CN209858494U - Online analysis pretreatment and offline analysis integrated liquid chromatograph - Google Patents
Online analysis pretreatment and offline analysis integrated liquid chromatograph Download PDFInfo
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- CN209858494U CN209858494U CN201920582275.9U CN201920582275U CN209858494U CN 209858494 U CN209858494 U CN 209858494U CN 201920582275 U CN201920582275 U CN 201920582275U CN 209858494 U CN209858494 U CN 209858494U
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Abstract
The utility model discloses an integrative liquid chromatograph of online analytical pretreatment and offline analysis, including autosampler, two chromatographic pumps, two six-way valves, online purification post, chromatographic fractionation post, ultraviolet detector, fluorescence detector, can switch between offline analysis and two kinds of modes of online pretreatment analysis as required. When the method is used for on-line pretreatment analysis, a sample can be directly detected after being simply extracted without pretreatment, so that the detection flow is greatly shortened, the manual operation is reduced, and the analysis cost is reduced. When off-line analysis is carried out, the system does not need to be changed, and the system can be directly switched, so that the use efficiency and the analysis capability of the instrument are effectively improved.
Description
Technical Field
The utility model relates to a liquid chromatograph especially relates to and can realize accomplishing the off-line analysis, can accomplish the integrative liquid chromatograph of online analysis pretreatment and off-line analysis of online analysis again.
Background
Liquid chromatography is a common separation and detection device, but some problems are found in application, such as complicated pretreatment process, time and labor waste and high material cost in liquid chromatography detection. And a set of on-line pretreatment system is established, samples can be injected after being simply extracted, the operation is very simple and convenient, the labor is greatly saved, and the problems of large sample amount and insufficient hands can be effectively solved. More importantly, the online purifying column only purifies a small amount of samples entering the system, so that the purifying amount is greatly reduced, and the experiment cost can be greatly saved.
Although on-line pretreatment liquid chromatography brings cost savings, it still faces a new problem of not being able to perform off-line detection, and if the sample is not suitable for on-line pretreatment or the sample matrix is simple and requires only conventional off-line analysis, the system flow path must be restored to the original state. In the face of the conditions of large sample detection quantity and more sample types, the treatment is more difficult. Often switch in two kinds of system states, the operation is very loaded down with trivial details, also unfavorable to the life-span of instrument, and dismouting system specialty is very strong moreover, and personnel that do not have the training can't operate. If can be to this defect of liquid chromatogram, can enough accomplish off-line liquid chromatogram analysis on a liquid chromatograph, can accomplish on-line chromatogram analysis again, realize the on-line switching of multiple liquid chromatogram system, just can make liquid chromatogram analysis operation simple and convenient, improve detection efficiency, use manpower and materials sparingly, reduce use cost.
SUMMERY OF THE UTILITY MODEL
The utility model aims to solve the technical problem that an online analysis pretreatment and integrative liquid chromatograph of off-line analysis are provided, based on the multivalve switching technique, realize accomplishing the off-line analysis, can accomplish the on-line analysis again. The online switching device can complete offline liquid chromatographic analysis and online chromatographic analysis on one liquid chromatograph, and realize online switching of various liquid chromatographic systems, so that the liquid chromatographic analysis is simple and convenient to operate, the detection efficiency is improved, manpower and material resources are saved, and the use cost is reduced.
In order to solve the technical problem, the utility model discloses a technical scheme is: an on-line pretreatment for analysis and off-line analysis integrated liquid chromatograph comprises an automatic sample injector, two chromatographic pumps, two six-way valves, an on-line purifying column, a chromatographic separation column, an ultraviolet detector and a fluorescence detector, wherein six interfaces of a first six-way valve for system switching are a, b, c, d, e and f in sequence, six interfaces of a second six-way valve are g, h, i, j, k and l in sequence, the automatic sample injector is connected with the first chromatographic pump and the second six-way valve interface g respectively, the second six-way valve interface k is connected with a second chromatographic pump, the second chromatographic pump is connected with a plurality of liquid storage bottles, a quantitative ring is connected between the second six-way valve interface h and the interface j, the second six-way valve interface i is connected with the first six-way valve interface d, the second six-way valve interface l is connected with the first six-way valve interface a, the first six-way valve interface c is connected with the chromatographic separation column, the ultraviolet detector and the fluorescence detector, The device comprises an ultraviolet detector and a fluorescence detector, wherein an online purification column is connected between a first six-way valve interface b and an interface e, a first six-way valve interface f is a waste liquid interface, and a plurality of liquid storage bottles are connected to a first chromatographic pump.
The use method of the on-line analysis pretreatment and off-line analysis integrated liquid chromatograph comprises the following steps:
step one, when the method is used for off-line analysis, the method sequentially comprises the following steps:
(1) and (3) a system balancing process: the mobile phase in the liquid storage bottle is brought into the second six-way valve by the second chromatographic pump, and enters the first six-way valve, the chromatographic separation column, the ultraviolet detector and the fluorescence detector through the quantitative ring to balance the whole system;
(2) sample introduction: the second six-way valve is switched, the automatic sample injector enables the sample injection system to enter the second six-way valve along with the mobile phase in the liquid storage bottle on the first chromatographic pump, and the sample is temporarily stored in the quantitative ring;
(3) elution analysis process: the second six-way valve is switched, the second chromatographic pump brings the mobile phase in the liquid storage bottle on the second chromatographic pump into the quantitative ring connected to the second six-way valve, and the sample enters the first six-way valve and the chromatographic separation column for separation and is analyzed by the ultraviolet detector and the fluorescence detector;
and step two, when the method is used for on-line pretreatment analysis, the method sequentially comprises the following steps:
(1) sample introduction and system balance process: the automatic sample injector enables a sample injection system to enter the second six-way valve along with the mobile phase in the first chromatographic pump, the sample is brought into the first six-way valve to be enriched in the online purification column, and impurities and matrixes are discharged into waste liquid along with the mobile phase; the second chromatographic pump brings the mobile phase into the first six-way valve, the chromatographic separation column, the ultraviolet detector and the fluorescence detector, and the whole system is balanced;
(2) elution analysis process: and the second six-way valve is switched, the second chromatographic pump enables the mobile phase to enter an online purification column connected to the first six-way valve, and a sample in the online purification column enters a chromatographic separation column for separation and is analyzed by an ultraviolet detector and a fluorescence detector.
And (2) after the elution analysis process, an on-line activation regeneration process is also included: the mobile phase in the stock solution bottle is taken into online purification post on with first chromatography pump to first chromatography pump, carries out activation regeneration to online purification post, and flow in first chromatography pump stock solution bottle is compared the column and is switched into the initial proportion of appearance, balances online purification post for the purification analysis of next sample afterwards.
The utility model has the advantages that: the online switching between the offline liquid chromatographic analysis and the online pretreatment liquid chromatographic analysis can be realized on one liquid chromatograph, the defect that the traditional liquid chromatograph only can perform the offline liquid chromatographic analysis or the online pretreatment liquid chromatographic analysis is overcome, an analysis system can be switched online according to the property of a sample to be analyzed, the convenience and the use efficiency of the instrument are greatly improved, and the use cost of the instrument is reduced. Meanwhile, the online purification analysis system saves the time and the complex operation process of sample pretreatment, and the online purification column is activated, regenerated and recycled, so that the online purification analysis system is more environment-friendly compared with the disposable use characteristic of the traditional purification column, and the analysis cost is greatly reduced.
Drawings
Fig. 1 is the structure diagram of the off-line detection system balance and elution process of the on-line pretreatment and off-line analysis integrated liquid chromatograph of the utility model.
Fig. 2 is the structure diagram of the off-line detection sample injection analysis process of the on-line analysis pretreatment and off-line analysis integrated liquid chromatograph of the utility model.
Fig. 3 is the structure diagram of the online pretreatment detection sample introduction and system balance and online activation regeneration process of the online pretreatment and offline analysis integrated liquid chromatograph of the utility model.
Fig. 4 is the structure diagram of the online pretreatment detection elution process of the online pretreatment and offline analysis integrated liquid chromatograph of the utility model.
Fig. 5 is a chromatogram of ochratoxin a in grain analysis according to an embodiment of the invention.
Fig. 6 is a calibration graph of ochratoxin a in accordance with an embodiment of the invention.
FIG. 7 is a chromatogram of a fluorescence detector for analyzing on-line pretreatment detection of benzo (a) pyrene in toner according to the embodiment of the present invention.
FIG. 8 is a chromatogram of an ultraviolet detector for analyzing benzo (a) pyrene on-line pretreatment detection in toner.
Fig. 9 is a calibration graph of benzo (a) pyrene according to an embodiment of the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description below:
as shown in fig. 1-4, the integrated liquid chromatograph for on-line pre-analysis and off-line analysis of the present invention comprises an automatic sample injector, two chromatographic pumps, two six-way valves, an on-line purification column, a chromatographic separation column, an ultraviolet detector, and a fluorescence detector, wherein six interfaces of a first six-way valve 3 for system switching are a, b, c, d, e, and f, six interfaces of a second six-way valve 8 are g, h, i, j, k, and l, the automatic sample injector 2 is connected with the first chromatographic pump 1 and the interface g of the second six-way valve 8, the interface k of the second six-way valve 8 is connected with a second chromatographic pump 10, the second chromatographic pump 10 is connected with a first liquid storage bottle 15, a second liquid storage bottle 16, a third liquid storage bottle 17, and a fourth liquid storage bottle 18, a quantitative ring 9 is connected between the interface h and the interface j of the second six-way valve 8, the interface i of the second six-way valve 8 is connected with the interface d of the first six-way valve 3, the interface l of the second six-way valve 8 is connected with the interface a of the first six-way valve 3, the interface c of the first six-way valve 3 is sequentially connected with a chromatographic separation column 5, an ultraviolet detector 6 and a fluorescence detector 7, an online purification column 4 is connected between the interface b and the interface e of the first six-way valve 3, the interface f of the first six-way valve 3 is a waste liquid interface, and the first chromatographic pump 1 is respectively connected with a fifth liquid storage bottle 11, a sixth liquid storage bottle 12, a seventh liquid storage bottle 13 and an eighth liquid storage bottle 14.
The use method of the on-line analysis pretreatment and off-line analysis integrated liquid chromatograph comprises the following steps:
step one, when the method is used for off-line analysis, the method sequentially comprises the following steps:
(1) and (3) a system balancing process: the second chromatographic pump 10 brings the mobile phase in the fourth liquid storage bottle 18 into the second six-way valve 8, and enters the first six-way valve 3, the chromatographic separation column 5, the ultraviolet detector 6 and the fluorescence detector 7 through the quantitative ring 9 to balance the whole system, as shown in fig. 1;
(2) sample introduction: the second six-way valve 8 is switched, the automatic sample injector 2 enables the sample injection system to enter the second six-way valve 8 along with the mobile phase in the eighth liquid bottle 18 in the first chromatographic pump 1, and the sample is temporarily stored in the quantitative ring 9, as shown in fig. 2;
(3) elution analysis process: the second six-way valve 8 is switched, the second chromatographic pump 10 brings the mobile phase in the fourth bottle 18 into the quantitative ring 9 connected to the second six-way valve, the sample enters the first six-way valve 3 and the chromatographic separation column 5 for separation, and is analyzed by the ultraviolet detector 6 and the fluorescence detector 7, as shown in fig. 1;
and step two, when the method is used for on-line pretreatment analysis, the method sequentially comprises the following steps:
(1) sample introduction and system balance process: the automatic sample injector 2 enables a sample injection system to enter the second six-way valve 8 along with the mobile phase in the first chromatographic pump 1, the sample is brought into the first six-way valve 3 to be enriched in the online purification column 4, and impurities and matrixes are discharged into waste liquid along with the mobile phase; the second chromatographic pump 10 brings the mobile phase into the first six-way valve 3, the chromatographic separation column 5, the ultraviolet detector 6 and the fluorescence detector 7, and balances the whole system, as shown in fig. 3;
(2) elution analysis process: the second six-way valve 8 is switched, the second chromatographic pump 10 enters the mobile phase into the on-line purifying column 4 connected to the first six-way valve, the sample in the on-line purifying column enters the chromatographic separation column 5 for separation, and the sample is analyzed by the ultraviolet detector 6 and the fluorescence detector 7, as shown in fig. 4.
Preferably, the elution analysis process of step two (2) further comprises an on-line activation regeneration process: first chromatogram pump 1 brings the mobile phase in fifth stock solution bottle 11 and sixth stock solution bottle 12 into online clean column 4, carries out activation regeneration to online clean column, and first chromatogram pump 1 compares the flow in fifth stock solution bottle 11 and the sixth stock solution bottle 12 and arranges the switching for the initial proportion of appearance of advancing afterwards, balances online clean column for the purification analysis of next sample, as shown in fig. 3.
Specifically, the automatic sample injector 2 is connected with a first chromatographic pump 1 and a second six-way valve 8, a quantitative ring 9 is arranged on the second six-way valve and is connected with a first six-way valve 3 and a second chromatographic pump 10, the first chromatographic pump 5 is connected with a fifth liquid storage bottle 11, a sixth liquid storage bottle 12, a seventh liquid storage bottle 13 and an eighth liquid storage bottle 14, the second chromatographic pump is connected with a first liquid storage bottle 15, a second liquid storage bottle 16, a third liquid storage bottle 17 and a fourth liquid storage bottle 18, the first six-way valve 3 is connected with a chromatographic separation column 5, and the chromatographic separation column 5 is sequentially connected with an ultraviolet detector 6 and a fluorescence detector 7 to form an offline liquid chromatographic analysis system;
the automatic sample injector 2 is connected with a first chromatographic pump 1 and a second six-way valve 8, a quantitative ring 9 is arranged on the second six-way valve and is connected with a first six-way valve 3 and a second chromatographic pump 10, a first chromatographic pump 5 is connected with a fifth liquid storage bottle 11, a sixth liquid storage bottle 12, a seventh liquid storage bottle 13 and an eighth liquid storage bottle 14, a second chromatographic pump is connected with a first liquid storage bottle 15, a second liquid storage bottle 16, a third liquid storage bottle 17 and a fourth liquid storage bottle 18, the first six-way valve 3 is connected with an online purification column 4 and a chromatographic separation column 5 connected with the first six-way valve 3, and the chromatographic separation column 5 is sequentially connected with an ultraviolet detector 6 and a fluorescence detector 7 to form an online purification pretreatment liquid chromatographic analysis system.
The first liquid storage bottle 15 is used for containing acetonitrile, the second liquid storage bottle 16 is used for containing water, the fourth liquid storage bottle 18 is used for containing water/acetonitrile/acetic acid solution water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V/V), and the third liquid storage bottle 17 is reserved.
The fifth liquid storage bottle 11 is used for containing acetonitrile, the second liquid storage bottle 12 is used for containing water, the fourth liquid storage bottle 14 contains water/acetonitrile/acetic acid solution water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V/V), and the third liquid storage bottle 13 is used for standby.
The specific implementation is as follows:
when the liquid chromatography system is used for off-line analysis of ochratoxin A in grains, the steps are as follows:
(1) and (3) a system balancing process: weighing 20g of sample, adding the extracting solution (sodium chloride-sodium bicarbonate solution) into a 25mL volumetric flask, fixing the volume to the scale, mixing uniformly, and clarifying the filtrate by using glass fiber filter paper. Adding 100mL of the extractive solution, homogenizing at high speed for 3min, quantitatively filtering with filter paper, transferring 10mL of the filtrate, adding 40mL of phosphate buffer solution, diluting to 50mL, and filtering with glass fiber filter paper. Transferring 10mL of the filtrate to an immunoaffinity column, injecting 10mL of washing solution (sodium chloride-sodium bicarbonate solution) 10mL of mycotoxin washing buffer solution and 10mL of water respectively for elution, eluting with 1mL of methanol, and measuring by HPLC. The second chromatographic pump 10 brings the water/acetonitrile/acetic acid solution water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V) in the fourth liquid storage bottle 18 into the second six-way valve 8 at the speed of 1mL/min, and the water/acetonitrile/acetic acid solution enters the first six-way valve 3, the chromatographic separation column 5 (model: Utimate XB-C18, 4.6 x 150mm, 5 μm), the ultraviolet detector 6 and the fluorescence detector 7 through the quantitative ring 9 to balance the whole system;
(2) sample introduction: the second six-way valve 8 is switched, the autosampler 2 enables a 10 μ L sample injection system to enter the second six-way valve 8 along with a water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V) in the eighth bottle 14 in the first chromatographic pump 1, 90 μ L (0.18mL/min injection speed is 0.5min) is injected in total, and the sample is temporarily stored in the quantification ring 9;
(3) elution analysis process: the second six-way valve 8 is switched, the second chromatographic pump 10 brings the water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V) in the fourth bottle 18 into the quantification ring 9 connected to the second six-way valve 8, the sample enters the first six-way valve 3, the chromatographic separation column 5 (model: Utimate XB-C18, 4.6 x 150mm, 5 μm) for separation, and the sample is analyzed by the ultraviolet detector 6 and the fluorescence detector 7 (excitation wavelength 333nm, emission wavelength 460 nm). Ochratoxin a has a weak uv response and this example of use uses only a fluorescence detector, the uv detector being reserved for other analyses.
(4) Drawing a standard curve: calibration curve solutions of 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL were prepared with water + acetonitrile + acetic acid solution (49.5+49.5+1, V/V/V) and linear regression was performed with mass concentration (X) and peak area (Y). The linear equation for obtaining the calibration curve is
Y is 108.9273X +98.1914, and the linear correlation coefficient R2 is 1.0. Ochratoxin A chromatogram is shown in FIG. 5, and ochratoxin A standard curve is shown in FIG. 6.
When the liquid chromatography system is used for carrying out online pretreatment and analysis on benzo (a) pyrene in toner, the steps are as follows:
(1) sample introduction and system balance process: weighing 0.5g of sample in a 10mL volumetric flask, fixing the volume to the scale with acetonitrile, ultrasonically extracting for 30min, centrifuging for 5min at 5000r/min, taking the supernatant, and filtering the supernatant through a 0.45um filter membrane for analysis. The automatic sample injector 2 injects 20 μ L of sample into the system and enters the second six-way valve 8 at a speed of 1.0mL/min with acetonitrile in the fifth stock bottle 11 and water in the sixth stock bottle 12 in the first chromatographic pump 1 (acetonitrile + water is 5+95) and transfers to the first six-way valve 3, benzo (a) pyrene in the sample is enriched in the online purification column 4 (model: Acclaim PolarAdvantage II 3 μm, 4.6 × 50mm), and impurities and matrix are discharged into waste liquid with isopropanol; the second chromatography pump 10 brought the mobile phase (acetonitrile + water 88+12) in the first and second vials 11 and 12 at a rate of 1.2mL/min into the six-way valve 3, the chromatography separation column 5 (model: Utimate XB-C18, 4.6 x 150mm, 5 μm), the ultraviolet detector 6 and the fluorescence detector 7, and the whole system was equilibrated.
(2) Elution analysis process: the first six-way valve 3 is switched, the second chromatographic pump 4 brings acetonitrile in the first bottle stock 15 and water in the second bottle stock 16 (acetonitrile + water: 88+12) into an online purifying column 4 (model: Acclaim polar additive II 3 μm, 4.6 μm, 50mm) on the six-way valve 3 at a speed of 1.2mL/min, the benzo (a) in the online purifying column 4 is eluted into a chromatographic separation column 5 (model: Utimate XB-C18, 4.6 μm, 150 μm), separated and analyzed by an ultraviolet detector 6 (wavelength 254nm) and a fluorescence detector 6 (excitation wavelength 370nm, emission wavelength 406 nm).
(3) And (3) washing and activating processes of the online purification column: the first six-way valve 3 is switched, the first chromatographic pump 1 brings acetonitrile in the fifth bottle stock solution 11 and water in the sixth bottle stock solution 12 into the online purifying column 7 (model: Acclaim PolarAdvantage II 3 μm, 4.6 mm) through the six-way valve 3 at a speed of 1.0mL/min (gradient ratio is shown in the following table), and then the acetonitrile in the fifth bottle stock solution 11 is switched to wash the online purifying column, and the acetonitrile in the fifth bottle stock solution 11 and the water in the sixth bottle stock solution 12 are switched to activate and regenerate the online purifying column for purification analysis of the next sample. Simultaneously, the second chromatographic pump 10 brought acetonitrile in the first bottle stock 15 and water in the second bottle stock 16 (acetonitrile + water: 88+12) into the first six-way valve 3, the chromatographic separation column 5 (model: Utimate XB-C18, 4.6 × 150mm, 5 μm), the ultraviolet detector 6 and the fluorescence detector 7 at a rate of 1.2mL/min, and equilibrated the chromatographic separation column 5.
(4) The first chromatography pump 1 reservoir 8, 9, 10 time gradient program is shown in the following table:
| time min | Liquid storage bottle 11 (%) | Liquid storage bottle 12 (%) |
| 0 | 5 | 95 |
| 0.3 | 5 | 95 |
| 1.8 | 5 | 95 |
| 8 | 5 | 95 |
| 8.5 | 100 | 0 |
| 22 | 100 | 0 |
| 23 | 5 | 95 |
(5) Drawing a standard curve: calibration curve solutions of 0.08ng/mL, 0.16ng/mL, 0.80ng/mL, 4.0ng/mL, 16.0ng/mL were prepared with acetonitrile and linear regression was performed with mass concentration (X) and peak area (Y). The linear equation for the calibration curve is obtained as Y29928.1330X-5.1980 and the linear correlation coefficient R2 is 0.9999. The chromatogram of benzo (a) pyrene is shown in figures 7 and 8, and the standard curve of benzo (a) pyrene is shown in figure 9, in the application example, the response of an ultraviolet detector is low, and the ultraviolet detector is only used for performing auxiliary qualitative determination on a sample when a positive result appears, so that the standard curve of benzo (a) pyrene is not made.
The above-mentioned embodiments are only used for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to implement the present invention accordingly, the scope of the present invention should not be limited by the embodiment, that is, all equivalent changes or modifications made by the spirit of the present invention should still fall within the scope of the present invention.
Claims (1)
1. An on-line pretreatment for analysis and off-line analysis integrated liquid chromatograph comprises an automatic sample injector, two chromatographic pumps, two six-way valves, an on-line purifying column, a chromatographic separation column, an ultraviolet detector and a fluorescence detector, and is characterized in that six interfaces of a first six-way valve (3) for system switching are a, b, c, d, e and f in sequence, six interfaces of a second six-way valve (8) are g, h, i, j, k and l in sequence, the automatic sample injector (2) is respectively connected with a first chromatographic pump (1) and a second six-way valve (8) interface g, the second six-way valve (8) interface k is connected with a second chromatographic pump (10), the second chromatographic pump (10) is connected with a plurality of liquid storage bottles, a quantitative ring (9) is connected between the second six-way valve (8) interface h and the interface j, the second six-way valve (8) interface i is connected with a first six-way valve (3) interface d, the interface l of the second six-way valve (8) is connected with the interface a of the first six-way valve (3), the interface c of the first six-way valve (3) is sequentially connected with a chromatographic separation column (5), an ultraviolet detector (6) and a fluorescence detector (7), an online purification column (4) is connected between the interface b and the interface e of the first six-way valve (3), the interface f of the first six-way valve (3) is a waste liquid interface, and the first chromatographic pump (1) is connected with a plurality of liquid storage bottles.
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| CN109959742A (en) * | 2019-04-25 | 2019-07-02 | 天津出入境检验检疫局动植物与食品检测中心 | A kind of on-line analysis pre-treatment and off-line analysis one liquid chromatograph and its application method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109959742A (en) * | 2019-04-25 | 2019-07-02 | 天津出入境检验检疫局动植物与食品检测中心 | A kind of on-line analysis pre-treatment and off-line analysis one liquid chromatograph and its application method |
| CN109959742B (en) * | 2019-04-25 | 2024-02-20 | 天津海关动植物与食品检测中心 | Online analysis pretreatment and offline analysis integrated liquid chromatograph and use method thereof |
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