Chemistry: analytical and immunological testing – Including chromatography
Reexamination Certificate
2000-02-10
2002-12-24
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Including chromatography
C073S061550, C073S061560, C073S061590, C210S198200, C210S656000, C210S662000, C324S307000, C422S070000, C436S173000, C436S178000
Reexamination Certificate
active
06498040
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
This invention relates to process and apparatus for mobile phase conversion in a high-performance liquid chromatography to achieve efficient separation and preparation of a sample for nuclear magnetic resonance (NMR) analysis.
2. Description of the Prior Art
The high-performance liquid chromatography (referred to hereinafter as HPLC, in certain cases) has usually been utilized as the technique to analyze a trace amount of ingredient contained in a sample.
Recently, the system has been also proposed, which comprises this HPLC combined with the mass spectrometer so that separation and identification of an ingredient can be achieved at once.
For example, Japanese Patent Application Disclosure Gazette No 1991-175355 describes the process and apparatus for mobile phase conversion in mass spectrometry utilizing the high-performance liquid chromatography. According to this disclosure, a target ingredient contained in the sample is trapped by the high-performance liquid chromatography and this target ingredient is transferred to the mass spectrometer by using the mobile phase which is different from the mobile phase for separation of the sample and suitable for the mass spectrometry.
Structure analysis utilizing NMR requires at least several hundred micrograms of sample and an initial sample as much as several hundred milligrams is required to separate, for example, 0.1% of impurities contained in drug substance.
Conventionally, separation of a NMR sample from a given initial sample has been carried out several times on each divided amount of said initial sample. Such procedure has disadvantageously taken much time to concentrate the NMR sample and, if the NMR sample is unstable, decomposition thereof has sometimes occurred during the step of concentration.
In NMR analysis, deuterated solvent such as deuterium oxide or deuterated methanol having every hydrogen atom replaced by deuterium which is its isotope. In view of the fact that such solvent is considerably expensive, there is a serious demand that the sample for NMR analysis should be prepared as efficiently as possible.
While the system comprising the high-performance liquid chromatography combined with NMR is well known as has already been described. However, it has been impossible for this system of well known art to inject an adequate amount of sample into the system because this system uses HPLC for analysis. Therefore, it has been difficult to isolate a trace amount, e.g., 0.1% of impurities contained in drug substance and to analyze its structure. Furthermore, a probe exclusively used for this purpose must be separately purchased and correspondingly increases a cost to use this system of prior art. From the viewpoint of the cost, there is a demand for a more simple and more convenient system.
In view of the problem of the prior art as has been described, it is a principal object of this invention to provide a system enabling separation, desalting, concentration and deuterium replacement of a trace amount of ingredient contained in a mixture to be achieved in on-line fashion particularly using an extremely small amount of expensive deuterated solvent such as deuterium oxide or deuterated methanol.
SUMMARY OF THE INVENTION
The object set forth above is achieved, according to one aspect of this invention, by a Process for mobile phase conversion in high-performance liquid chromatography to carry out separation and preparation of a sample for NMR analysis comprising steps of: separating ingredients contained in the sample by the high-performance liquid chromatography; trapping said ingredients in a trapping column using a different mobile phase; replacing water by deuterium oxide; and eluting target ingredients from said trapping column using a deuterated solvent other than said deuterium oxide.
The object set forth above is achieved, according to another aspect of this invention, by a high-performance liquid chromatography apparatus for separation and preparation of a sample for NMR analysis comprising; a separation/sampling section serving for separation of ingredients contained in the sample using high-performance liquid chromatography; a trapping section serving to trap said ingredients in a trapping column using a different mobile phase; a deuterium oxide replacement section serving to replace water by deuterium oxide; and a deuterated solvent supplying section serving to supply a deuterated solvent other than said deuterium oxide and thereby to elute the target ingredients from said trapping column.
The novel process enables separation and preparation of a target compound suitable for NMR analysis to be simultaneously as well as efficiently achieved. This is owing to the steps of concentration and mobile phase conversion carried out during separation of a trace amount of ingredient contained in a sample by high-performance liquid chromatography.
According to one preferred embodiment of the high-performance liquid chromatography apparatus provided in the form of a system including pumps, injector, separation column, switching valves, distribution valve, sampling loop and trapping column, said system comprising: a separation/sampling section comprising a pump (P
1
) actuated to supply a mobile phase for separation of the sample, a detector to detect a peak of a desired compound, and valves (V
1
), (V
2
), (V
3
), wherein said desired compound flows through said valve (V
1
) into a loop (LOOP) extending between said valve (V
2
) and said valve (V
3
) so as to be accumulated in said loop (LOOP); a trapping section comprising a pump (P
2
) actuated to supply a mobile phase for concentration of the sample so as to flow through said valve (V
1
), said valve (V
2
), said loop (LOOP), said valve (V
3
), a valve (V
4
), a valve (V
5
), a trapping column (TC) and said valve (V
4
) in this order, and a distribution valve (R) serving to mix the flow with said mobile phase for concentration of the sample between said valve (V
3
) and said valve (V
4
) so that the desired compound may be concentrated in said trapping column (TC); a deuterium oxide replacement section comprising a pump (P
3
) actuated to supply deuterium oxide so as to flow said valve (V
5
), said trapping column (TC) and said valve (V
4
) in this order and to replace water by deuterium oxide; and a deuterated solvent supplying section comprising a pump (P
4
) actuated to supply deuterated solvent other than deuterium oxide so as to flow through said valve (V
4
), said trapping column (TC), said valve (V
5
), said valve (V
4
), said analytical column (AC) and a detector (D
2
) so that the desired compound may be obtained for NMR analysis.
The term “pump” used herein should be understood to be the liquid pump exclusively used for high-performance liquid chromatography. The term “valve” used herein should be understood to be the 6-way valve or the like usually used for high-performance liquid chromatography.
The term “analytical column” used herein should be understood to include various separation columns usually used for high-performance liquid chromatography. Such preparative column is not limited to so-called normal column, reverse column, GPC column or the like. The column may be selected from the various columns so far it is effective to separate a trace amount of target ingredient contained in a sample.
The term “trapping column” used herein should be understood to be. the column adapted to trap and concentrate the target ingredient. This is not limited to the column exclusively used for high-performance liquid chromatography but any other column may be used so far as it has a desired pressure resistance .
The term “sampling loop” used herein should be understood to be a capillary tube adapted to accumulate ingredient separated from the sample by the separation column and, in most cases, stainless tube is used for this purpose. Length of this tube may be adjusted depending on factors such as a concentration of the ingredient.
The pumps, the columns, the valves and the like are usually connected one to another by the s
Asakawa Naoki
Kishi Naoya
Tanaka Masayuki
Yokoyama Yumi
Birch & Stewart Kolasch & Birch, LLP
Eisai Co. Ltd.
Ludlow Jan
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