Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
1999-09-20
2001-06-05
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200, C210S502100
Reexamination Certificate
active
06241891
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of using, in a reversed phase chromatography, a stationary phase comprising an alkyl group as a main component thereof, with a mobile phase comprising water as a main component thereof, and also relates to a reversed phase chromatograph apparatus comprising a stationary phase comprising an alkyl group as a main component thereof and a mobile phase comprising water as a main component thereof.
2. Related Art Statement
There are known a reversed phase chromatography in which water is used as a main component of a mobile phase as a solvent for dissolving a sample including a water soluble compound to be separated, and a non-polar compound is used as a main component of a liquid or solid stationary phase supported by a solid support filled in a column, and a reversed phase chromatograph apparatus comprising the mobile phase and the stationary phase. In view of hydrophobicity and compound-retention capacity, an alkyl group having 8 carbon atoms (octyl group) to 18 carbon atoms (octadecyl group; hereinafter, referred to the “ODS”) has been widely used as the stationary phase.
However, in the case where an alkyl group having 8 to 18 carbon atoms is used as the stationary phase, if 100% of water is used as the mobile phase for the purpose of separating a water soluble compound, a time-wise change occurs that the sample-retention or compound-retention time between the time when a sample is injected into the column and the time when the compound is detected under the column gradually decreases.
FIG. 1
shows three chromatograms for illustrating a time-wise change that was observed when an identical sample is used at three different times, each for separating five sorts of compounds. The ODS (octadecyl group) is used as the stationary phase, and 100% of water is used as the mobile phase. The three chromatograms of
FIG. 1
prove that as time passes, the compound-retention time of the column, i.e., the stationary phase gradually decreases, which leads to an incomplete separation of the compound. Thus, in the case where 100% of water is used as the mobile phase, the ODS cannot be used as the stationary phase. It is thought that the above time-wise change would result from the “slipping” phenomenon that as the 100% water mobile phase iteratively passes through the column, the carbon chains (ligands) gradually lie on their sides, as illustrated in
FIG. 2
, and the interaction between the stationary phase and the mobile phase gradually decreases.
For preventing the above time-wise change, it has been proposed to use, as the stationary phase, an alkyl group having a smaller number of carbon atoms that is physically thought to less likely lie on its side.
FIG. 3
shows three chromatograms in which trimethylsilyl group is used as the stationary phase, 100% water is used as the mobile phase, and the same sample as used in those shown in
FIG. 1
is used at three different times. The three chromatograms of
FIG. 3
prove that the compound-retention time of trimethylsilyl group used as the stationary phase does not decrease even though it may be used for a long time. It has also been proved that in the case where phenyl group is used as the stationary phase, its compound-retention time does not decrease. Those proofs also support that the above time-wise decrease in the compound-retention time of the alkyl group having 8 to 18 carbon atoms used as the stationary phase results from the “slipping” phenomenon.
However, in the case where an alkyl group having a small number of carbon atoms is used as the stationary phase, the stationary phase cannot completely separate different isomers from each other, because the alkyl group has only a poor steric selectivity. In addition, since the alkyl group has only a low hydrophobicity, it cannot sufficiently separate certain compounds from samples. Meanwhile, a stationary phase in which ODS is bonded at a lower density to the solid support and trimethylsilyl group is additionally bonded with the solid support, has been developed. This stationary phase enjoys the advantage of excellent compound-retention capacity of ODS and the advantage of trimethylsilyl group that a solution including water as a main component can be used as the mobile phase. This stationary phase has solved the problem of the time-wise decrease of compound-retention time to some degree, but not a satisfactory degree. In addition, since the amount of trimethylsilyl group is more, i.e., the amount of long alkyl group is less, the hydrophobicity of this stationary phase is lower than that of the prior stationary phase in which only the long alkyl group is used. Thus, this stationary phase suffers from the problems of low compound-retention capacity, insufficient compound-separate capability, and low steric selectivity.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method of using, in a reversed phase chromatography, a stationary phase exhibiting a high hydrophobicity and a great compound-retention capacity, with a mobile phase comprising water as a main component thereof.
It is another object of the present invention to provide a reversed phase chromatograph apparatus comprising a stationary phase exhibiting a high hydrophobicity and a great compound-retention capacity, and a mobile phase comprising water as a main component thereof.
In the above-indicated background, the Inventor has performed various studies and has found that in the case where an alkyl group having a greater number of carbon atoms than that of the alkyl group whose compound-retention time time-wise decreases is used as the stationary phase and a solution including water as its main component is used as the mobile phase, the compound-retention time of the stationary phase does not time-wise decrease. As indicated above, it has been thought that the time-wise decrease of compound-retention time of the stationary phase would result from the “slipping” phenomenon that the carbon chains time-wise lie on their sides because of their hydrophobicity. Accordingly, it has also been thought that even though an alkyl group having a greater number of carbon atoms may be used as the stationary phase, with the mobile phase including water as its main component, the “slipping” phenomenon would naturally occur because the carbon chains time-wise lie on their slides and accordingly the compound-retention time of the stationary phase would time-wise decrease. In fact, however, no time-wise decrease of the compound-retention time of the stationary phase occurs. The present invention has been developed based on this finding.
The present invention provides a reversed-phase-chromatography stationary-phase using method and a reversed phase chromatograph apparatus which have one or more of the technical features that are described below in respective paragraphs given parenthesized sequential numbers (1) to (17). Any technical feature which includes another technical feature shall do so by referring, at the beginning, to the parenthesized sequential number given to that technical feature.
(1) According to a first feature of the present invention, there is provided a method of using, in a reversed phase chromatography, a stationary phase comprising, as a main component thereof, an alkyl group having not less than 24 carbon atoms, with a mobile phase comprising water as a main component thereof. In the present chromatograph method, the compound-retention time of the stationary phase does not change as time passes. The alkyl group having 24 or more carbon atoms as the main component of the stationary phase has great hydrophobicity and steric selectivity that have not been available from a conventional stationary phase which is used with a mobile phase comprising water as its main component. Thus, the present method enjoys a great compound-retention ability or capacity, and accordingly can easily separate a certain compound from a sample, or separate different isomers from each other, though it has been impossible
Nomura Chemical Co., Ltd.
Oliff & Berridg,e PLC
Therkorn Ernest G.
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