CN107589190B - Large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combined equipment - Google Patents
Large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combined equipment Download PDFInfo
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Abstract
The invention relates to a large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combination device. The apparatus of the present invention comprises: three chromatographic pumps, a large-volume automatic sample injector, an automatic sample injector with a common sample injection amount, a high-pressure ten-way switching valve, two solid-phase extraction columns, a column incubator, a chromatographic column and a detector, wherein the simplest hardware equipment and flow path connection are used through the combined equipment, the direct sample injection of a large-volume liquid sample larger than 1mL is allowed, the two parallel-processed solid-phase extraction columns are equipped and are combined with high-performance liquid chromatography for carrying out overlapped sample injection, pretreatment and separation, the occupied time of a single sample in the whole batch processing sample is greatly reduced, in addition, the combined equipment can be conveniently switched into a common high-performance liquid chromatography system, and the free switching between an online system and a common separation system is realized.
Description
Technical Field
The invention belongs to the field of analytical chemistry sample pretreatment, and particularly relates to design, analysis and application of a large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combined device, which is suitable for sample introduction, online extraction, enrichment, impurity removal, desorption, separation and detection of trace organic matters in liquid samples in environment and food.
Background
For the analysis of trace compounds, there are generally two strategies: small volume sampling (e.g., biological samples), high sensitivity detector detection; or sampling in large volume (such as environmental sample), enriching and removing impurities, and then injecting sample for analysis.
Environmental samples, such as water trace pollutants, are analyzed and detected by increasing the sampling volume and the sample injection volume, then enriching and removing impurities by using Solid Phase Extraction (SPE), concentrating, re-dissolving the analytical solution and analyzing by sample injection. However, the offline solid-phase extraction has the defects of Large manual error, tedious operation, time and labor consumption and the like, so that an online 'Large Volume Injection (LVI) -solid-phase extraction-online combined method' needs to be developed to improve the reproducibility and automation of the method and shorten the analysis time.
High Performance Liquid Chromatography (HPLC) is the most common analytical means at present, the technology can accurately quantify a target substance, accurate semi-quantitation can be achieved by matching with detectors with strong qualitative abilities such as a diode array and a mass spectrum, and most compounds can be subjected to High-efficiency separation and quantitative quantitation by injecting HPLC after pretreatment. The hardware principle of the Ultra High Performance Liquid Chromatography system (Ultra High Performance Liquid Chromatography, UHPLC or Ultra Performance Liquid Chromatography, UPLC) is the same as that of a common HPLC, and the Ultra High Performance Liquid Chromatography system also conforms to the technical background of the present patent.
However, the on-line combination of solid phase extraction and high performance liquid chromatography has two limitations: firstly, the sample introduction volume is small, generally less than 1mL, and the sample is concentrated and then introduced in a manual off-line mode; secondly, the time for SPE sample loading and sample processing and the time for HPLC target separation are both long, resulting in prolonged whole analysis time. The existing on-line solid-phase extraction coupling system generally uses a six-way switching valve to connect a single solid-phase extraction column, and realizes the conversion of two states of sample loading extraction and desorption separation by the switching of the six-way switching valve; in addition, the sample injection speed of a common sample injector is about 1mL/min, if the sample injection volume is as high as several milliliters or even tens of milliliters, the sample injection time is difficult to ignore, and in addition, the solid phase extraction column also needs activation, impurity removal and balance time, the whole analysis time is very long, and the analysis flux is greatly reduced.
Disclosure of Invention
Based on this, the present invention aims to provide an online combined device, which uses the simplest hardware device and flow path connection, allows direct sample injection of large volume samples larger than 1mL, is equipped with two parallel processed solid phase extraction columns, is combined with high performance liquid chromatography, and performs overlapped sample injection, pretreatment and separation, thereby greatly reducing the occupied time of a single sample in the whole batch of processed samples, and the combined device can be conveniently switched to a common high performance liquid chromatography system, so as to realize the purpose of a multifunctional system.
The invention is realized by the following technical scheme: a large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combination device comprises a large-volume sample introduction-double solid phase extraction online combination device and a high performance liquid chromatography separation device;
the large-volume sample injection-double solid phase extraction online combination device comprises a first liquid chromatography pump for pushing a solvent required by solid phase extraction, a first automatic sample injector for loading a sample, a ten-way switching valve for switching a flow path, and a first solid phase extraction column and a second solid phase extraction column for enriching a target and removing impurities;
the high performance liquid chromatography separation device comprises a second chromatographic pump and a third chromatographic pump which are used for pushing and separating a mobile phase, a second automatic sample injector used for loading a sample and a chromatographic column used for chromatographic separation;
the ten-way switching valve is sequentially provided with ten interfaces according to a clockwise sequence: the first automatic sample injector is connected with a first interface, the two ends of the first solid-phase extraction column are respectively connected with a second interface and a ninth interface, the chromatographic column is connected with a third interface, the two ends of the second solid-phase extraction column are respectively connected with a fourth interface and a seventh interface, a sixth interface is connected with a waste liquid pipe, and the second automatic sample injector is connected with an eighth interface, a fifth interface and a tenth interface.
Compared with the prior art, the sample injection-double solid phase extraction device and the high performance liquid chromatography separation device are connected by adopting the ten-way switching valve as a connecting center, and the ten-way switching valve has only two states, so that only two flow paths are arranged in the system, and the operation is simple; the simultaneous switching of two solid phase extractions and two flow paths is realized through the state switching of the ten-way switching valve, and the overlapped sampling, pretreatment and separation are carried out, so that the overall analysis time is shortened.
Further, when the ten-way switching valve is in a state that the first interface is communicated with the second interface, the third interface is communicated with the fourth interface, the fifth interface is communicated with the sixth interface, the seventh interface is communicated with the eighth interface, and the ninth interface is communicated with the tenth interface, the first liquid chromatography pump, the first automatic sample injector and the first solid-phase extraction column are connected to form an extraction flow path, and the second chromatography pump, the third chromatography pump, the second automatic sample injector, the second solid-phase extraction column and the chromatography column are connected to form a desorption separation flow path.
When the state of the ten-way switching valve is that the second interface is communicated with the third interface, the fourth interface is communicated with the fifth interface, the sixth interface is communicated with the seventh interface, the eighth interface is communicated with the ninth interface, and the tenth interface is communicated with the first interface, the first liquid chromatography pump, the first automatic sample injector and the second solid-phase extraction column are connected into an extraction flow path, and the second chromatography pump, the third chromatography pump, the second automatic sample injector, the first solid-phase extraction column and the chromatography column are connected into a desorption separation flow path.
Therefore, the two parallel solid phase extraction columns are fully automatically switched through the state switching of the ten-way switching valve, the two solid phase extraction columns are in a cleaning, balancing, loading or desorption separation stage at any time, when one of the solid phase extraction columns is in the cleaning, balancing and loading stage, the other solid phase extraction column is in the desorption separation stage, and the analysis processes of the two solid phase extraction columns are overlapped and alternated.
Further, the flow directions of the extraction flow path and the desorption separation flow path in the solid phase extraction column are opposite. Therefore, the migration path of the target object in the solid phase extraction column is reduced, and the desorption efficiency and the chromatographic separation performance are improved.
Further, online combination equipment still includes column incubator and is used for the detector of detected signal, first solid phase extraction post, second solid phase extraction post, ten logical diverter valve and chromatographic column are located in the column incubator, the detector is connected in the export of chromatographic column.
Furthermore, a low-pressure gradient proportional valve is arranged in the first chromatographic pump, and at least three solvents with different components are selectively pushed. The elution capacity of the sample loading or equilibrium solvent is low, the sample loading sample can be pushed to flow through the solid phase extraction column, but the target substance enriched in the solid phase extraction column cannot be eluted; the cleaning solution with moderate elution capacity can elute a part of impurities but not elute the target; the cleaning solution with high elution capacity is responsible for cleaning the whole extraction pipeline and eluting the target substance and the matrix remained on the solid phase extraction column, thereby not causing the cross contamination of the next analysis.
Further, the sample amount of the first auto-sampler is greater than 1 mL.
Further, the first autosampler is provided with a dosing ring switchable out of the flow path. After the extraction is completed, the solid phase extraction column needs to be cleaned and equilibrated, at which time the quantification loop of the first autosampler with a large volume sample loading is switched out of the flow path to reduce the time to clean and equilibrate through the quantification loop.
The invention also provides a high performance liquid chromatography system which comprises the second chromatographic pump, the third chromatographic pump, the second automatic sample injector, the chromatographic column, the ten-way switching valve and the two-way connector; the ten-way switching valve is sequentially provided with ten interfaces according to a clockwise sequence: the second autosampler accesses the eighth interface, the second interface and the ninth interface are accessed respectively at two ends of the double-way connector, and the chromatographic column accesses the third interface.
The invention also provides a high performance liquid chromatography system which comprises the second chromatographic pump, the third chromatographic pump, the second automatic sample injector, the chromatographic column, the ten-way switching valve and the two-way connector; the ten-way switching valve is sequentially provided with ten interfaces according to a clockwise sequence: the second autosampler is connected with the eighth interface, the fourth interface and the seventh interface are connected with the two ends of the double-way connector respectively, and the chromatographic column is connected with the third interface.
Compared with the prior art, when one of the first solid-phase extraction column and the second solid-phase extraction column is replaced by the double-way joint, the connected flow path is changed into a common high-performance liquid chromatography system, so that simple switching between an online analysis system and the common high-performance liquid chromatography system is realized, and multifunctional application of the system is met.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic view of an on-line coupling apparatus according to example 1;
FIG. 2 is a schematic view showing the flow passage communication in the state of "0" in example 1;
FIG. 3 is a schematic view showing the flow passage communication in the state of "1" in example 1;
FIG. 4 is a schematic view showing the flow passage communication in example 2;
FIG. 5 is a chromatogram of an acidic loading assay of 80 drugs and personal care products from experiment 1;
FIG. 6 is a chromatogram of an alkaline loading assay of 80 drugs and personal care products from experiment 1;
FIG. 7 is a chromatogram of the analysis of 7 pesticides in experiment 2;
FIG. 8 is a chromatogram of a common HPLC profile analysis of 25 drugs and personal care products in experiment 3.
Detailed Description
In order to further illustrate the technical means and technical effects of the present invention adopted to achieve the predetermined objects, the following description, in combination with the embodiments and the accompanying drawings, will explain the structure, characteristics and specific embodiments of the large-volume sample injection-dual solid phase extraction-high performance liquid chromatography online combination device provided by the present invention, and the detailed description will be given below.
Example 1
Please refer to fig. 1, which is a schematic diagram of the large-volume sample injection-dual solid phase extraction-high performance liquid chromatography online combination apparatus of the present embodiment, which includes a large-volume sample injection-dual solid phase extraction online combination apparatus and a high performance liquid chromatography separation apparatus.
The large-volume sample introduction-double solid phase extraction online combination device comprises a first liquid chromatography pump 1 used for pushing different solvents required by solid phase extraction, a first automatic sample injector 4 used for loading samples and having large-volume sample introduction amount, a high-pressure ten-way switching valve 7 used for switching flow paths, and a first solid phase extraction column 8 and a second solid phase extraction column 9 used for enriching targets and removing impurities.
Specifically, the first automatic sampler 4 has a sample volume of more than 1mL, and a quantitative loop that can be switched to leave the flow path is provided therein, and after the extraction is completed, the extraction column needs to be cleaned and equilibrated, and at this time, the quantitative loop of the first automatic sampler having a large volume sample volume is switched to leave the flow path, so as to reduce the time for cleaning and equilibrating the quantitative loop.
The high performance liquid chromatography separation apparatus comprises a second 2 and a third 3 chromatographic pump for pushing a separation mobile phase, a second autosampler 5 for loading a common volume of sample, a chromatographic column 10 for chromatographic separation and a detector 11 for detecting a signal.
The large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combination equipment also comprises a column temperature box 6 which is used for loading a first solid phase extraction column 8, a second solid phase extraction column 9, a high-pressure cross-pass switching valve 7 and a chromatographic column 10 and has a temperature control function.
The high-pressure cross-connection switching valve 7 is used as a connection center to connect the large-volume sample introduction-double solid phase extraction online combination device and the high-performance liquid chromatography separation device, and is sequentially provided with a first interface a, a second interface b, a third interface c, a fourth interface d, a fifth interface e, a sixth interface f, a seventh interface g, an eighth interface h, a ninth interface i and a tenth interface j in a clockwise sequence. The first automatic sample injector 4 is connected with a first interface a, two ends of the first solid-phase extraction column 8 are respectively connected with a second interface b and a ninth interface i, the chromatographic column 10 is connected with a third interface c, two ends of the second solid-phase extraction column 9 are respectively connected with a fourth interface d and a seventh interface g, the sixth interface f is connected with a waste liquid pipe, the second automatic sample injector 5 is connected with an eighth interface h, and the fifth interface e and the tenth interface j are connected with each other by a short pipe.
In the present embodiment, the high-pressure ten-way switching valve 7 includes two communication states, i.e., the state "0" and the state "1", respectively.
When the high-pressure ten-way switching valve 7 is in the state of "0" (as shown in fig. 2), the first port a communicates with the second port b, the third port c communicates with the fourth port d, the fifth port e communicates with the sixth port f, the seventh port g communicates with the eighth port h, the ninth port i communicates with the tenth port j, the first liquid chromatography pump 1, the first autosampler 4, and the first solid-phase extraction column 8 are connected to form one flow path, and the second chromatography pump 2, the third chromatography pump 3, the second autosampler 5, the second solid-phase extraction column 9, and the chromatography column 10 are connected to form another flow path. Specifically, the solvent flowing out of the first liquid chromatography pump 1 sequentially flows through the first autosampler 4, enters the first interface a of the high-pressure ten-way switching valve 7, flows out of the second interface b, is communicated with the first solid-phase extraction column 8, enters the ninth interface i, flows out of the tenth interface j, enters the fifth interface e through a communication pipeline, and flows out of the waste liquid from the sixth interface f; on the other hand, the mobile phase pushed by the second chromatographic pump 2 and the third chromatographic pump 3 flows through the second autosampler 5, enters the eighth interface h of the high-pressure ten-way switching valve 7, flows out of the seventh interface g, enters the second solid-phase extraction column 9, enters the fourth interface d, flows out of the third interface c, enters the chromatographic column 10, and finally flows into the detector 11 to complete analysis.
When the high-pressure ten-way switching valve 7 is in the state of "1" (as shown in fig. 3), when the second port b is communicated with the third port c, the fourth port d is communicated with the fifth port e, the sixth port f is communicated with the seventh port g, the eighth port h is communicated with the ninth port i, and the tenth port j is communicated with the first port a, the first liquid chromatography pump 1, the first autosampler 4, and the second solid-phase extraction column 9 are connected to form one flow path, and the second chromatography pump 2, the third chromatography pump 3, the second autosampler 5, the first solid-phase extraction column 8, and the chromatography column 10 are connected to form another flow path. Specifically, the solvent flowing out of the first liquid chromatography pump 1 sequentially flows through the first autosampler 4, enters the first interface a of the high-pressure ten-way switching valve 7, flows out of the tenth interface j, enters the fifth interface e through the communication pipeline, flows out of the fourth interface d, enters the second solid-phase extraction column 9, enters the seventh interface g, and flows out of the waste liquid through the sixth interface f; on the other hand, the mobile phase pushed by the second chromatographic pump 2 and the third chromatographic pump 3 flows through the second autosampler 5, enters the eighth interface h of the high-pressure ten-way switching valve 7, flows out of the ninth interface i, enters the first solid-phase extraction column 8, flows into the second interface b, flows out of the third interface c, enters the chromatographic column 10, and finally flows into the detector 11 to complete analysis.
The working principle of the embodiment is as follows: when the high-pressure ten-way switching valve 7 is in a state of '0', the first liquid chromatography pump 1 pushes the target solution to the first solid-phase extraction column 8, after the extraction process is finished, the high-pressure ten-way switching valve 7 is switched to a state of '1', the first solid-phase extraction column 8 is connected with the chromatography column 10 in series, the desorption separation process is finished by utilizing the mobile phase pushed by the second chromatography pump 2 and the third chromatography pump 3, at the moment, the second solid-phase extraction column 9 is cleaned, balanced and loaded in the desorption separation time period, then the high-pressure ten-way switching valve 7 is switched again, and desorption and separation are carried out again. In the first half of the analysis process, the cleaning, balancing and sample loading of the first solid-phase extraction column 8 and the desorption and separation of the second solid-phase extraction column 9 are synchronously carried out, the high-pressure ten-way switching valve 7 enters the second half of the analysis process after switching, and the desorption and separation of the first solid-phase extraction column 8 and the cleaning, balancing and sample loading of the second solid-phase extraction column 9 are synchronously carried out in the first half of the analysis process, so that the overlapping analysis is realized.
Example 2
Please refer to fig. 4, which is a schematic view of the flow path communication in the present embodiment. The structural design and connection mode of the present embodiment are basically the same as those of embodiment 1, except that one of the first solid phase extraction column 8 and the second solid phase extraction column 9 of embodiment 1 is replaced by a zero dead volume double pass 12, so that the conventional high performance liquid chromatography separation system is switched.
In this embodiment, the first solid-phase extraction column 8 in embodiment 1 is replaced with a zero dead volume double-pass 12, so that the switched common high performance liquid chromatography separation system includes a second chromatography pump 2, a third chromatography pump 3, a second autosampler 5, a chromatography column 10, a high-pressure ten-pass switching valve 7, and the zero dead volume double-pass 12. The second autosampler 5 is connected to the eighth interface h, the two ends of the zero dead volume bi-pass 12 are connected to the second interface b and the ninth interface i respectively, and the chromatographic column is connected to the third interface 7. Specifically, the mobile phase pushed by the second chromatographic pump 2 and the third chromatographic pump 3 flows through the second autosampler 5, enters the eighth interface h of the high-pressure ten-way switching valve 7, flows out of the ninth interface i, enters the zero dead volume two-way 12, flows into the second interface b, flows out of the third interface c, enters the chromatographic column 10, and finally flows into the detector 11 to complete analysis.
Compared with the prior art, the invention uses the simplest hardware equipment and flow path connection, allows the direct sample introduction of large-volume samples larger than 1mL, and does not need to concentrate the samples in a manual off-line mode and then carry out sample introduction; meanwhile, two solid phase extraction columns which are processed in parallel are equipped and are used together with the high performance liquid chromatography for overlapped sample introduction, pretreatment and separation, so that the overall analysis time is shortened, and the occupation time of a single sample in the whole batch of processed samples is greatly reduced; moreover, the combined equipment can be conveniently switched to a common high performance liquid chromatography system, and the aim of a multifunctional system is fulfilled.
The invention is further illustrated by several experiments below.
This experiment used the online combination device described in example 1 for online analysis of 80 drugs and personal care products in water. In the present experiment, the first solid-phase extraction column 8 and the second solid-phase extraction column 9 are both high-pressure resistant reverse-phase extraction columns, and the chromatographic column 10 is an ultrahigh-pressure reverse-phase chromatographic column. The target substances are 80 medicines and personal care products, and are divided into an acidic sampling mode and a basic sampling mode according to the retention characteristics of the target substances, and the specific experimental conditions are as follows:
liquid chromatography conditions (solid phase extraction conditions)
The first liquid chromatography pump 1 is equipped with a low-pressure gradient proportional valve, and comprises three optional solutions, wherein the flow path A is 0.05% formic acid aqueous solution, the flow path B is acetonitrile/methanol/acetone (1: 1V/V/V), and the flow path C is 0.1% ammonia aqueous solution. The flow rate of the first liquid chromatography pump 1 was 4 mL/min.
When the sample loading condition is acidic, the equilibrium solution is an A flow path solution, the high-pressure cross-channel switching valve 7 is in a state of No. 0 during sample loading, the first liquid chromatography pump 1 pushes the target solution to the first solid phase extraction column 8 at a flow rate of 4mL/min, the extraction process is completed after 2.6min, the high-pressure cross-channel switching valve 7 is switched to a state of No. 1, the first solid phase extraction column 8 and the chromatography column 10 are connected in series, the first liquid chromatography pump 1 is switched to a B flow path, the second solid phase extraction column 9 is cleaned at a flow rate of 4mL/min, the first liquid chromatography pump 1 is switched to an A flow path after 1min, and the second solid phase extraction column 9 is balanced at a flow rate of 4 mL/min. On the other hand, the high-pressure ten-way switching valve 7 is set to the state of "1" at the time of loading, the first liquid chromatography pump 1 pushes the target solution to the second solid-phase extraction column 9 at the flow rate of 4mL/min, the extraction process is completed after 2.6min, the high-pressure ten-way switching valve 7 is set to the state of "0", the second solid-phase extraction column 9 and the chromatography column 10 are connected in series, the first liquid chromatography pump 1 is switched to the flow path B, the first solid-phase extraction column 8 is washed at the flow rate of 4mL/min, and the first liquid chromatography pump 1 is switched to the flow path a after 1min, and the first solid-phase extraction column 8 is balanced at the flow rate of 4 mL/min. If the loading conditions are alkaline, the cleaning solution is a C flow path solution, and the loading process is the same as described above.
Liquid chromatography conditions (separation conditions)
A chromatographic column: inertsustatin AQ-C18(2.10mm I.D.. times.50 mm L.,1.9 μm)
Flow rate: 0.4mL/min
Column temperature: 40 deg.C
Sample introduction amount: 5mL
After the high-pressure ten-way switching valve 7 is switched, the mobile phases pushed by the second chromatographic pump 2 and the third chromatographic pump 3 start desorption and separation processes, the initial proportion of the organic phase is 5%, the linear gradient change starts after the maintenance of 0.2min, the organic phase ratio is 80% after 8min, the organic phase ratio is 100% after 0.1min and is maintained for 2.1min, and the organic phase ratio is changed to 5% within 0.1min and is maintained until the analysis is finished.
Chromatograms obtained by acid sample loading and alkaline sample loading in the experiment are respectively shown in fig. 5 and fig. 6, peak shapes of all compounds are symmetrical and sharp, the recovery rate is between 60-120%, the overall analysis time is shortened by half compared with that of a single SPE online system, and the flux is doubled.
The on-line combination apparatus described in example 1 was used for the on-line analysis of 7 pesticides in water for this experiment. In the present experiment, the first solid-phase extraction column 8 and the second solid-phase extraction column 9 are both high-pressure resistant reverse-phase extraction columns, and the chromatographic column 10 is an ultrahigh-pressure reverse-phase chromatographic column. The target is 7 kinds of pesticides, and the specific experimental conditions are as follows:
liquid chromatography conditions (solid phase extraction conditions)
Cleaning solution: a-20mM ammonium acetate in water; b-acetonitrile
Flow rate: 2mL/min
When the sample is loaded, the high-pressure cross-flow switching valve 7 is in a state of No. 0, the first liquid chromatography pump 1 pushes the target solution to the first solid-phase extraction column 8 at a flow rate of 2mL/min, the extraction process is completed after 4min, meanwhile, the high-pressure cross-flow switching valve 7 is switched to be in a state of No. 1, the first solid-phase extraction column 8 is connected with the chromatography column 10 in series, the first liquid chromatography pump 1 is switched to be a flow path B, the second solid-phase extraction column 9 is cleaned at a flow rate of 2mL/min, and after 3min, the first liquid chromatography pump 1 is switched to be a flow path A, and the second solid-phase extraction column 9 is balanced at a flow rate of 2 mL/min. On the other hand, when the sample is loaded, the high-pressure ten-way switching valve 7 is in the state of "1", the first liquid chromatography pump 1 pushes the target solution to the second solid-phase extraction column 9 at the flow rate of 2mL/min, and after 4min, the extraction process is completed, and at the same time, the high-pressure ten-way switching valve 7 is switched to the state of "0", the second solid-phase extraction column 9 and the chromatography column 10 are connected in series, the first liquid chromatography pump 1 is switched to the flow path B, the first solid-phase extraction column 8 is cleaned at the flow rate of 2mL/min, and after 3min, the first liquid chromatography pump 1 is switched to the flow path a, and the first solid-phase extraction column 8 is balanced at the flow rate of 2.
Liquid chromatography conditions (separation conditions)
A chromatographic column: inertsustatin AQ-C18(2.10mm I.D.. times.50 mm L.,3 μm)
Mobile phase: phase A-20mM ammonium acetate aqueous solution; phase B-acetonitrile
Flow rate: 0.4mL/min
Column temperature: 40 deg.C
Sample introduction amount: 2mL
After the high-pressure cross-pass switching valve 7 is switched, the mobile phases pushed by the second chromatographic pump 2 and the third chromatographic pump 3 start desorption and separation processes, the initial proportion of the organic phase is 10%, the linear gradient change starts after the organic phase is kept for 0.5min, the proportion of the organic phase is 60% after 1min, the proportion of the organic phase is 95% after 3.6min and is kept for 0.5min, and the proportion of the organic phase is changed into 5% within 0.1min and is kept unchanged until the analysis is finished.
The chromatogram obtained in the experiment is shown in FIG. 7, the peak patterns of all compounds are symmetrical and sharp, the recovery rate is between 60 and 120 percent, the overall analysis time is shortened by half compared with that of a single SPE online system, and the flux is doubled.
This experiment used the common high performance liquid chromatography functionality of the on-line combination apparatus described in example 2 for on-line analysis of 21 drugs and personal care products. In this experiment, the first solid phase extraction column 8 was replaced with a zero dead volume double pass 12 and the column 10 was an ultra high pressure reverse phase column using the flow path shown in fig. 4. The target is 21 medicines, 10 mu L of sample is directly injected after a sample is concentrated, and the specific experimental conditions are as follows:
liquid phase conditions
A chromatographic column: shim-pack XR-ODS III 2.0mm I.D. times 75mm L, 1.6 μm
Flow rate: 0.3mL/min
Column temperature: 40 deg.C
Sample introduction amount: 10 μ L
Mobile phase A: 0.1% aqueous formic acid solution
Mobile phase B: methanol
After the sample injection is finished, the mobile phases pushed by the second chromatographic pump 2 and the third chromatographic pump 3 start desorption and separation processes, the initial proportion of the organic phase is 5%, the proportion of the organic phase is 25% after 0.5min, the proportion of the organic phase is 35% after 4.5min, the proportion of the organic phase is 95% after 1min and is kept for 3min, and the proportion of the organic phase is changed into 5% within 0.1min and is kept unchanged until the analysis is finished.
The chromatogram obtained in the experiment is shown in fig. 8, the obtained chromatographic separation result and method index have no significant difference from the common ultra-high performance liquid chromatography system, and the system can be simply switched to the common ultra-high performance liquid chromatography system.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (8)
1. A large-volume sample introduction-double solid phase extraction-high performance liquid chromatography online combined device is characterized in that: comprises a large-volume sample introduction-double solid phase extraction online coupling device and a high performance liquid chromatography separation device;
the large-volume sample injection-double solid phase extraction online combination device comprises a first liquid chromatography pump for pushing a solvent required by solid phase extraction, a first automatic sample injector for loading a sample, a ten-way switching valve for switching a flow path, and a first solid phase extraction column and a second solid phase extraction column for enriching a target and removing impurities;
the high performance liquid chromatography separation device comprises a second chromatographic pump and a third chromatographic pump which are used for pushing and separating a mobile phase, a second automatic sample injector used for loading a sample and a chromatographic column used for chromatographic separation;
the ten-way switching valve is sequentially provided with ten interfaces according to a clockwise sequence: the first automatic sample injector is connected with a first interface, the two ends of the first solid-phase extraction column are respectively connected with a second interface and a ninth interface, the chromatographic column is connected with a third interface, the two ends of the second solid-phase extraction column are respectively connected with a fourth interface and a seventh interface, the sixth interface is connected with a waste liquid pipe, the second automatic sample injector is connected with an eighth interface, and the fifth interface is connected with a tenth interface;
the high performance liquid chromatography separation device further comprises a double-way connector, wherein the double-way connector can replace the first solid-phase extraction column, and two ends of the double-way connector are respectively connected to the second interface and the ninth interface; or the second solid-phase extraction column can be replaced by the double-way joint, and two ends of the double-way joint are respectively connected to the fourth interface and the seventh interface.
2. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 1, characterized in that: when the ten-way switching valve is in a state that the first interface is communicated with the second interface, the third interface is communicated with the fourth interface, the fifth interface is communicated with the sixth interface, the seventh interface is communicated with the eighth interface, and the ninth interface is communicated with the tenth interface, the first liquid chromatography pump, the first automatic sample injector and the first solid-phase extraction column are connected into an extraction flow path, and the second chromatography pump, the third chromatography pump, the second automatic sample injector, the second solid-phase extraction column and the chromatography column are connected into a desorption separation flow path.
3. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 2, characterized in that: when the state of the ten-way switching valve is that the second interface is communicated with the third interface, the fourth interface is communicated with the fifth interface, the sixth interface is communicated with the seventh interface, the eighth interface is communicated with the ninth interface, and the tenth interface is communicated with the first interface, the first liquid chromatography pump, the first automatic sample injector and the second solid-phase extraction column are connected into an extraction flow path, and the second chromatography pump, the third chromatography pump, the second automatic sample injector, the first solid-phase extraction column and the chromatography column are connected into a desorption separation flow path.
4. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 3, characterized in that: the extraction flow path and the desorption separation flow path have opposite flow directions in the solid phase extraction column.
5. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 1, characterized in that: the online combination equipment further comprises a column temperature box and a detector for detecting signals, the first solid-phase extraction column, the second solid-phase extraction column, the ten-way switching valve and the chromatographic column are located in the column temperature box, and the detector is connected to an outlet of the chromatographic column.
6. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 1, characterized in that: the first liquid chromatography pump is internally provided with a low-pressure gradient proportional valve for selectively pushing at least three solvents with different components.
7. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 1, characterized in that: the sample amount of the first automatic sample injector is more than 1 mL.
8. The large-volume sample introduction-dual solid phase extraction-high performance liquid chromatography online combined device according to claim 1, characterized in that: the first autosampler is provided with a dosing ring switchable out of the flow path.
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| CN109900840A (en) * | 2019-04-16 | 2019-06-18 | 李宜珊 | Multidimensional liquid chromatographic separation system based on double two position four-way valves |
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| US6989129B2 (en) * | 2001-04-05 | 2006-01-24 | The President And Fellows Of Harvard College | Automated capillary liquid chromatography small volume analysis system |
| JP3816883B2 (en) * | 2003-03-06 | 2006-08-30 | 株式会社日立ハイテクノロジーズ | Liquid chromatograph mass spectrometer |
| JP4166165B2 (en) * | 2004-01-30 | 2008-10-15 | 株式会社島津製作所 | Liquid chromatograph |
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