systems, and the structure is complex and expensive; the second, more common and simple method, is to use a grating to switch rapidly to realize two well-defined wavelengths for time-sharing collection. The sampling frequency of the whole process adopting the dual wavelengths is lower, and the accuracy of the analysis result is influenced. The dual-wavelength detector can obtain two independent spectra (each wavelength corresponds to one) in an acquisition process, the grating is always in a reciprocating switching state (generally, the wavelength is switched once in 0.5 second) under the control of a transmission mechanism in the process, the grating slightly shakes at the moment of switching to a set wavelength in rotation, and the shaking brings great noise to spectrum acquisition and affects a baseline and a signal-to-noise ratio.
At present, the ultraviolet detector of the liquid chromatograph is sensitive to the wavelength change due to the flow when the wavelength-changing program is operated, and can cause the base line to obviously change. As shown in fig. 3, a spectrum without any baseline adjustment is shown when the wavelength is changed, and a significant "step" appears on the baseline during the switching process of the wavelength. The common method needs to stop collecting and continue collecting after the wavelength is stable. Fig. 4 shows a spectrogram which stops collecting when the wavelength is changed and continues to collect after the wavelength is stabilized, because the collection is interrupted during the wavelength changing process, which results in the discontinuity of the actually collected data. Other procedures have been used to force the baseline straight, but have destroyed the original continuity of the data. Furthermore, the processing method of stopping the collection and waiting for the stabilization thereof may cause analysis errors due to differences between the collection time and the gradient setting time when the gradient elution program is used. If the requirement of data acquisition continuity can be met, the wavelength-variable program can realize more stable noise and higher sensitivity.
Existing multi-wavelength solutions on the market mainly use DAD diode array detectors, but are expensive. The variable wavelength application related to pharmacopoeia or other national standards only analyzes an isocratic system with constant mobile phase conditions in consideration of the obvious change of a base line caused by the variable wavelength during switching, and belongs to simpler application analysis. While for complex components gradient elution is required, if multi-wavelength analysis is involved, instead of using expensive diode array detectors, the variable wavelength function of the uv detector can be considered instead of the DAD detector if the separation of the components is satisfactory. However, when the wavelength-variable operation and the gradient elution function are operated in a combined manner, the baseline can be significantly changed due to the superposition effect of the change of the wavelength-variable and the gradient conditions, so that the analysis is influenced, and therefore, the combined application of the wavelength-variable and the gradient elution is not available at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a variable wavelength data acquisition and processing method of an ultraviolet detector of a liquid chromatograph, which can keep the originality and consistency of detection data through continuous data processing and improve the baseline stability.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for acquiring and processing variable wavelength data of an ultraviolet detector of a liquid chromatograph comprises the following steps:
s1, starting to pass through the fixed wavelength lambdanAs initial wavelength, data acquisition is carried out at a higher sampling frequency;
s2, in step S1, tnAfter x time length, the transmission mechanism controls the grating to start rotating and switches to lambdanAnd subsequent wavelength lambdan+1A dual-wavelength acquisition mode with a lower sampling frequency is carried out alternately, the acquisition time is x, and a background software algorithm carries out wavelength lambdan+1At tnPost-acquired data baseline translationTo and wavelength lambdanThe baseline level of the collected data is consistent, so that lambda is enabledn+1At tnData spectrum and lambda in later timenAt tnSeamless connection is carried out on the data spectrogram collected before;
s3, go to t in step S2nWhen the grating stops rotating, the transmission mechanism controls the grating to switch to a fixed wavelength lambdan+1Continuously acquiring data at a higher sampling frequency until a preset second wavelength-variable time point is reached;
s4, let n be n +1, and repeat steps S1 to S3.
Further, the higher sampling frequency in the fixed wavelength mode is 20Hz, the dual-wavelength sampling is adopted in the variable wavelength switching process, and the lower sampling frequency in the dual-wavelength mode is 2 Hz.
Further, in steps S2 and S3, the transmission mechanism is a servo motor equipped with a planetary reducer, which can meet the requirements of high precision and good rigidity.
Further, the variable wavelength data acquisition and processing method of the liquid chromatograph ultraviolet detector is applied to a gradient elution program.
The working principle of the invention is that the ultraviolet detector of the liquid chromatograph realizes the switching between the fixed wavelength acquisition mode and the dual wavelength acquisition mode through program control.
The invention adopts a dual-wavelength mode to process a wavelength changing process, the wavelength changing function is to collect data of different wavelengths and different time periods and present the data on a spectrogram according to the condition that the retention time of each component in the wavelength band near the maximum absorption wavelength has an obvious time region.
The method is applied to a gradient elution program, and can effectively avoid the influence on the baseline and the data integrity caused by wavelength switching and gradient elution in the process of matching the variable wavelength with the gradient elution program.
In summary, compared with the prior art, the invention has the beneficial effects that:
(1) the variable wavelength data acquisition and processing method provided by the invention eliminates the adverse effect of grating vibration on spectrum acquisition during wavelength switching, effectively ensures the data integrity in the variable wavelength process, and improves the baseline level.
(2) The variable wavelength data acquisition processing method can realize seamless connection of spectrograms of different wavelength bands, abandons a subsequent wavelength pre-acquisition part, and is sequentially connected with data of the previous wavelength, thereby avoiding distortion and base line mutation caused by real-time data acquisition and display, ensuring the base line consistency and data rationality of the data in each wavelength acquisition time period, and enabling the spectrogram data to be collected in a uniform base line and coordinate range for convenient processing and analysis.
(3) The variable wavelength data acquisition and processing method can meet the regional requirement of gradient elution time, can realize more stable noise and higher sensitivity by variable wavelength acquisition, and expands the combined application of variable wavelength and gradient elution.
(4) The invention adopts the wavelength-variable function of the ultraviolet detector to replace a DAD detector in certain application occasions, thereby saving the cost for multi-component experiments.
(5) The solution of the invention can effectively reduce the influence on the baseline during the variable wavelength switching, so that the baseline and the fixed wavelength during the multi-wavelength gradient elution are close.
Drawings
FIG. 1 is a schematic diagram of a method for acquiring and processing variable wavelength data according to the present invention.
FIG. 2 is a diagram illustrating the wavelength λ of step S2 in the method for acquiring and processing variable wavelength data according to the present inventionn+1Collected data and wavelength lambdanAnd the collected data are subjected to splicing processing.
FIG. 3 is a spectrum of the prior art without any baseline adjustment at variable wavelength.
FIG. 4 is a spectrogram obtained by stopping collecting the medium wavelength and continuing to collect the medium wavelength after the medium wavelength is stabilized in the prior art.
FIG. 5 is a diagram showing the effect of the variable wavelength data acquisition and processing method of the present invention on the application of the traditional Chinese medicine rhizoma corydalis Decumbentis.
FIG. 6 is a graph showing the effect of the variable wavelength stacking gradient elution procedure of the present invention on the use of a more abundant "polycyclic aromatic hydrocarbon".
FIG. 7 shows the gradient elution conditions of the variable wavelength stacking gradient elution procedure of the present invention when applied to a "polycyclic aromatic hydrocarbon" with a high content of components.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples. The objects, aspects and advantages of the present invention will become more apparent from the following description. It should be understood that the described embodiments are preferred embodiments of the invention, and not all embodiments.
Referring to fig. 1 and fig. 2, the method for acquiring and processing variable wavelength data of the ultraviolet detector of the liquid chromatograph includes the following steps:
s1, starting to pass through the fixed wavelength lambdanAs initial wavelength, data acquisition is carried out at a higher sampling frequency;
s2, in step S1, tnAfter x time length, the transmission mechanism controls the grating to start rotating and switches to lambdanAnd subsequent wavelength lambdan+1A dual-wavelength acquisition mode with a lower sampling frequency is carried out alternately, the acquisition time is x, and a background software algorithm carries out wavelength lambdan+1At tnThe baseline shift of the later collected data to λnThe baseline level of the collected data is consistent, so that lambda is enabledn+1At tnData spectrum and lambda in later timenAt tnSeamless connection is carried out on the data spectrogram collected before;
s3, go to t in step S2nWhen the grating stops rotating, the transmission mechanism controls the grating to switch to a fixed wavelength lambdan+1Continuously acquiring data at a higher sampling frequency until a preset second wavelength-variable time point is reached;
s4, let n be n +1, and repeat steps S1 to S3.
The higher sampling frequency in the fixed wavelength mode is 20Hz, the dual-wavelength sampling is adopted in the variable wavelength switching process, and the lower sampling frequency in the dual-wavelength mode is 2 Hz.
As shown in fig. 2, in step S2, λ is discardedn+1Pre-collecting the formed data spectrogram in x time length and comparing the data spectrogram with the wavelength lambdanThe data are connected, so that distortion or base line mutation caused by secondary processing of the data is avoided. By processing, the baselines collected for both wavelengths are translated to the same horizontal line,the step jump of the base line is avoided, and the analysis is conveniently carried out on a spectrogram in a centralized manner.
In steps S2 and S3, the transmission mechanism is a servomotor with a planetary reducer, which can meet the requirements of high precision and good rigidity.
The working principle of the invention is that the ultraviolet detector of the liquid chromatograph realizes the switching between the fixed wavelength acquisition mode and the dual wavelength acquisition mode through program control.
The invention adopts a dual-wavelength mode to process a wavelength changing process, the wavelength changing function is to collect data of different wavelengths and different time periods and present the data on a spectrogram according to the condition that the retention time of each component in the wavelength band near the maximum absorption wavelength has an obvious time region.
As can be seen from fig. 5 and 6, the specific application of the present invention is that no jump or mutation occurs in the baseline of the data spectrogram during the wavelength-varying switching, so as to ensure the baseline consistency and data reasonableness of the data in each wavelength acquisition time period.
The method is applied to a gradient elution program, and can effectively avoid the influence on the baseline and the data integrity caused by wavelength switching and gradient elution in the process of matching the variable wavelength with the gradient elution program.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the scope of the present invention, and it is obvious that any person skilled in the art can easily conceive of alternative or modified embodiments based on the above embodiments and these should be covered by the present invention.
Claims (4)
1. A method for acquiring and processing variable wavelength data of an ultraviolet detector of a liquid chromatograph is characterized by comprising the following steps:
s1, starting to pass through the fixed wavelength lambdanAs initial wavelength, data acquisition is carried out at a higher sampling frequency;
s2, in step S1, tnAfter x time length, the transmission mechanism controls the grating to start rotating and switches to lambdanAnd subsequent wavelength lambdan+1Dual wavelength at lower sampling frequency with alternationAn acquisition mode with acquisition duration of x and background software algorithm for converting wavelength lambdan+1At tnThe baseline shift of the later collected data to λnThe baseline level of the collected data is consistent, so that lambda is enabledn+1At tnData spectrum and lambda in later timenAt tnSeamless connection is carried out on the data spectrogram collected before;
s3, go to t in step S2nWhen the grating stops rotating, the transmission mechanism controls the grating to switch to a fixed wavelength lambdan+1Continuously acquiring data at a higher sampling frequency until a preset second wavelength-variable time point is reached;
s4, let n be n +1, and repeat steps S1 to S3.
2. The method for acquiring and processing the variable wavelength data of the ultraviolet detector of the liquid chromatograph according to claim 1, wherein:
the higher sampling frequency in the fixed wavelength mode is 20Hz, the dual-wavelength sampling is adopted in the variable wavelength switching process, and the lower sampling frequency in the dual-wavelength mode is 2 Hz.
3. The method for acquiring and processing the variable wavelength data of the ultraviolet detector of the liquid chromatograph according to claim 1, wherein:
in steps S2 and S3, the transmission mechanism is a servo motor with a planetary reducer.
4. A method for processing data acquisition of variable wavelength of a liquid chromatograph ultraviolet detector according to any of claims 1 to 3, characterized in that:
the variable wavelength data acquisition processing method can be applied to a gradient elution program.
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