JP3719407B2 - Preparative liquid chromatograph - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a preparative liquid chromatograph for separating fraction components separated by a separation column and detected by a detector.
[0002]
[Prior art]
In order to fractionate and extract several desired components from a sample containing a plurality of components, a liquid chromatograph such as a high performance liquid chromatograph is used to separate and elute each component by a separation column.
[0003]
In the off-line method, components eluted from the separation column are collected by a fraction collector together with the mobile phase. Since the components collected by the fraction collector are collected together with the mobile phase solvent, the mobile phase solvent must be removed in order to concentrate the collected components. For this reason, the fractionated components are dried and concentrated by nitrogen purging by blowing nitrogen into the collection solution containing the sample components and mobile phase solvent, or by evaporating the mobile phase solvent by heating. are doing.
[0004]
In the on-line fractionation method, a trap column is provided downstream of the separation column, the desired components eluted from the separation column are concentrated by capturing in the trap column, and then another solvent is passed through the trap column. The captured fraction components are eluted.
[0005]
[Problems to be solved by the invention]
When the fraction component is concentrated and collected offline, if the mobile phase solvent collected together with the fraction component contains a component that does not easily evaporate, it takes a long time to evaporate the solvent. In addition, when the desired fraction component is a substance that is easily decomposed, there is a possibility that it is decomposed by heat when the solvent is heated to volatilize it.
[0006]
If processing is performed online using a trap column, fraction components can be collected in a short time, and decomposition of the fraction components can be suppressed. However, since only one trap column has been used so far, only one component can be captured. In the fraction component trapping operation, the fraction component eluted from the separation column is directly introduced to the trap column together with the mobile phase solvent. Therefore, if the fraction component is difficult to trap depending on the mobile phase solvent, it is recovered. There arises a problem that the rate decreases.
[0007]
An object of the present invention is to increase the recovery rate by increasing the number of fraction components that can be collected in online fractionation using a trap column.
[0008]
[Means for Solving the Problems]
In the present invention, in order to fractionate the fraction components separated by the separation column and detected by the detector, a plurality of trap columns connected in parallel to each other via the channel selection means downstream of the separation column, and the trap A diluent supply channel that is connected to the inlet channel of the column and supplies a diluent for diluting the eluate from the separation column, and a desorption that has the property of desorbing the fraction components captured by the trap column from the trap column. A desorption mobile phase supply channel for individually supplying the desorption mobile phase to the trap column is provided, and the diluting solution supply channel can mix the desorption mobile phase.
[0009]
By connecting a plurality of trap columns in parallel to each other, it is possible to collect as many components as the number of trap columns.
When the fraction components are introduced to the trap column, they are captured by the trap column while being diluted with a diluent, making it easier to capture and improving the recovery rate compared to capturing the trap column using only the mobile phase solvent for separation. To do. At that time, the concentration of the organic solvent in the diluent can be freely adjusted by mixing the mobile phase for desorption into the diluent.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The channel selection means is a switching valve or other appropriate means for selecting a channel.
As the detector, an ultraviolet-visible absorptiometer, a fluorimeter, a refractometer, an electrochemical detector, an electrical conductivity detector, a mass spectrometer, etc. that are generally used in liquid chromatographs can be used. .
[0011]
The diluent is selected to have a characteristic that facilitates the capture of the fraction components by the trap column by replacing the mobile phase solvent of the eluate.
In order to collect the fraction components once trapped in the trap column and then desorbed from the trap column in the downstream of the trap column, it is preferable to include a collection means such as a fraction collector. The mobile phase solvent used to desorb the fraction components from the trap column has a wider range of choices than the mobile phase solvent used to separate the sample with the separation column, so it was collected in a collection container. When concentrating the fraction components, a solvent that can be easily evaporated can be selected.
[0012]
When the eluate from the separation column is detected by the detector and the desired fractional component is eluted, the time required for the component to reach the trap column from the detector is determined in advance, The flow path selection means is operated so as to lead to the trap column. In order to desorb and elute the components captured by the trap column from the trap column, a desorption mobile phase solvent is supplied to the trap column. The component desorbed from the trap column can be supplied online as a sample of another analyzer, or can be collected in each collection container using means such as a fraction collector. .
[0013]
【Example】
FIG. 1 shows a comparative example. Here, a detector having an LC / MS configuration in which a mass spectrometer is connected in addition to a UV-visible absorptiometer detector is shown. A high-pressure gradient high-performance liquid chromatograph is used to separate sample components.
[0014]
H ₂ O and ACN (acetonitrile) are prepared as mobile phase solvents. In order to supply the mobile phase from each of the mobile phase containers 2 and 4, liquid feed pumps 6 and 8 are provided to constitute a mobile phase supply flow path using a high-pressure gradient. The liquid feed pumps 6 and 8 are controlled so as to have a flow rate set with time, and the supplied solvent is mixed by the mixer 10 and supplied to the separation column 14 as a mobile phase having a predetermined composition.
[0015]
An injector 12 for supplying a sample is provided in a flow path between the mixer 10 and the column 14. A sample can be automatically injected into the injector 12 by an autosampler.
An ultraviolet-visible absorptiometer is provided as a detector 16 downstream of the column 14. The eluent flow path that has passed through the detector 16 is connected to the switching valve 20 and also led to the mass spectrometer 18. Most of the eluate that has passed through the detector 16 is guided to the valve 20 side, but a part of the eluate is separated by a splitter and guided to the mass spectrometer 18, and either or both of the detector 16 and the mass spectrometer 18 are connected. It can be used to detect fraction components. By providing two types of detectors, both qualitative and quantitative can be performed.
[0016]
The valve 20 guides the eluate flowing through the detector 16 by switching to the trap column side or the drain 22 side. A plurality of flow paths are connected in parallel to each other through the switching valve 24 on the trap column side, and a trap column 28 is provided in each of the flow paths 26-1 to 26-n. These flow paths 26-1 to 26-n merge into one flow path, and are switched to the drain 22 or the fraction collector 30 through the switching valve 20 and led again. Although the flow path 26-1 is a drain flow path, the same trap as the trap flow paths 26-2 to 26-n is provided in the flow path 26-1 in order to always keep the diversion ratio constant by the diversion by the splitter. A column 28 is provided.
[0017]
Channels are connected to the channels 26-1 to 26-n so that a mobile phase solvent for desorption for desorbing and eluting the components captured by the diluent or the trap column 28 is supplied. Yes. H ₂ O is provided as a diluent, supplied from the container 32 by the pump 34, and led to the respective flow paths 26-1 to 26-n via the switching valves 36 to 38. On the other hand, acetonitrile (ACN) is provided as a mobile phase solvent for desorption, supplied from the container 40 by the pump 42, and led to the respective flow paths 26-1 to 26-n via the switching valves 36 and 38. It has become. The switching valve 36 selects either the diluent H ₂ O or the desorption mobile phase solvent ACN and guides it to the flow paths 26-1 to 26-n. For discharging to the drain 22 through the flow path 44, the switching valve 38 selectively supplies the selected diluent or mobile phase solvent to any of the flow paths 26-1 to 26-n. It is.
[0018]
Next, the operation of the comparative example will be described.
The mobile phase is supplied by a high pressure gradient by the pumps 6 and 8, and the mobile phase is supplied to the column 14 through the mixer 10. The switching valve 20 is set so as to guide the eluate that has passed through the detector to the valve 24 side, and the switching valve 36 is set so as to guide the diluent, and the diluent 34 is supplied from the container 32 by the pump 34. Yes. The flow rate of the diluent can be arbitrarily set in accordance with the fractional component, for example, 10 times the flow rate of the eluate sent through the detector 16. The switching valve 24 is set to guide the eluate to the flow path 26-1, and the switching valve 38 is set to guide the diluted liquid to the flow path 26-1. Pump 42 is not operating.
[0019]
In this state, when a sample is injected from the injector 12 by the autosampler, separation is performed by being guided to the column 14 by the mobile phase. The eluate from the column 14 is subjected to peak detection by either or both of the detector 16 and the mass spectrometer 18.
[0020]
When the peak of the desired component to be collected is detected by the detector 16 or the mass spectrometer 18, after a predetermined delay time, the switching valves 24 and 38 are connected to the flow paths 26-2 to 26-of the same trap column 28. It is switched to select any one of n. Thereby, the component is captured by the trap column 28 of the selected flow paths 26-2 to 26-n while being diluted with the diluent. When the peak of the fractional component ends, the switching valves 24 and 38 are returned to the train channel 26-1 side after a predetermined delay time.
[0021]
Thereafter, each time the second and third fractional component peaks are detected, the switching valves 24 and 38 are switched and led to the flow paths 26-2 to 26-n of the separate trap columns 28, respectively. The collection of painting components is repeated.
[0022]
When the preparative analysis is completed, the switching valve 20 is switched, the trap column 28 is connected to the fraction collector 30, and the flow path downstream of the detector 16 is connected to the drain 22 side. The switching valve 36 is also switched so that the flow path of the desorption mobile phase solvent ACN is connected to the trap column 28, and the pump 42 is operated to feed the desorption mobile phase solvent. The diluent 32 is discharged from the switching valve 36 to the drain 22 through the flow path 44.
[0023]
The mobile phase solvent for desorption is sequentially switched to the flow paths 26-2 to 26-n of the predetermined trap column 28 by the switching valve 38, and sent to the traps of the respective flow paths 26-2 to 26-n. The fraction components captured by the column 28 are desorbed and collected by the fraction collector 30 in separate collection containers. Since the time to elute the components captured by the trap column 28 is short, all captured components can be collected while the high performance liquid chromatograph is in a conditioned state for the next analysis.
[0024]
FIG. 2 shows an embodiment in which only the detector 16 is provided as means for detecting separated components and no mass spectrometer is provided. By not providing a mass spectrometer, it is not necessary to guide a fixed proportion of the eluate from the detector 16 to the mass spectrometer with a splitter. Therefore, in order to keep the diversion ratio constant by the diversion by the splitter, as shown in FIG. It is not necessary to arrange the trap column 28 in all the flow paths 26-1 to 26-n, and the flow path 26-1 for the drain is only a pipe as shown in FIG. Can do.
[0025]
The diluting liquid in the container 32 and the mobile phase solvent for desorption in the container 40 can be sent by a common liquid feeding pump 34. Open / close valves 46 and 48 for selecting the liquid feed are provided in the respective flow paths of the diluting liquid connected to the liquid feed pump 34 and the mobile phase solvent for desorption.
[0026]
In this embodiment, the fraction component is captured in each trap 28 while being diluted with a diluent, and the fraction component captured in each trap 28 is desorbed and eluted with a mobile phase solvent for desorption, and the fraction collector The operation of collecting by 30 is basically the same as the comparative example of FIG. The only difference is that the opening / closing of the on-off valves 46, 48 is switched in place of the switching operation of the switching valve 36 of FIG.
[0027]
In the case where the diluent and the mobile phase solvent for desorption are supplied by the same pump as in the embodiment of FIG. 2, the concentration of the organic solvent in the dilute solution is set to the mobile phase solvent for desorption in order to suppress the precipitation of fraction components. It can be freely adjusted by adding.
[0028]
【The invention's effect】
In the present invention, a plurality of trap columns are connected in parallel to the downstream of the separation column, and the desired fraction components are sequentially guided to the respective trap columns for measurement, so that only the number of trap columns is captured. can do.
In addition, when the fraction component is introduced to the trap column, it is trapped on the trap column while diluting with a diluent, making it easier to capture than when trapping on the trap column using only the mobile phase solvent for separation. Recovery rate is improved. At that time, since the mobile phase for desorption can be mixed into the diluted solution, the organic solvent concentration of the diluted solution can be freely adjusted by adding the mobile phase solvent for desorption, and the fraction components can be precipitated. Can be suppressed.
[Brief description of the drawings]
FIG. 1 is a flow chart showing a preparative liquid chromatograph of a comparative example.
FIG. 2 is a flow chart showing a preparative liquid chromatograph of another embodiment.
[Explanation of symbols]
2,4 Mobile phase vessel 6,8 Liquid feed pump 10 Mixer 12 Injector 14 Separation column 16 Detector 18 Mass spectrometer 20, 24, 36, 38 Switching valve 26-1 to 26-n Flow path 28 Trap column 32 Dilution Liquid container 34, 42 Pump 40 Desorption mobile phase solvent container 46, 48 On-off valve

Claims (2)

In a preparative liquid chromatograph that separates fraction components separated by a separation column and detected by a detector,
A plurality of trap columns connected in parallel to each other via a flow path selection means downstream of the separation column;
A diluent supply channel that is connected to the inlet-side channel of the trap column and supplies a diluent for diluting the eluate from the separation column;
A desorption mobile phase supply flow path for individually supplying a desorption mobile phase having a characteristic of desorbing the fraction components captured by the trap column from the trap column;
The preparative liquid chromatograph, wherein the diluting liquid supply channel can be mixed with the desorption mobile phase.   2. The preparative liquid chromatograph according to claim 1, further comprising means for collecting a fraction component desorbed from the trap column in a respective collection container downstream of the trap column.

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