WO2006046468A1 - 液体クロマトグラフィー装置 - Google Patents
液体クロマトグラフィー装置 Download PDFInfo
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- WO2006046468A1 WO2006046468A1 PCT/JP2005/019325 JP2005019325W WO2006046468A1 WO 2006046468 A1 WO2006046468 A1 WO 2006046468A1 JP 2005019325 W JP2005019325 W JP 2005019325W WO 2006046468 A1 WO2006046468 A1 WO 2006046468A1
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- 239000007788 liquid Substances 0.000 title abstract description 61
- 238000005194 fractionation Methods 0.000 claims abstract description 49
- 239000012141 concentrate Substances 0.000 claims abstract description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 36
- 238000001514 detection method Methods 0.000 claims description 27
- 238000004458 analytical method Methods 0.000 claims description 23
- 239000003085 diluting agent Substances 0.000 claims description 17
- 238000010828 elution Methods 0.000 claims description 17
- 230000000717 retained effect Effects 0.000 claims description 5
- 230000005855 radiation Effects 0.000 abstract 1
- 238000000862 absorption spectrum Methods 0.000 description 25
- 239000003960 organic solvent Substances 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 238000000034 method Methods 0.000 description 17
- 238000000926 separation method Methods 0.000 description 17
- 238000010586 diagram Methods 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 238000002211 ultraviolet spectrum Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000006172 buffering agent Substances 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000282376 Panthera tigris Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- -1 for example Substances 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000012464 large buffer Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/78—Detectors specially adapted therefor using more than one detector
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
- G01N2030/085—Preparation using an enricher using absorbing precolumn
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/468—Flow patterns using more than one column involving switching between different column configurations
Definitions
- the present invention relates to a liquid chromatography apparatus.
- UV detector first ultraviolet light detector
- a liquid chromatographic apparatus that captures and concentrates on a trap column, sends it to a second analytical column, separates it, and detects it with a second UV detector (see, for example, Patent Documents 1 and 2). .
- the components detected by the first UV detector Since the retention times of the components detected by the second UV detector are different, it was difficult to reliably identify whether they were the same component.
- components other than the components to be concentrated such as back-and-forth components, contaminant components, and components remaining in the fractionation flow path, may be detected by the second UV detector.
- the trap column may not be trapped in the trap column. In such a case, the component detected by the second UV detector is used as the target concentration target. Sometimes it was mistakenly identified as a component.
- Patent Document 1 Japanese Patent No. 2892795
- Patent Document 2 Pamphlet of International Publication No.99Z61905
- the present inventors ensured that the components separated and concentrated by the first analytical column and the components separated by the second analytical column were the same, and In order to develop an easily determinable liquid chromatography device, a photodiode array detector, an infrared detector, a radioisotope detector, or a fluorescence detector can be used as the first and second detection means.
- a vacuum vessel separation was performed with the first analytical column that was not affected by knock ground components, contaminant components, components remaining in the flow path for fractionation, etc., and was separated with the concentrated target component and the second analytical column. The inventors have found that the components are the same and can be determined easily and have reached the present invention.
- the present invention provides a first analytical column for separating components in a sample guided by a first mobile phase, a first detection means for detecting the components, and the first detection means.
- the fractionation flow path for fractionating the components detected in step (1) and holding them in the fractionation section, and the components retained in the fractionation section are sent to the trap column, and the components are trapped in the trap column and concentrated.
- a second detection means for detecting the separated components are a photodiode array detector, an infrared detector, a radioisotope detector.
- the first and second detection means have a photodiode array detector, an infrared detector, a radioisotope detector, and a fluorescence detector force which are the same detectors selected from each other. .
- a plurality of trap columns are provided, and the trapping and concentrating operation for trapping and concentrating components in one trap column of the trap channel, and eluting the trapped components from other trap columns. It is preferable to further include a flow path switching mechanism for simultaneously performing the elution operation.
- each of the first and second detection means further includes an ultraviolet light detector.
- the components held in the fractionation unit are sent out to the trap column while being diluted with a diluent.
- liquid chromatography apparatus according to item 1 or 2, wherein the components separated by the first analytical column are fractionated and held together with the diluent in the fractionation unit.
- a photodiode array detector In the liquid chromatography apparatus of the present invention, a photodiode array detector, an infrared light detector, a radioisotope detector, or a fluorescence detector is used as the first and second detection means. Make sure that the target component detected by the first detection means and the component detected by the second detection means are the same as those that are not affected by background components, contaminant components, components remaining in the fractionation channel, etc. Moreover, it can be easily determined.
- FIG. 1 is a diagram showing a liquid chromatography apparatus according to one embodiment of the present invention and its operation, in which a component separation step and a separated component fractionation step in a sample are performed. Indicates the state.
- FIG. 2 is a diagram showing a liquid chromatography apparatus according to one embodiment of the present invention and its operation, showing a state in which a component is trapped and concentrated in a trap column.
- FIG. 3 is a diagram showing a liquid chromatography apparatus according to one embodiment of the present invention and its operation, in which an analysis step in a second analysis column of components captured and concentrated in the trap column is performed. It shows the state!
- FIG. 4 is a diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, showing a state in which the components in the sample are separated and the separated components are separated.
- FIG. 5 is a diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, in which a component is trapped on a trap column and concentrated and an elution step of the concentrated component and a second analysis of the component The state where the analysis process in a column is performed simultaneously is shown.
- FIG. 6 is a diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, in which components are trapped in another trap column and concentrated in a trap column and trapped in the trap column. In this state, the elution of the components and the analysis process in the second analytical column are performed simultaneously.
- FIG. 7 A diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, showing a state in which the components in the sample are separated and the separated components are separated.
- FIG. 8 is a diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, in which a component is trapped on a trap column and the concentrated component is eluted and a second analysis column of the component This shows a state in which the analysis process is performed simultaneously.
- FIG. 9 is a diagram showing a liquid chromatography apparatus according to another embodiment of the present invention and its operation, in which components are trapped in another trap column and concentrated, and trapped in the trap column and concentrated.
- Elution ⁇ Shows the state in which the analysis process in the second analytical column is performed simultaneously.
- FIG. 1 shows one embodiment of the liquid chromatography apparatus of the present invention.
- the liquid chromatography apparatus shown in FIG. 1 uses a photodiode array detector (hereinafter abbreviated as PDA detector) as the first and second detection means, and a liquid chromatography equipped with one trap column.
- PDA detector photodiode array detector
- the liquid feed pumps 2a and 2b may be any pumps capable of feeding a solvent that can be used as a mobile phase such as an organic solvent and water.
- a liquid feed pump is preferably one that can arbitrarily set the flow rate.
- a switching valve 10 is connected to the upstream side of the liquid feed pump 2a by a flow path L1, and the switching valve 10 and the organic solvent 4a constituting the first mobile phase are connected by a flow path L2.
- An online degasser 8 is provided in the middle of the flow path L2.
- the online degasser 8 has a function of preventing air bubbles from getting into the organic solvent 4a and the diluent 6a flowing in the flow path, and may be provided in order to maintain a stable liquid feeding state. preferable.
- the switching nozzle 10 is connected to a flow path L3 to the diluent 6a.
- a switching valve 10 is connected to the upstream side of the liquid feed pump 2b by a flow path L4.
- the switching valve 10 and the water 4b constituting the first mobile phase are connected by a flow path L5, and the switching valve 10 and the carrier liquid 6b are connected by a flow path L6.
- an online degasser 8 is provided in the middle of the flow paths L5 and L6.
- the diluent 6a is a liquid that is sent out to the trap column 30 while diluting components pushed out from the fractionation sections 25a to 25e described later, and the carrier liquid 6b is held in the fractionation sections 25a to 25e described later.
- This is a liquid that pushes out the components into the trap column 30, which may be the same solvent or different solvents, depending on the organic solvent 4a and water 4b that constitute the first mobile phase, depending on the components, etc.
- the diluting liquid 6a and the carrier liquid 6b water or an aqueous solution not containing a buffer such as a nonvolatile salt can be used.
- a desalting treatment can be performed when the components are trapped and concentrated in the trap column by using a carrier solution that does not contain a large buffer. .
- the flow paths L7 and L8 on the downstream side of the liquid feed pumps 2a and 2b are connected to the mixer 14 that mixes the liquid flowing in both flow paths via the switching valve 12, and the solution mixed in the mixer 14 Are connected to the first analytical column 18 via an auto sampler 16 which is a sample injection portion.
- the flow rates of the liquid feed pumps 2a and 2b may be appropriately selected according to the sample, the first analytical column, etc.
- the flow rates of the respective liquid feed pumps may be constant, or may be independent of each other. It may be changed over time.
- the organic solvent 4a and water 4b constituting the first mobile phase are fed by two liquid feed pumps, mixed by the mixer 14, and the first composition having a predetermined composition is obtained.
- the composition of the first mobile phase is not limited to a mixed solution of an organic solvent and water, but may be a single organic solvent or a mixed solution of two different organic solvents. What is necessary is just to select suitably according to a component, an analysis column, etc.
- a buffer solution in which a buffering agent such as a non-volatile salt is dissolved may be used as a solvent constituting the first mobile phase.
- a sample means a sample in any form as long as it contains a component to be concentrated.
- a sample component-containing preparation or the like in solution for example, blood Sample components using plasma, urine and the like as a medium can also be mentioned.
- the first analytical column 18 various columns such as normal phase column, reverse phase column, ion exchange column, affiliation column, gel permeation chromatography (GPC) column can be used. What is necessary is just to select suitably according to the component in the sample to do.
- the inner diameter and length of such an analytical column are not particularly limited.
- a PDA detector 20 as a first detection means is connected to the downstream side of the first analytical column 18, and the components in the sample separated by the first analytical column 18 are connected to the PDA detector 20. It is designed to be detected.
- the PDA detector is a detector that continuously detects the absorption spectrum of each wavelength in the ultraviolet region (about 190 to about 400 nm) and also in the visible region (about 300 to about 800 nm), and is separated by the first analytical column 18. Absorption spectra of various components at various wavelengths from the ultraviolet region to the visible region can be obtained.
- the detected absorption spectrum is stored in a storage means (not shown).
- an infrared detector hereinafter abbreviated as an IR detector
- a radioisotope is used instead of the force PDA detector in which a PDA detector is used.
- a detector hereinafter abbreviated as RI detector
- RI detector a detector or a fluorescence detector
- the IR detector is used.
- the component to be concentrated is a compound containing a radioisotope
- the RI detector is used.
- the component can be reliably and more easily identified by using a fluorescence detector.
- a fractionation flow path 24 is connected to the PDA detector 20 via a switching nozzle 22.
- Fractionation channel 24 is a channel that has a fractionation section between two distribution valves 26a and 26b.
- the components separated by the first analytical column 18 were fractionated and fractionated by the distribution operation of the distribution valve.
- the components are held in the fractionating sections 25a to 25e together with the mobile phase.
- the switching valve 22 is also connected to a flow path L22 connected to the drain. In Fig. 1, there are five powers provided in the sorting section, and the number is not limited.
- Two flow paths L11 and L12 are connected between the switching valve 12 and the switching valve 22, and one of the flow paths L11 is branched and connected to the trap flow path.
- the trap flow path is a flow path for sending the components held in the sorting sections 25a to 25e to the trap column, trapping the components in the trap column and concentrating them, and providing one trap force ram 30. It has been.
- the trap column 30 is connected to the switching valve 28 by flow paths L16 and L17.
- a flow path L13 branched from the flow path L11 is connected to a switching valve 28.
- the switching valve 28 includes a second analytical column 32 and a second analytical column 32 that separate components captured and concentrated in the trap column 30.
- a PDA detector 33 is provided as a second detection means for detecting the components separated by the analysis column 32 of the above.
- the trap column 30 As the trap column 30, a column whose inner diameter is usually smaller than the inner diameter of the first analytical column 18 is used, and the force depending on the inner diameter of the first analytical column 18 is usually 0.03 to 6 mm. A column with an inner diameter of.
- the trap column 30 for example, a packed column, a monolithic column, or the like in which a cylindrical member is filled with a filler can be used.
- a packed column When a packed column is used as a trap column, it is preferable to use a packed column packed with a filler having a particle size of 10 to 60 m in order to reduce the pressure in the trap column.
- the length of the trap column 30 is not particularly limited, but is usually 10 to: L00 mmfe degrees.
- the second analytical column 32 from the viewpoint of further concentrating the components eluted from the trap column 30 to a higher concentration, for example, a column having an inner diameter of 0.03 to 0.3 mm such as a micro column or a nano column should be used. Is preferred.
- the length of the second analytical column 32 is usually 10-30 cm.
- the component eluted from the trap column 30 is detected by the PDA detector 33 as the second detection means, and the absorption spectrum of the component at various wavelengths from the ultraviolet region to the visible region is detected. You can get it.
- the detected absorption spectrum is stored in a storage means (not shown), and the absorption spectrum detected by the PDA detector 20 and the PDA detector 3 stored in the storage means.
- PDA detectors can acquire absorption spectra at various wavelengths in the ultraviolet region and visible region, it is possible to obtain more detailed spectral information than an ultraviolet detector that can acquire absorption spectra at a single wavelength. This makes it easy to identify the components.
- Liquid supply pumps 36a and 36b for supplying an organic solvent 38a and water 38b constituting the second mobile phase are connected to the switching valve 28 via a mixer 40.
- An online degasser 39 is provided in the flow path connecting the organic solvent 38a and water 38b to the liquid feed pumps 36a and 36b. Further, the switching valve 28 is connected to a discharge channel to the drain.
- the second mobile phase may be appropriately determined according to the component and the trap column 30 in order to facilitate the elution of the component from the trap column 30.
- a buffer such as a non-volatile salt
- the first analysis column 18 and the trap column 30 are provided in a column oven 41, and are maintained at a substantially constant temperature.
- the force that the first analytical column 18 and the trap column 30 are provided in one column oven may be provided with a column oven for each column.
- the second analytical column 32 is also provided in the power ram oven 41 or another column oven (not shown), and is maintained at a substantially constant temperature.
- Fig. 1 shows the state in which the separation process of the components in the sample and the separation process of the separated components are performed.
- the flow path used in the powerful process is a thick line, and the flow of the liquid is indicated by arrows.
- Fig. 2 shows the state in which components are trapped and concentrated in the trap column.
- the flow path used in the powerful step is a thick line, and the flow of the liquid is indicated by the arrow.
- Fig. 3 shows a state in which the second analytical column is separating the components that have been captured and concentrated in the trap column, and is the same as in Figs. 1 and 2.
- the flow path used in the intensive process is indicated by a thick line, and the flow of the liquid is indicated by an arrow.
- the switching knob 10 is operated to connect the flow path L1 and the flow path L2, and connect the flow path L4 and the flow path L5.
- the organic solvent 4a and water 4b are fed by the feed pumps 2a and 2b, respectively, passed through the flow paths L7 and L8, respectively, and mixed in the mixer 14 via the switching valve 12.
- the first mobile phase is transferred to the first analytical column 18 via the autosampler 16.
- the sample is injected by the autosampler 16
- the injected sample is guided to the first analytical column 18 by the first mobile phase, and the components in the sample are separated by the first analytical column 18.
- the separated component elutes from the first analytical column 18, is detected by the PDA detector 20, passes through the switching valve 22, passes through the flow path L9, and flows to the fractionation flow path 24.
- the distribution valves 26a and 26b work according to the detection signal, and any one of the fractionation sections 25a to 25e in the fractionation flow path 24 is selected,
- the separated components are fractionated, and the fractionated components are held together with the first mobile phase in the selected fractionation unit.
- the spectrum detected by the PDA detector 20 is stored in storage means (not shown). In FIG. 1, the sorting unit 25e is selected, and the components are fractionated into the sorting unit 25e.
- the distribution valves 26a and 26b are switched, and one of the fractionation sections in the separation flow path 24 is selected, and a fractionation operation is performed for each separated component. And the fractionated components are held in the selected fractionation section together with the first mobile phase. What is not held in the fractionation section of the fractionation flow path 24 by the first mobile phase flowing out from the first analytical column 18 passes through the distribution valve 26b, the flow path L10, the switching valve 22, and the flow path L22. The drain force is discharged.
- liquid feed pumps 36a and 36b are also activated, and the organic solvent 38a and water 38b constituting the second mobile phase are fed by the liquid feed pumps 36a and 36b, respectively, and mixed by the mixer 40.
- the second mobile phase is sent to trap column 30 via switching nozzle 28. And conditioning is now underway! /
- the switching knob 10 is operated to connect the flow path L1 and the flow path L3, and connect the flow path L4 and the flow path L6.
- Dilution liquid 6a and carrier liquid 6b are fed by liquid feed pumps 2a and 2b, and liquid carrier 6b flows through flow paths L6, L4, and L8, and flows through switching valve 12, flow path L12, and switching valve 22.
- Distributing valves 26a and 26b are operated to select one of the fractionation units in which the fractionated components are retained, and the transport liquid 6b passes through the fractionation unit selected from the distribution valve 26b and passes through the fractionation unit.
- the trap column 30 Along with the component and the first mobile phase held in the section, it goes to the trap column 30 through the distribution valve 26a, the flow path L9, the switching valve 22, the flow path L11, the flow path L13, the switching valve 28, and the flow path L16.
- the diluent 6a passes through the flow paths L3, L1, and L7, passes through the switching valve 12, passes through the flow path L11, and the components, the first mobile phase, and the carrier that are held in the selected fractionation unit. It joins with the flow of liquid 6b and is led to trap column 30.
- the component guided to the trap column 30 is captured by the trap column 30 and concentrated.
- the first mobile phase, the diluent 6a, and the carrier liquid 6b that have passed through the trap column 30 pass through the flow path L17 and are discharged from the drain valve via the switching valve 28.
- the organic solvent 38a and the water 38b constituting the second mobile phase are fed by the feed pumps 36a and 36b through the online degasser 39 with the bubbles removed, mixed by the mixer 40, and mixed with the second mobile phase.
- the mobile phase passes through the switching valve 28, passes through the flow path L16, and is led to the trap column 30.
- the components already captured and concentrated in the trap column 30 are eluted by the second mobile phase, and the eluted components pass through the flow path L17 together with the second mobile phase through the switching valve 28 and the second analysis.
- second analytical column Separated at 32.
- the separated components are detected by the PDA detector 33, which is the second detector, and an absorption spectrum is acquired.
- the acquired absorption spectrum is stored in storage means (not shown).
- the PDA detector 20 detects the obtained absorption spectrum and PDA detection.
- the components separated in the first analytical column and concentrated in the trap column and the components separated in the second analytical column are identical. Can be determined reliably and easily.
- an IR detector, RI detector, or fluorescence detector instead of a PDA detector, characteristic infrared absorption can be detected, and radioactive isotopes can be included.
- the components that are labeled with fluorescent compounds can be reliably and easily detected and the spectra can be compared, so the components separated in the first analytical column and concentrated in the trap column It is possible to reliably and easily determine whether or not the components separated by the second analytical column are the same.
- a combination of PDA detector 20 and PDA detector 33 instead of the combination of PDA detector 20 and PDA detector 33, a combination of infrared detector 20 and infrared detector 33, or a combination of radioisotope detector 20 and radioisotope detector 33 Alternatively, it is preferable to employ a combination of the fluorescence detector 20 and the fluorescence detector 33.
- the components held in the fractionation sections 25a to 25e in the fractionation flow path 24 are diluted with the diluent 6a and the carrier liquid 6b, and are pushed out to the trap column 30.
- the fractionated components may be held in the sorting sections 25a to 25e together with the carrier liquid 6b.
- the liquid chromatography apparatus shown in FIGS. 4 to 6 is a liquid chromatography apparatus in which two trap columns are provided in parallel and a flow path switching mechanism is provided.
- the trapping and concentration of components and the elution operation of components are performed alternately in one trap column. May be mixed with the components to be captured and concentrated next, so it may be necessary to extend the time required for elution of the components, for example. Then tiger Since two trap columns are provided in parallel, the trap column for capturing and concentrating the components can be replaced, so that processing can be performed more efficiently.
- Fig. 4 shows a state in which the separation process of the components in the sample and the separation process of the separated components are performed, and the flow path used in the powerful process is a thick line, and the flow of the liquid Is represented by an arrow.
- Figure 5 and Figure 6 show the situation where the components are trapped in the trap column 'concentration step and trapped in the trap column' eluted component elution 'and the analysis step is performed in the second analytical column at the same time.
- the flow path used in the powerful process is indicated by a thick line, and the liquid flow is indicated by arrows!
- the switching knob 10 is operated to connect the flow path L1 and the flow path L3, and connect the flow path L4 and the flow path L6.
- Dilution liquid 6a and carrier liquid 6b are fed by liquid feed pumps 2a and 2b, and liquid carrier 6b flows through flow paths L6, L4, and L8, and flows through switching valve 12, flow path L12, and switching valve 22.
- Distributing valves 26a and 26b are operated to select one of the fractionation units in which the fractionated components are retained, and the transport liquid 6b passes through the fractionation unit selected from the distribution valve 26b and passes through the fractionation unit.
- the diluent 6a passes through the flow paths L3, L1, and L7, passes through the switching valve 12, passes through the flow path L11, and the components, the first mobile phase, and the components retained in the selected fractionation unit. It merges with the flow of carrier liquid 6b and is led to trap column 30b.
- the component guided to the trap column 30b is captured by the trap column 30b and concentrated.
- the first mobile phase, the diluent 6a, and the carrier liquid 6b that have passed through the trap column 30b pass through the flow path L16 and pass through the switching nozzle 28a and the flow path L23. After that, drain force is discharged.
- the organic solvent 38a and the water 38b constituting the second mobile phase are fed by the feed pumps 36a and 36b through the online degasser 39 with the bubbles removed, respectively, and are fed by the mixer 40.
- the second mobile phase After being mixed, it becomes the second mobile phase, passes through the switching valve 28a, passes through the flow path L14, and is guided to the trap column 30a.
- the components already captured and concentrated in the trap column 30a are eluted by the second mobile phase, and the eluted components pass through the flow path L15 together with the second mobile phase through the switching valve 28b and the second mobile phase. It is led to the analytical column 32 and separated by the second analytical column 32.
- the separated components are detected by the PDA detector 33 which is the second detector, and an absorption spectrum is acquired.
- the acquired absorption spectrum is stored in a storage means (not shown).
- a PDA detector By using a PDA detector, it becomes possible to acquire an absorption spectrum at an arbitrary wavelength in the ultraviolet region and visible region for each component.
- the absorption spectrum detected by the PDA detector 20 and the PDA detection are obtained.
- the components separated by the first analytical column and concentrated in the trap column and the components separated by the second analytical column are identical. Whether or not can be determined reliably and easily.
- an IR detector, RI detector, or fluorescence detector instead of a PDA detector, characteristic infrared absorption can be detected, radioisotopes can be contained, or fluorescence can be obtained.
- the compounds that are labeled with a compound can be reliably and easily detected and the spectra can be compared. Therefore, the components separated in the first analytical column and concentrated in the trap column can be compared with the second analytical column. It is possible to reliably and easily determine whether or not the force is the same as the component separated in step (b).
- the combination of the PDA detector 20 and the PDA detector 33 instead of the combination of the PDA detector 20 and the PDA detector 33, the combination of the infrared detector 20 and the infrared detector 33, the combination of the radioisotope detector 20 and the radioisotope detector 33, or It is preferable to employ a combination of the fluorescence detector 20 and the fluorescence detector 33.
- the component is trapped in another trap column and concentrated, and the component is eluted and concentrated, and the component is eluted.
- the analysis step in the second analytical column is performed simultaneously. The state will be described with reference to FIG.
- the component captured and concentrated in the trap column 30b is eluted by the second mobile phase that has passed through the flow path L16 via the switching valve 28a, and the eluted component together with the second mobile phase It passes through the flow path L17, passes through the switching nozzle 28b, is guided to the second analytical column 32, and is separated by the second analytical column 32.
- the separated components are detected by the PDA detector 33, which is the second detector, and an absorption spectrum is acquired.
- the acquired absorption spectrum is stored in storage means (not shown).
- a PDA detector it is possible to acquire an absorption spectrum at an arbitrary wavelength in the ultraviolet region and visible region for each component, and the absorption spectrum and PDA detection detected by the PDA detector 20 are detected.
- the components separated in the first analytical column and concentrated in the trap column and the components separated in the second analytical column are identical. Can be determined reliably and easily.
- the components separated in the first analytical column and concentrated in the trap column are the same as the components separated in the second analytical column. It is possible to determine whether or not the trap is trapped by multiple trap columns that can be easily determined, and trap and concentrate the components in one trap column of the trap flow path. Since the column force is also equipped with a flow path switching mechanism that simultaneously performs the elution operation to elute the captured component, trap the component in the trap column • Concentration operation and the concentration of the component that was captured and concentrated in another trap column Since elution can be performed simultaneously and continuously, the processing efficiency is also improved.
- the component analyzed by the second analytical column is in a concentrated state as compared with the sample injected from the autosampler 16, the amount of the component contained in the sample is not shown. Even if it is not so, highly sensitive data can be acquired and the measurement efficiency can be improved by analyzing the components analyzed by the second analytical column by, for example, mass spectrometry, NMR or the like.
- the salt is also desalted by using a diluent or carrier solution that does not contain a buffer such as a non-volatile salt.
- FIGS. 7 to 9 The liquid chromatography apparatus shown in FIGS. 7 to 9 is provided with two trap columns in parallel and a flow path switching mechanism in the same manner as the liquid chromatography apparatuses shown in FIGS. Furthermore, as the first detection means, two types of detectors, a UV detector 20a and an IR detector 20b, and as the second detection means, two types of detectors, a UV detector 33a and an IR detector. 33b is a liquid chromatograph provided respectively.
- the first and second detection means there are at least two types of detector forces, one of which is a UV detector and the other is a PDA detector, IR detector, RI detector or fluorescence detector.
- the components are identified by two types of detectors, more information can be obtained and the components can be identified more reliably.
- a combination of IR detector 20b and IR detector 33b instead of the combination of IR detector 20b and IR detector 33b, a combination of PDA detector 20b and PDA detector 33b, or a combination of radioactive isotope detector 20b and radioactive isotope detector 33b, Or, it is preferable to use a combination of the fluorescence detector 20b and the fluorescence detector 33b.
- Fig. 7 shows a state in which the separation process of the components in the sample and the separation process of the separated components are performed, and the flow path used in the powerful process is a thick line, and the flow of the liquid Is represented by an arrow.
- Fig. 8 and Fig. 9 show the state in which components are trapped in the trap column 'concentration step and trapped in the trap column' concentrated component elution 'and the analysis step in the second analytical column is performed simultaneously.
- the flow path used in the powerful process is indicated by a thick line, and the flow of the liquid is indicated by an arrow.
- the same operation as described in the state shown in FIGS. 1 and 4 is performed.
- UV spectrum and IR spectrum Can be acquired. Capture the components shown in Fig. 8 and Fig. 9 into the trap column and concentrate them in the trap column.
- the switching valve 10 is provided, and the organic solvent 4a and water 4b constituting the first mobile phase are provided by the liquid feed pumps 2a and 2b, and the diluent 6a and carrier liquid 6b are switched and sent to each other through the same channel and sent to the fractionation channel 24!
- the diluting liquid 6a and the carrier liquid 6b pass through a different flow path from the flow path through which the organic solvent 4a and the water 4b constituting the first mobile phase flow.
- the separation of the components in the sample and the separation process of the separated components and the elution and extraction process of the components or the analysis process of the components in the second analytical column And capture of the components to the trap column 'concentration and elution of the concentrated components' It can also be performed in the analysis step in analytical ram simultaneously.
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE112005002632T DE112005002632T5 (de) | 2004-10-26 | 2005-10-20 | Flüssigchromatograph |
US11/718,073 US20080044309A1 (en) | 2004-10-26 | 2005-10-20 | Liquid Chromatograph |
GB0708434A GB2433901B (en) | 2004-10-26 | 2007-05-01 | Liquid chromatography apparatus comprising dual columns, dual detectors, and a component trap |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004310673A JP2006125856A (ja) | 2004-10-26 | 2004-10-26 | 液体クロマトグラフィー装置 |
JP2004-310673 | 2004-10-26 |
Publications (1)
Publication Number | Publication Date |
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WO2006046468A1 true WO2006046468A1 (ja) | 2006-05-04 |
Family
ID=36227708
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2005/019325 WO2006046468A1 (ja) | 2004-10-26 | 2005-10-20 | 液体クロマトグラフィー装置 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20080044309A1 (ja) |
JP (1) | JP2006125856A (ja) |
DE (1) | DE112005002632T5 (ja) |
GB (1) | GB2433901B (ja) |
WO (1) | WO2006046468A1 (ja) |
Cited By (3)
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JP2014534055A (ja) * | 2011-10-04 | 2014-12-18 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung | クロマトグラフィー精製のための方法および装置 |
CN108037233A (zh) * | 2017-12-28 | 2018-05-15 | 大连博迈科技发展有限公司 | 基于同一检测器的全在线检测的多维液相色谱分离系统 |
CN108603864A (zh) * | 2016-01-15 | 2018-09-28 | 高丽大学校产学协力团 | 非连续样品分级和级联装置以及具有该装置的双在线多功能液相色谱系统 |
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DE102014226481B3 (de) * | 2014-12-18 | 2016-06-02 | Siemens Aktiengesellschaft | Gaschromatograph |
DE102016121519B4 (de) * | 2016-11-10 | 2019-07-11 | Dionex Softron Gmbh | System und Verfahren zum Verbinden von Komponenten, insbesondere in der HPLC |
DE102016121515A1 (de) | 2016-11-10 | 2018-05-17 | Dionex Softron Gmbh | Verfahren zum Einführen einer Probe in eine Trennsäule und entsprechendes System |
JP6790963B2 (ja) * | 2017-03-30 | 2020-11-25 | 株式会社島津製作所 | 液体クロマトグラフ |
US10041914B1 (en) * | 2017-06-02 | 2018-08-07 | Shimadzu Corporation | Degassing device |
US11119079B2 (en) * | 2017-08-17 | 2021-09-14 | Daylight Solutions, Inc. | Liquid chromatography analyzer system with on-line analysis of eluting fractions |
US20210405001A1 (en) * | 2017-08-17 | 2021-12-30 | Daylight Solutions, Inc. | Liquid analyzer system with on-line analysis of samples |
US11275066B2 (en) * | 2017-09-14 | 2022-03-15 | Shimadzu Corporation | Liquid chromatograph |
EP3885764A4 (en) * | 2018-11-20 | 2022-08-17 | Hitachi High-Tech Corporation | ANALYZER WITH A VARIETY OF CHROMATOGRAPHS AND CONTROL METHODS FOR ANALYZER |
WO2020236471A1 (en) * | 2019-05-17 | 2020-11-26 | Illumina, Inc. | Linear peristaltic pumps for use with fluidic cartridges |
DE102019123373A1 (de) * | 2019-08-30 | 2021-03-04 | Dionex Softron Gmbh | Verfahren und System für zweidimensionale Chromatografie |
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- 2005-10-20 US US11/718,073 patent/US20080044309A1/en not_active Abandoned
- 2005-10-20 DE DE112005002632T patent/DE112005002632T5/de not_active Withdrawn
- 2005-10-20 WO PCT/JP2005/019325 patent/WO2006046468A1/ja active Application Filing
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JP2014534055A (ja) * | 2011-10-04 | 2014-12-18 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung | クロマトグラフィー精製のための方法および装置 |
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CN108603864A (zh) * | 2016-01-15 | 2018-09-28 | 高丽大学校产学协力团 | 非连续样品分级和级联装置以及具有该装置的双在线多功能液相色谱系统 |
CN108037233A (zh) * | 2017-12-28 | 2018-05-15 | 大连博迈科技发展有限公司 | 基于同一检测器的全在线检测的多维液相色谱分离系统 |
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Also Published As
Publication number | Publication date |
---|---|
DE112005002632T5 (de) | 2008-07-17 |
JP2006125856A (ja) | 2006-05-18 |
GB0708434D0 (en) | 2007-06-06 |
GB2433901B (en) | 2010-01-27 |
US20080044309A1 (en) | 2008-02-21 |
GB2433901A (en) | 2007-07-11 |
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