A solid sample online pretreatment device and its application

Abstract

The present invention discloses an online pretreatment device for solid samples, comprising a high-performance liquid chromatography (HPLC), a column oven device, a condensing device, an enrichment device and a computer, wherein the outlet of the HPLC is connected to the inlet of the column oven device, the outlet of the column oven device is connected to the inlet of the condensing device, and the outlet of the condensing device is connected to the inlet of the enrichment device; the HPLC device, the column oven device and the enrichment device are respectively connected to the computer; the present invention also discloses an application of the device in solid sample detection, wherein the present invention combines pressurized hot water extraction with a dispersed liquid-liquid microextraction system to detect trace target compounds in solid samples; and the present invention can realize online pretreatment and detection of solid samples, and the method has good reproducibility.

Description

Technical Field

[0001] The present invention specifically relates to an online solid sample pre-treatment device and application thereof. Background Art

[0002] Pressurized hot water extraction (PHE) is an environmentally friendly extraction method suitable for solid samples. Under high temperature and pressure, water has low viscosity, dielectric constant, surface tension, and polarity, resulting in a high affinity and solubility for hydrophobic substances. Furthermore, the solubility of water in a subcritical state for hydrophobic compounds varies with temperature. Therefore, PHE is suitable for extracting a variety of compounds from different matrices. However, due to the dilution effect, PHE tends to dilute the compounds, resulting in low concentrations that are not conducive to subsequent testing.

[0003] Currently, most solid sample pretreatment is done manually. This offline manual pretreatment is not only time-consuming and labor-intensive, but also reduces the reproducibility of the detection method. Therefore, the development of online pretreatment devices and detection methods for solid samples is a current research hotspot. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an online pretreatment device for solid samples and applications thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A solid sample online pretreatment device comprises a high performance liquid chromatography (HPLC), a column oven device, a condensing device, an enrichment device, and a computer, wherein the HPLC outlet is connected to the inlet of the column oven device, the outlet of the column oven device is connected to the inlet of the condensing device, and the outlet of the condensing device is connected to the inlet of the enrichment device; the HPLC device, the column oven device, and the enrichment device are respectively connected to the computer;

[0007] The high performance liquid chromatography device includes an eluent bottle, a liquid chromatography pump, a six-way valve, a quantitative loop, a ten-way valve, a chromatographic column, and a UV detector. The eluent bottle is connected to the liquid chromatography pump, the quantitative loop is installed on the six-way valve, the liquid chromatography pump and the ten-way valve are respectively connected to the six-way valve, and the chromatographic column and the UV detector are both installed on the ten-way valve; the six-way valve, the ten-way valve, and the liquid chromatography pump are all connected to a computer;

[0008] The column oven device includes a column oven, a preheating tube and an extraction tube, the outlet of the ten-way valve is connected to the inlet of the preheating tube, the outlet of the preheating tube is connected to the inlet of the extraction tube, and the extraction tube and the preheating tube are installed in the column oven;

[0009] The condensation device includes a condenser, a beaker and a pressure reducing valve, the condenser is placed in the beaker containing the condensate, the extraction tube outlet is connected to the condenser inlet, and the condenser outlet is connected to the pressure reducing valve inlet;

[0010] The enrichment device includes an enrichment solvent bottle, a binary pump, a tee and an enrichment bottle, the binary pump and the pressure reducing valve outlet are respectively connected to the inlet of the tee, the outlet of the tee is connected to the inlet of the enrichment bottle, and the outlet of the enrichment bottle is connected to the six-way valve.

[0011] Furthermore, the enrichment bottle is a bottle made of glass.

[0012] Furthermore, the inlet and outlet of the enrichment bottle contain latex cylindrical stoppers.

[0013] Furthermore, the connecting pipes of the device are all stainless steel capillaries.

[0014] An application of an online solid sample pretreatment device in solid sample detection, wherein the pretreatment device is the pretreatment device described above.

[0015] The working process of the pre-treatment device of the present invention is as follows:

[0016] (1) Sample loading: Weigh an appropriate amount of solid powder sample and mix it with diatomaceous earth or quartz sand, place it evenly in the extraction tube, and contain glass wool at both ends of the extraction tube to prevent clogging of the extraction tube and loss of sample; then, set the temperature of the column oven, and under the heating action of the column oven, the extraction tube and the preheating column are heated; other components of the device do not start working, at this time the six-way valve is in the sample testing state, that is, the first interface of the six-way valve is connected to the second interface, the third interface is connected to the fourth interface, and the fifth interface is connected to the sixth interface; the ten-way valve is in the sample testing state, that is, the first interface of the ten-way valve is connected to the tenth interface, the sixth interface is connected to the seventh interface, the second interface is connected to the third interface, the fourth interface is connected to the fifth interface, and the eighth interface is connected to the ninth interface;

[0017] (2) Extraction and enrichment: At this time, the six-way valve is in the sampling state, that is, the first interface of the six-way valve is connected to the second interface, the third interface is connected to the fourth interface, and the fifth interface is connected to the sixth interface; the ten-way valve is in the injection state, that is, the first interface of the ten-way valve is connected to the second interface, the third interface is connected to the fourth interface, the fifth interface is connected to the sixth interface, the seventh interface is connected to the eighth interface, and the ninth interface is connected to the tenth interface. Add a little organic matter to water as the extract, and place the extract in the eluent bottle of the liquid chromatography pump; select an organic solvent with a density lower than that of water as the enrichment solvent, and place it in the enrichment solvent bottle. The extract and enrichment liquid should be selected according to the properties of the target compound, and the corresponding reagents are placed in the enrichment bottle to facilitate subsequent liquid-liquid microextraction; under the action of the liquid chromatography pump, the extract comes out of the eluent bottle, passes through the six-way valve and the ten-way valve, and enters the preheating pipeline. The extract is preheated in the preheating pipeline, and then the extract comes out of the preheating pipeline and infiltrates the extraction tube at a speed of 1.0mL / min. Under high temperature and high pressure, the extract is a subcritical solution, which extracts the solid sample in the extraction tube, so that the target compound is extracted into the extract; then, the extract c (The extract here c The extract and the target compound) enter the tee through the condenser; the enriched solvent enters the tee at a rate of 0.07 mL / min under the action of the binary pump; the extract c The enrichment solvent is premixed in the tee and transferred to the enrichment bottle together; liquid-liquid microextraction occurs in the enrichment bottle, at this time the enrichment solvent is extracted and the enriched extract is c The target compound in the enrichment bottle enters the enrichment solvent, and after extraction and enrichment, the concentration of the target compound reaches the monitoring requirement of the detector; finally, under the action of gravity, the liquid in the enrichment bottle is divided into two layers. Due to density reasons, the mixture containing the enrichment solvent and the target compound is located in the upper layer;

[0018] (3) Injection: At this time, the six-way valve is in the injection state, that is, the first interface of the six-way valve is connected to the sixth interface, the second interface is connected to the third interface, and the fourth interface is connected to the fifth interface; the ten-way valve is in the injection state, that is, the first interface of the ten-way valve is connected to the second interface, the third interface is connected to the fourth interface, the fifth interface is connected to the sixth interface, the seventh interface is connected to the eighth interface, and the ninth interface is connected to the tenth interface; the liquid chromatography pump and the binary pump are in the running state, and under the action of pressure, the upper layer solution in the enrichment bottle (containing a mixture of enrichment solvent and target compound) is transported to the quantitative loop;

[0019] (4) Sample analysis (sampling measurement): At this time, the six-way valve is in the sampling state, that is, the first interface of the six-way valve is connected to the second interface, the third interface is connected to the fourth interface, and the fifth interface is connected to the sixth interface; the ten-way valve is in the sampling state, that is, the first interface of the ten-way valve is connected to the tenth interface, the sixth interface is connected to the seventh interface, the second interface is connected to the third interface, the fourth interface is connected to the fifth interface, and the eighth interface is connected to the ninth interface. Under the action of the liquid chromatography pump, the mobile phase enters the quantitative loop, the ultraviolet detector is started, and the mixture of the enrichment solvent and the target compound in the quantitative loop is transferred to the high performance liquid chromatography system for separation and analysis.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention establishes an online solid sample pretreatment device coupled with high performance liquid chromatography based on pressurized hot water extraction and dispersive liquid-liquid microextraction. Dispersive liquid-liquid microextraction is a simple and rapid enrichment method for concentrating organic analytes in aqueous samples. Dispersive liquid-liquid microextraction can increase the contact area between the extraction solvent and the aqueous solution, accelerating the extraction rate. Compared with other classical methods, dispersive liquid-liquid microextraction has lower organic matter consumption and higher enrichment factors. The combination of pressurized hot water extraction and dispersive liquid-liquid microextraction systems enables the detection of trace target compounds in solid samples.

[0022] (2) The solid sample pretreatment device of the present invention does not require offline manual operation and can realize online pretreatment and detection of solid samples, and the method has good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a structural schematic diagram of the pre-treatment device of the present invention.

[0024] Figure 2 Schematic diagram of the pretreatment device of the present invention in the sample loading step (arrows represent the direction of liquid flow).

[0025] Figure 3 Schematic diagram of the pretreatment device of the present invention in the extraction and enrichment steps (arrows represent the direction of liquid flow).

[0026] Figure 4 Schematic diagram of the pretreatment device of the present invention in the injection step (arrows represent the direction of liquid flow).

[0027] Figure 5 Schematic diagram of the pre-treatment device of the present invention in the sample testing step (arrows represent the direction of liquid flow). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings. It should be noted that the specific embodiments are only detailed descriptions of the present invention and should not be regarded as limitations of the present invention. The reagents and components used in the following examples can all be purchased from commercial sources.

[0029] like Figure 1 As shown, a solid sample online pretreatment device includes a high-performance liquid chromatography device 1, a column oven device 2, a condensing device 3, an enrichment device 4 and a computer 5. The outlet of the high-performance liquid chromatography device 1 is connected to the inlet of the column oven device 2, the outlet of the column oven device 2 is connected to the inlet of the condensing device 3, and the outlet of the condensing device 3 is connected to the inlet of the enrichment device 4. All of the above connecting pipes are stainless steel capillaries; the high-performance liquid chromatography device 1, the column oven device 2, and the enrichment device 4 are respectively connected to the computer 5, and the computer can control the high-performance liquid chromatography device 1, the column oven device 2, and the enrichment device 4. The computer used is an existing conventional computer, and the software used is Chromeleon, which is matched with the high-performance liquid chromatography device.

[0030] The high performance liquid chromatography device 1 is a Thermo Fisher Ultimate 3000 high performance liquid chromatograph, such as Figure 1 As shown, it includes an eluent bottle 11, a liquid chromatography pump 12, a six-way valve 13, a quantitative loop 14, a ten-way valve 15, an ultraviolet detector (UV) 16 and a chromatographic column 17. In some preferred embodiments, the eluent bottle includes multiple eluent bottles, which can be used to store mobile phase or extraction liquid, etc. Each eluent bottle 11 can be connected to the liquid chromatography pump 12 through a plastic connecting tube. The six-way valve 13 contains six interfaces, such as Figure 1-5 As shown, they are respectively the first interface A1, the second interface A2, the third interface A3, the fourth interface A4, the fifth interface A5, and the sixth interface A6; the liquid chromatography pump 12 is connected to the first interface A1 of the six-way valve 13, the quantitative loop 14 is a spiral tubular hollow stainless steel tube, and when in use, the quantitative loop 14 is connected to the second interface A2 and the fifth interface A5 of the six-way valve 13, the fourth interface A4 of the six-way valve 13 is connected to the waste liquid tank, and the ten-way valve 15 has ten interfaces, as shown Figure 1-5As shown, they are respectively the first interface B1, the second interface B2, the third interface B3, the fourth interface B4, the fifth interface B5, the sixth interface B6, the seventh interface B7, the eighth interface B8, the ninth interface B9, and the tenth interface B10; the first interface B1 of the ten-way valve 15 is connected to the sixth interface A6 of the six-way valve 13, the second interface B2 of the ten-way valve 15 is connected to the eighth interface B8, the chromatographic column 17 is connected to the sixth interface B6 and the tenth interface B10 of the ten-way valve 15, and the ultraviolet detector 16 is connected to the third interface B3 and the seventh interface B7 of the ten-way valve 15; the ninth interface B9 of the ten-way valve 15 is connected to the waste liquid tank, and the six-way valve 13, the ten-way valve 15 and the liquid chromatography pump 12 are all connected to the computer 5.

[0031] like Figure 1 As shown, the column temperature box device 2 includes a preheating tube 21, a column temperature box 22 and an extraction tube 23. The fourth interface B4 of the ten-way valve 15 is connected to the inlet of the preheating tube 21, and the outlet of the preheating tube 21 is connected to the inlet of the extraction tube 23. The extraction tube 23 and the preheating tube 21 are both installed and placed in the column temperature box 22.

[0032] The condensation device 3 includes a condenser 31, a beaker 32 and a pressure reducing valve 33. The condenser 31 is placed in the beaker 32. The beaker 32 is filled with condensate, which is generally water. The outlet of the extraction tube 23 is connected to the inlet of the condenser 31, and the outlet of the condenser 31 is connected to the inlet of the pressure reducing valve 33.

[0033] The enrichment device 4 includes an enrichment solvent bottle 41, a binary pump 42, a tee 43 and an enrichment bottle 44. The binary pump 42 and the outlet of the pressure reducing valve 33 are respectively connected to the first interface and the second interface of the tee 43, the third interface of the tee 43 is connected to the inlet of the enrichment bottle 44, and the outlet of the enrichment bottle 44 is connected to the third interface A3 of the six-way valve 13; the enrichment bottle 44 is a glass bottle, and its inlet and outlet contain latex cylindrical stoppers.

[0034] Taking the extraction of nitrosamines from cured meat as an example, the working process of the pre-treatment device of the present invention is as follows: Figure 2-5 The workflow of the device controlled by the computer software Chromeleon is shown in Table 1:

[0035] (1) Sample loading: Cut the solid sample into small particles of similar size and sieve them with a 50-mesh sieve. Figure 2As shown, 3.0 g of sample particles and 2.0 g of quartz sand (particle size of 1-2 mm) are uniformly mixed and loaded into the extraction tube 23. Glass wool is placed at both ends of the extraction tube 23 to prevent clogging of the extraction tube 23 and sample loss. After the solid sample is loaded, the temperature of the column oven 22 is set to 111.7° C. The column oven 22 starts heating, and the extraction tube 23 and the preheating tube 21 are heated. The ten-way valve 15 is in the sample measurement state; the six-way valve 13 is in the sample measurement state; and the pressure reducing valve 33 is adjusted to control the system pressure at 100 bar.

[0036] (2) Extraction and enrichment: Figure 3 As shown, the ten-way valve 15 is in the injection state; the six-way valve 13 is in the sample measurement state; the extract is placed in the eluent bottle of the liquid chromatography pump; an organic solvent with a density lower than that of water is selected as the enrichment solvent and placed in the enrichment solvent bottle, and the corresponding reagent is placed in the enrichment bottle 44 to facilitate subsequent liquid-liquid microextraction; under the action of the liquid chromatography pump 12, the extract is infiltrated into the extraction tube 23 at a speed of 1.0 mL / min, and the extract is a subcritical solution under high temperature and high pressure, which extracts the solid sample in the extraction tube 23, and then the extract is c The extract enters the tee through the condensing device, and the enriched solvent enters the tee under the action of the binary pump; c The enriched solvent is premixed at the three-way connection and transported to the enrichment bottle 44; liquid-liquid microextraction occurs in the enrichment bottle 44, at which time the enriched solvent is extracted and the enriched extract is extracted. c The target compound in the mixture enters the enrichment solvent, and finally the mixed liquid is divided into two layers. Due to density reasons, the mixture containing the enrichment solvent and the target compound is in the upper layer;

[0037] Specifically, the computer program controlled the binary pump to pump at a rate of 0.07 mL min -1 The flow rate of n-octanol (enriched solvent) is used to transport n-octanol and the extract c Premix in a three-way pipe to form an emulsion, and all the solutions are collected in an enrichment bottle 44, which contains 13 mL of a mixture of 0.2 g / mL potassium sulfate and 0.4 mg / mL sodium hydroxide. After collecting for 11.8 minutes, the mixture gradually becomes clear and separates into two phases, an upper and lower layer, with n-octanol and the target compound in the upper layer. The extract is a mixture of water and an organic solvent. In this embodiment, the extract is a mixture of water and acetonitrile (water:acetonitrile = 83.3:16.7, v / v); the extract c Includes extract and target compound.

[0038] (3) Injection: Figure 4 As shown, the ten-way valve 15 is in the injection state; the six-way valve 13 is in the injection state; the binary pump is in the running state, and under the action of pressure, the upper layer solution in the enrichment bottle 44, i.e., n-octanol rich in N-nitrosamine, is transferred to the quantitative loop 14.

[0039] (4) Sample measurement: Figure 5 As shown, the ten-way valve 15 is in the sampling state; the six-way valve 13 is in the sampling state; under the action of the liquid chromatography pump 12, the mobile phase enters the quantitative loop 14. In this embodiment, the mobile phase is a mixture of water and acetonitrile (water:acetonitrile = 60:40, v / v); the ultraviolet detector 16 is started with a wavelength of 230 nm; the target compound N-nitrosamine in the quantitative loop 14 is separated and quantified in the high performance liquid chromatography system.

[0040] Table 1 Workflow of the device controlled by computer software

[0041]

[0042]

[0043] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

Claims

1. A solid sample online pretreatment device, characterized in that: The invention comprises a high performance liquid chromatography (HPLC), a column oven device, a condensing device, an enrichment device and a computer, wherein the outlet of the HPLC is connected to the inlet of the column oven device, the outlet of the column oven device is connected to the inlet of the condensing device, and the outlet of the condensing device is connected to the inlet of the enrichment device; the HPLC device, the column oven device and the enrichment device are respectively connected to the computer; The high performance liquid chromatography device comprises an eluent bottle, a liquid chromatography pump, a six-way valve, a quantitative loop, a ten-way valve, a chromatographic column and an ultraviolet detector, wherein the eluent bottle is connected to the liquid chromatography pump, the quantitative loop is installed on the six-way valve, the liquid chromatography pump and the ten-way valve are respectively connected to the six-way valve, and the chromatographic column and the ultraviolet detector are both installed on the ten-way valve; the six-way valve, the ten-way valve and the liquid chromatography pump are all connected to a computer; the column oven device comprises a column oven, a preheating tube and an extraction tube, the outlet of the ten-way valve is connected to the inlet of the preheating tube, the outlet of the preheating tube is connected to the inlet of the extraction tube, and the extraction tube and the preheating tube are installed and placed in the column oven; The condensation device includes a condenser, a beaker and a pressure reducing valve, the condenser is placed in the beaker containing the condensate, the extraction tube outlet is connected to the condenser inlet, and the condenser outlet is connected to the pressure reducing valve inlet; The enrichment device includes an enrichment solvent bottle, a binary pump, a tee and an enrichment bottle, the binary pump and the pressure reducing valve outlet are respectively connected to the inlet of the tee, the outlet of the tee is connected to the inlet of the enrichment bottle, and the outlet of the enrichment bottle is connected to the six-way valve; The enrichment bottle is made of glass; the inlet and outlet of the enrichment bottle contain latex cylindrical stoppers; The working process of the pre-processing device is as follows: (1) Sample loading: Weigh an appropriate amount of solid powder sample and mix it with diatomaceous earth or quartz sand, place it evenly in the extraction tube, set the temperature of the column oven, heat the extraction tube and preheating column, and at this time, the six-way valve and the ten-way valve are both in the sample measurement state; (2) Extraction and enrichment: At this time, the six-way valve is in the sample measurement state, and the ten-way valve is in the injection state; under the action of the liquid chromatography pump, the extract comes out of the eluent bottle, passes through the six-way valve and the ten-way valve, and enters the preheating pipeline. The extract is preheated in the preheating pipeline. Then, the extract comes out of the preheating pipeline. Under high temperature and high pressure, the extract is a subcritical solution, which extracts the solid sample in the extraction tube, so that the target compound is extracted into the extract; The extract then passes through a condenser into a tee, and an enrichment solvent enters the tee under the action of a binary pump. The extract and enrichment solvent are premixed in the tee and transferred together to an enrichment flask. Liquid-liquid microextraction occurs in the enrichment flask, where the enrichment solvent extracts and enriches the target compound in the extract. The target compound then enters the enrichment solvent and, after extraction and enrichment, the concentration of the target compound reaches the detection requirement of the detector. Finally, under the action of gravity, the liquid in the enrichment flask separates into two layers, with the mixture containing the enrichment solvent and the target compound located in the upper layer. (3) Injection: At this time, the six-way valve is in the injection state, the ten-way valve is in the injection state, the liquid chromatography pump and the binary pump are in the running state, and under the action of pressure, the upper layer solution in the enrichment bottle is transported to the quantitative loop; (4) Sampling: At this time, the six-way valve and the ten-way valve are in the sampling state. Under the action of the liquid chromatography pump, the mobile phase enters the quantitative loop, and the mixture containing the enriched solvent and the target compound is transferred to the high performance liquid chromatography system for separation and analysis.

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