Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2001-03-09
2003-03-18
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S656000, C210S198200, C210S502100, C502S402000, C502S439000
Reexamination Certificate
active
06533939
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a packing agent for reversed phase liquid chromatography, a production process of the packing agent, a column for reversed phase liquid chromatography using the packing agent, and an analysis method by reversed phase liquid chromatography using the packing agent.
BACKGROUND OF THE INVENTION
The separation system of liquid chromatography includes gel permeation, partition adsorption, ion exchange, ion pair, ligand exchange and affinity. An optimal system is selected according to the properties of the substance to be separated. Among these, the partition adsorption is most widely used and this includes a normal phase mode and a reversed phase mode. In the normal phase mode, the separation occurs due to the hydrophilic interaction between the packing agent and the substance to be separated, and in the reversed phase mode, the separation takes place due to the hydrophobic interaction between the packing agent and the substance to be separated. In actual practice, however, the reversed phase mode is preferred because the mobile phase can be selected from a wide range and good resolution can be easily obtained. In many cases, the partition adsorption may be considered to indicate the reversed phase mode. The column used for the separation in this reversed phase mode is a column for reversed phase liquid chromatography and the separating agent filled thereinto is a packing agent for reversed phase liquid chromatography.
The column for reversed phase liquid chromatography includes silica type and polymer type columns. Most commercially available products are silica type and among these, a column called ODS column obtained by chemically bonding an octadecyl group to the surface of silica gel is overwhelmingly predominant, and is being used in various industrial and academic fields over a wide range. However, the silica-type packing agent used in the ODS column is found to have problems in that the acid resistance/alkali resistance is not sufficiently high and a basic substance readily adsorbs thereto. In recent years, investigations have been made on various techniques to overcome this problem. One of these methods is to use a polymer-type packing agent in place of the silica-type packing agent. Since the above-described problem is greatly attributable to the base material silica gel, the exchange of the base material itself is expected as a most essential countermeasure.
Examples of the polymer-type column for reversed phase liquid chromatography heretofore available on the market or reported include the following:
(1) styrene-divinylbenzene type crosslinked polymer particle (e.g., Shodex (trademark of Showa Denko K.K.) RSpak RP18-413 produced by Showa Denko K.K.);
(2) methacrylate-type crosslinked polymer particle (e.g., Shodex (trademark of Showa Denko K.K.) RSpak DE-413 produced by Showa Denko K.K.);
(3) poly(vinyl alcohol)-type crosslinked polymer particle having chemically bonded thereto a long-chain acyl group (e.g., Shodex (trademark of Showa Denko K.K.) Asahipak ODP-50 4D produced by Showa Denko K.K.); and
(4) hydroxyl group-containing methacrylate-type crosslinked polymer particle having chemically bonded thereto a long chain acyl group (see, JP-A-4-58154 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”)).
These polymer-type columns for reversed phase liquid chromatography are generally excellent in the acid resistance/alkali resistance, and therefore, one of the problems of the ODS column is solved. However, another problem that a basic substance readily adsorbs still remains unsolved. In addition, the polycyclic aromatic compound is liable to have a broad peak.
The polycyclic aromatic group of the polycyclic aromatic compound is very popular as a partial structure of naturally occurring products or medicaments and it is disadvantageous if the polycyclic aromatic group cannot be sharply separated. Unless separation performance equal to or higher than that of the ODS column can be obtained for many compounds, the polymer-type column for reversed phase liquid chromatography cannot have a truly elevated value in practical use despite its excellent acid resistance/alkali resistance. Therefore, it is necessary for separation performance to be equal to or higher than that of the ODS column.
In conventional polymer-type columns for reversed phase liquid chromatography, polycyclic aromatic compounds are considered to give a broad peak because of the following reasons. The particle has an internal structure such that flat and slit-like hydrophobic micropores are readily formed and a polycyclic aromatic compound easily fits into the pore present. Furthermore, the hydrophobicity is almost the same between the outside and the inside of the particle. Therefore, a great difference is generated in the migration speed between the molecule that permeated into the inside and is “fitted” and the molecule “slipped” on the outside. This is individually verified below for the above-described four conventional techniques (1) to (4).
In the styrene-divinylbenzene type crosslinked polymer particle of (1), the skeleton itself of the packing agent is an aromatic ring. Therefore, the interaction between the hydrophobic micropore and the polycyclic aromatic compound is highest and the peak is very largely broadened.
In the methacrylate-type crosslinked polymer particle of (2), the skeleton does not contain an aromatic ring; however, an interaction is still present between the hydrophobic micropore surrounded by a carbonyl group and the polycyclic aromatic compound. Therefore, the peak is also broadened.
In the poly(vinyl alcohol)-type crosslinked polymer particle having chemically bonded thereto a long chain acyl group of (3), a non-saponified acetyl group remains in some hydrophobic micropores, and therefore, the peak is also broadened.
In the hydroxyl group-containing methacrylate-type crosslinked polymer particle having chemically bonded thereto a long-chain acyl group of (4), the inside is uniformly hydrophilized by the unreacted hydroxyl group. Therefore, even if the polycyclic aromatic compound migrates into the inside, the residence time is probably short because of low interaction. As a result, the hydrophobic interaction is limited to occur only between the polycyclic aromatic compound and the long-chain acyl group on the surface, and the peak is inhibited from broadening.
However, when the method of (4) was actually performed by varying the conditions in several ways, it was difficult to form a sharp peak for the polycyclic aromatic compound. From this result, the present inventors thought that another important factor must be present which was not taken into account in conventional techniques.
The object of the present invention is to elucidate the factor for inhibiting the aromatic compound from forming a sharp peak and provide specific means capable of actually forming a sharp peak for polycyclic aromatic compounds.
As described above, JP-A-4-58154 describes a crosslinked copolymer particle obtained by chemically bonding a long-chain acyl group to a hydroxyl group-containing methacrylate-type crosslinked polymer particle, which is excellent in the acid resistance and the alkali resistance and exhibits very small difference in the swelling/shrinkage among various solvents. However, this crosslinked polymer particle is not disclosed as forming a sharp peak for polycyclic aromatic compounds, and in the tests performed by the present inventors, such a phenomenon was not verified.
As a result of extensive investigations to overcome the above-described problems, the present inventors concluded that the pore size of the base particle is generally too large and this is one of the factors causing the problems. More specifically, if in the base particle used, pores have a sufficiently large size for the bulk of the long-chain acyl group-introducing reagent to occupy almost all of the pores, the long-chain acyl group is introduced into the inside (deeply) of the particle. As a result, the object that on
Mano Keisuke
Shimbo Kuniaki
Showa Denko Kabushiki Kaisha
Therkorn Ernest G.
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