Screening method and apparatus for identifying a reactive compound bonding with a target compound

Abstract

An screening method and apparatus for online identification of a reactive compound bonding with a target compound includes a first dimension analysis step whereby a sample including a target compound and a reactive compound are injected together into a sample injection port. The sample is led to a first dimension column using a first dimension mobile phase and is separated into components. An isolation step isolates the sample portion into a loop passage. A reactive compound concentration step includes leading the reaction products into a trap column that selectively traps the reactive compound while dissociating it from the reaction products isolated by the isolation step. A second dimension analysis step leads the sample trapped in the trap column to a second dimension column using a second dimension mobile phase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the passages in one aspect of the screening apparatus.

FIGS. 2A and 2B are the passage diagrams for the first dimension analysis step of the two-dimensional liquid chromatograph in according to the screening apparatus of FIG. 1, wherein FIG. 2A is the case wherein the reaction products containing protein is isolated, and FIG. 2B is the case wherein the unnecessary components are discharged.

FIG. 3 is a passage diagram showing one embodiment of the reactive compound concentration step according to the apparatus of FIG. 1.

FIG. 4 is a diagram showing the passages in one embodiment of the second dimension analysis step according to the apparatus of FIG. 1.

FIG. 5A is a schematic diagram of the passages used in another aspect of the screening apparatus according to FIG. 1.

FIG. 5B is a sample supply unit according to the screening apparatus of FIG. 1.

FIGS. 6A-6C illustrate the movement of the sample in the size-excluding column according to the screening apparatus of FIG. 1.

FIG. 7 is a waveform illustration showing one example of the chromatograms obtained from the eluate in the size-excluding column according to the screening apparatus of FIG. 1.

FIGS. 8A and 8B are charts showing the results of the analysis of the components eluted from the first dimension column of the screening apparatus according to FIG. 1, wherein FIG. 8A is the case wherein the target and reactive compounds are mixed beforehand and injected as a reaction solution to the sample injection port, and FIG. 8B is the case wherein the target and reactive compounds in the same example are injected separately into the sample injection port.

Claims

1. A screening method for identifying a reactive compound bonding with a target compound, comprising: a first dimension sample analyzing step including injecting a sample comprising a target compound and a reactive compound into a sample injection port, leading the sample to a first dimension column, and separating the sample into components using a first dimension mobile phase;an isolating reaction step for isolating products resulting from a bonding between the target compound and the reactive compound in a loop passage;a reactive compound concentration step for leading an eluant into a trap column, said eluant dissociating said reactive compound from said reaction products as a concentration mobile phase, for selectively trapping said reactive compound while dissociating it from the reaction products isolated by the isolation of the sample; anda second dimension analysis step for analyzing the sample trapped in said trap column while leading the sample to a second dimension column using a second dimension mobile phase for analysis.

2. The screening method according to claim 1, wherein the sample injected into said sample injection port is a reaction solution that the target and reactive compounds are reacted beforehand.

3. The screening method according to claim 1, wherein the sample injected into said sample injection port is a target compound, by itself, and a reactive compound, by itself, having a smaller molecular weight than the target compound; wherein the injection into said sample injection port is performed in an order of the reactive compound first, followed by the target compound;wherein said first dimension column is a size-excluding column selected to vary an elution rate based on a molecular weight so that said target compound elutes before said reactive compound;whereby the target compound injected after the reactive compound is allowed to flow out of said first dimension column in order to allow the reaction between the target and reactive compounds to occur within said first dimension column.

4. The screening method according to claim 1, wherein said reactive compound concentration step includes mixing in said concentration mobile phase while leading the isolated reaction products to said trap column.

5. The screening method according to claim 1, wherein injecting the target compound comprises injecting a protein.

6. A screening apparatus configured to identify a reactive compound bonding with a target compound, comprising: a first dimension passage configured to lead a sample injected into a sample injection port to a first dimension column using a first dimension mobile phase to separate the sample into components,a sample supply unit configured to inject the sample into said sample injection port,an isolation passage configured to isolate a portion of separated sample components containing reaction products resulting from bonding between the target and reactive compounds into a loop passage,a reactive compound concentration passage for leading an eluant into a trap column, said eluant dissociating said reactive compound from said reaction products as a concentration mobile phase, for selectively trapping said reactive compound while dissociating it from the reaction products isolated by the isolation of the sample; anda second dimension passage configured to lead the sample trapped in said trap column to a second dimension column using a second dimension mobile phase.

7. The screening apparatus according to claim 6, wherein said sample supply unit is configured to inject the sample comprising a reaction solution of the target compound and the reactive compound.

8. The screening apparatus according to claim 6, wherein said sample supply unit is configured to inject a target compound, by itself, and a reactive compound, by itself, the reactive compound comprising a smaller molecular weight than the target compound, said reactive compound being injected first and followed by said target compound; and wherein said first dimension column is a size-excluding column configured to vary an elution rate based on a molecular weight so that said target compound elutes before said reactive compound, and the target compound injected after the reactive compound is allowed to flow out of said first dimension column before said reactive compound in order to allow a reaction between the target and reactive compounds to occur within said first dimension column.

9. The screening apparatus according to claim 6, wherein said reactive compound concentration passage includes a passage for transferring the reaction products isolated by the isolation by the concentration mobile phase, and another passage for further mixing said concentration mobile phase.

10. The screening apparatus according to claim 6, further comprising a two-dimensional liquid chromatograph.