1. Field of the Invention
The present invention relates to a liquid chromatography apparatus. More specifically, the present invention relates to a three-dimensional liquid chromatography apparatus including, for example, a normal-phase, ion-exchange, and reversed-phase separation columns.
2. Description of the Related Art
In a complex biotic sample, a hydrophilic component, a hydrophobic component, and an ionic component are mixed and the molecular weight of each component is distributed over a wide range. Accordingly, there is a limit in separating the components by one type of column. To overcome such a limit, two-dimensional liquid chromatography apparatuses each using a combination of two types of columns operating based on different separation modes are proposed (see Non-Patent Document 1: A. J. Link et al, Nat. Biotechnol. 17, 676 (1999), Non-Patent Document 2: Y. Shen et al, Anal. Chem. 77, 3090 (2005), Non-Patent Document 3: T. Wehr, L C. G C Europe Mar. 2 (2003), and Non-Patent Document 4: P. Dugo et al, Anal. Chem. 76, 2525 (2004)). A column of first stage (first dimension) and a column of second stage (second dimension) used in those known techniques are restricted to a combination of the ion-exchange column (size exclusion column in some cases) and the reversed-phase column.
As a result of conducting intensive studies, the inventors have found the following.
Table 1 represents the relationships between three kinds of separation modes (i.e., normal-phase, ion-exchange, and reversed-phase modes) and samples. In Table 1, a mark ◯ means that the sample can be retained (separable), and a mark × means that the sample cannot be retained (non-separable). Although there are in practice samples having intermediate properties, those samples are omitted here for simplicity of the description. As seen from Table 1, in the case of employing the above-mentioned column combination, separation of hydrophilic components such as indicated by sample groups C and D cannot be successfully performed.
A combination of the normal-phase column and the reversed-phase column is required to perform separation and analysis of the biotic sample including the sample groups A-D. In that case, however, an organic solvent used for the component separation in the normal-phase column impedes the component separation in the reversed-phase column. More specifically, when the component separated in the normal-phase column is introduced to the reversed-phase column together with the organic solvent, the component is eluted as it is without being retained on the reversed-phase column or being further separated. In other words, the so-called “solution interference” occurs. For that reason, it is essential to devise some means or contrivance for realizing “solution non-interference” so that the solution used for the component separation in the column of first stage (first dimension) will not impede the component separation in the column of second stage (second dimension).
The simplest method of avoiding the “solution interference” is to perform the component separation and analysis by introducing a solution sample to each of two liquid chromatography apparatuses including the normal-phase column and the reversed-phase column, respectively, or to temporarily fraction a component separated by a liquid chromatography apparatus including the normal-phase column at intervals of a certain time, and after removing an organic solvent, to perform further separation and analysis of the separated component again by using a liquid chromatography apparatus including the reversed-phase column.
As an alternative method, it is also proposed to, instead of removing the organic solvent, dilute the organic solvent eluted from the normal-phase column at a flow rate ratio of 400:1 and to introduce the diluted organic solution into the reversed-phase column (see Patent Document 4). However, that method is not suitable for a high-sensitivity analysis because the separated component is also diluted at a ratio of 400:1.
An object of the present invention is to avoid the “solution interference” in a more satisfactory manner.
In a liquid chromatography apparatus of the present invention, a separation column of intermediate stage is additionally connected between a separation column of first stage and a separation column of second stage. Preferably, a switching unit and a liquid feed unit for mixing and feeding a plurality of solutions are added to improve a separation capability.
According to the present invention, a three-dimensional liquid chromatography apparatus capable of avoiding the “solution interference” can be realized. As a result, even a complex sample containing a hydrophilic component and a hydrophobic component in a mixed state can be separated and analyzed satisfactorily on-line.
The above-mentioned and other novel features of the present invention will be described below with reference to the drawings. Note that the drawings are attached merely for the sake of explanation and should not be construed to limit the scope of the present invention.
The three-dimensional liquid chromatography apparatus of the first embodiment comprises a gradient pump 4, a sample injection unit (means), a normal-phase column 7 serving as a separation column of first stage, a reversed-phase column 10 serving as a separation column of second stage, a 6-way flow passage switching valve 8 serving as a switching unit (means), and a mass spectrometer 11 serving as a detection unit (means) for detecting separated components. In addition, an ion-exchange column 9 serving as a separation column of intermediate stage is connected between the switching unit and the separation column of second stage.
The gradient pump 4 serves as a liquid feed unit (means) for mixing and feeding a plurality of solutions. More specifically, the gradient pump 4 is able to mix an aqueous solution A 1, an organic solvent solution B 2, and an aqueous solution C 3 at a predetermined ratio, and to feed the mixed solution to a flow passage.
The sample injection unit is made up of an auto-sampler 5 and a sample introducing unit 6.
The 6-way flow passage switching valve 8 is a switching unit for introducing a component separated by the separation column of first stage to the separation column of second stage.
The operation of the three-dimensional liquid chromatography apparatus according to the first embodiment will be described below.
The performance of the three-dimensional liquid chromatography apparatus of the first embodiment was verified as follows. The solutions were fed at a flow rate of 0.2 mL/min while changing the solution composition with time according to a gradient program shown in
According to this first embodiment, the combination of the normal-phase column and the reversed-phase column, for which the “solution interference” is unavoidable in principle, can be realized with an improvement of a two-dimensional liquid chromatography apparatus.
The separation column of intermediate stage may be a cation- or anion-exchange column. Also, the separation column of intermediate stage may consist of a cation (anion)-exchange column and an anion (cation)-exchange column connected in series. Further, another 6-way flow passage switching valve and a second gradient pump, i.e., a liquid feed unit (means) for mixing and feeding a plurality of solutions, may be additionally connected between the cation (anion)-exchange column and the anion (cation)-exchange column.
A three-dimensional liquid chromatography apparatus of the second embodiment comprises a first gradient pump 27, a second gradient pump 28, an auto-sampler 30 serving as a sample injection unit (means), a normal-phase column 31 serving as a separation column of first stage, a cation (anion)-exchange column 32 serving as a separation column of intermediate stage, a reversed-phase column 36 serving as a separation column of second stage, a first 6-way flow passage switching valve 34, and a mass spectrometer 37. In addition, a second 6-way flow passage switching valve 35 and a third gradient pump 29 are connected between the separation column of intermediate stage and the separation column of second stage.
The first gradient pump 27 is able to mix an aqueous solution A 21 and an organic solvent solution B 22 at a predetermined ratio for the normal-phase column, and to feed the mixed solution to a flow passage.
The second gradient pump 28 is able to mix an aqueous solution A 23 and an aqueous solution C 24 at a predetermined ratio for the ion-exchange column, and to feed the mixed solution to a flow passage.
The third gradient pump 29 is able to mix an aqueous solution D 25 and an organic solvent solution E 26 at a predetermined ratio for the reversed-phase column, and to feed the mixed solution to a flow passage.
The first 6-way flow passage switching valve 34 is able to switch over the flow passage between a flow passage A connecting the first gradient pump 27, the normal-phase column 31 and the ion-exchange column 32 in series and a flow passage B connecting the second gradient pump 28, the ion-exchange column 32 and the second 6-way flow passage switching valve 35 (reversed-phase trap column 33) in series.
The second 6-way flow passage switching valve 35 is able to switch over the flow passage between a flow passage A connecting the third gradient pump 29, the reversed-phase trap column 33 and the reversed-phase column 36 in series, and a flow passage B connecting the first 6-way flow passage switching valve 34 (ion-exchange column 32), the reversed-phase trap column 33 and the reversed-phase column 36 in series.
The operation of the three-dimensional liquid chromatography apparatus according to the second embodiment will be described below.
According to this second embodiment, the solution having a high salt concentration and eluted from the ion-exchange column can be prevented from being introduced to the reversed-phase column. When a mass spectrometer is employed as a detector, this second embodiment is effective in increasing detection sensitivity and improving maintainability of the apparatus. Incidentally, the component not retained in the ion-exchange column may flow out to the drain 38.
Number | Date | Country | Kind |
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2006-026488 | Feb 2006 | JP | national |