JP3298260B2 - Chromatographic packing material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatographic packing material, and is mainly used for high performance liquid chromatography (HPLC) in the fields of pharmacy, medicine and science and engineering.

[0002]

2. Description of the Prior Art Hitherto, silica gel fillers in which a dye system has been introduced have not been known. It has recently been found that glass can be easily colored with organic dyes by the sol-gel coating method. (Nakazumi
Chem. Express 6,607 (1991)) However, few attempts have been made to bind organic dyes covalently to silica gel, which is structurally very similar to glass. So far, a gel in which Rhodan B (RB) is simply physically adsorbed on a silica gel surface (CMKrishna et al. Photochem. Photobiol 45, 1 (198
7)) and a gel in which halogen was introduced into silica gel with a silane coupler and then RB was bonded in anticipation of the development of the sensitizing oxidation effect of the dye -O ₂ (Tamagaki et al., J.org.chem. 45, 1).
573 (1980)). Moreover, none of these are aimed at developing a filler for HPLC.

[0003]

Under these circumstances, the present inventors prepared a new HPLC filler having a dye-based π-electron system and examined its performance. As a result, 3-aminopropylsilyl silica gel and two kinds of organic dyes (5-amino-2,3-dicyano-1,4-naphthoquinone and 1,4-diamino-9,10-anthraquinone-2,
3-dicarboxylic anhydride) and the resulting chromatographic packings have been found to have an excellent effect on the separation of nucleic acid-related substances, thereby completing the present invention. That is, the present invention provides a compound represented by the general formula (I)

[0004]

Embedded image (Wherein R represents a group of the following formula (II) or (III))

[0005]

Embedded image

[0006]

Embedded image A chromatographic filler characterized in that the amino derivative represented by the formula (1) is grafted on an inorganic carrier having a hydroxyl group on its surface, and further comprising a chromatographic method for separating nucleic acid-related substances using the chromatographic filler. Is provided.

[0007] The chromatographic filler of the present invention can be produced as follows. That is, by heating 3-aminopropyltriethoxysilane, which is a so-called silane coupling agent, with an inorganic carrier having a hydroxyl group on the surface thereof in the presence or absence of an organic solvent, an aminopropylsilyl group is formed on the surface. The resulting inorganic carrier is obtained. An inorganic carrier having an aminopropylsilyl group on the surface is treated with 5-amino-2,3-dicyano-1,4-naphthoquinone (organic dye (I)) or 1,4-diamino-9,10-anthraquinone-2,3 By heating in the presence of an organic solvent together with -dicarboxylic anhydride (organic dye (II)), the desired chromatographic filler is obtained.

As the inorganic carrier having an aminopropylsilyl group on the surface used in the present invention, silica gel, alumina gel, zirconia gel, glass beads and the like can be mentioned. Among them, silica gel is preferable. The aminopropylsilyl group on the surface reacts with the organic dyes (I) and (II) to be useful for grafting the organic dyes (I) and (II) to the inorganic carrier. The shape of the inorganic carrier is spherical,
Although any form such as a crushed form may be used, fine particles having a uniform particle size are preferred in order to obtain a highly efficient chromatography column. Usually the average particle size is 4 to 310μ
m, preferably 4.5 to 44 μm.

As the organic solvent, benzene, toluene,
Xylene, chlorobenzene, hexane, cyclohexane, chloroform, tetrahydrofuran, dimethylformamide, dimethylsulfoxide and the like can be mentioned. The reaction temperature is preferably in the range of 160C to 180C. After the reaction, the reaction mixture is cooled to room temperature, filtered, washed with an organic solvent such as methanol, toluene, acetone or the like or pure water, and dried under normal pressure or reduced pressure to obtain the chromatographic filler of the present invention.

The filler having a π-electron system of the present invention is packed in a chromatography column according to a conventional method and used as a stationary phase in liquid chromatography. In liquid chromatography using this stationary phase, by selecting appropriate elution conditions, separation and analysis of nucleic acid-related substances can be performed with high resolution in a short time. As the mobile phase used in the chromatography method of the present invention, water or an aqueous solution in which acetonitrile, methanol, ethanol, or the like is dissolved in water, or a phosphate buffer in which phosphate-phosphate or the like is further dissolved in water or water. And the like, and among them, 10 to 60 in the chromatography method of the present invention.
(V / V%) It is preferable to use a 0.015M to 0.065M phosphate buffer containing methanol. The aqueous phosphate buffer solution is usually an aqueous solution comprising phosphoric acid, sodium hydrogen phosphate, and sodium dihydrogen phosphate, and can be arbitrarily adjusted within the range of pH 2 to 9 by changing each component.

The nucleic acid-related substances which can be separated by the chromatography method of the present invention include nucleic acid bases such as adenine, guanine, cytosine, uracil and thymine; guanosine;
Nucleosides such as adenosine, cytidine, uridine, deoxyguanosine, deoxyadenosine, deoxycytidine, thymidine, etc., adenosine 2 'monophosphate, adenosine 3' monophosphate, adenosine 5 'monophosphate, adenosine 5' diphosphate, adenosine 5 'Triphosphate, cytidyl 5' monophosphate, cytidyl 5 'diphosphate, cytidyl 5' triphosphate, uridine 5 'monophosphate, uridine 5' diphosphate, uridine 5 'triphosphate, guanyl 2' Monophosphate, guanyl 3'-monophosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, coenzyme A and the like.

[0012]

EXAMPLES The present invention will be described below with reference to examples. % Of cases
Is by weight unless otherwise specified. Example 1 1.109 g of 5-amino-2,3-dicyano-1,4-naphthoquinone was placed in a pressure decomposition vessel in dimethylformamide (DMF) 3
Aminopropylsilyl silica gel (manufactured by Daiso Co., Ltd .;
m, average pore size 120 angstrom) and heated in an oven at 160 ° C for 3 hours. After cooling, the reaction product was collected by filtration, washed with DMF and then with methanol,
Drying under reduced pressure (1 mmHg) at 0 ° C. for 3 hours gave a modified silica gel. Yield 6.422g. The elemental analysis value of the obtained gel was C
= 8.80%, H = 1.10%, N = 2.70%, and the modification rate was 36%.

Example 2 Except that 1.460 g of 1,4-diamino-9,10-anthraquinone-2,3-dicarboxylic anhydride was used instead of 5-amino-2,3-dicyano-1,4-naphthoquinone It was reacted with 5.000 g of aminopropylsilyl silica gel in the same manner as in Example 1 to obtain a modified silica gel. Yield 5.022g. The elemental analysis value of the obtained gel was C = 13.52%, H = 1.
14%, N = 2.70%, and the modification rate was 51%.

Example 3 The filler obtained in Example 1 was used with an inner diameter of 4.6 mm and a length of 150 mm.
Was packed in a slurry using a glycerin-methanol (volume ratio of 1: 1) solution and analyzed under the following conditions to obtain a chromatogram of FIG. Temperature Room temperature Mobile phase Methanol / 1 / 30M phosphate buffer (pH7) =
27/73 (V / V%) Flow rate 1 ml / min Detector Ultraviolet absorption spectrometer (wavelength 254 nm) A mixture of the following compounds was used as an analyte. 1. adenosine 5 'monophosphate, 2. adenosine 5' diphosphate, 3. adenosine 5 'triphosphate, 4. adenosine,
5. Adenine

Example 4 The filler obtained in Example 2 was used with an inner diameter of 4.6 mm and a length of 150 mm.
A glycerin-methanol (volume ratio: 1: 1) solution was used to slurry-fill a stainless steel column with methanol / 1 / 30M phosphate buffer (pH 7) = 20 as the mobile phase.
Analysis was performed under the same conditions as in Example 3 except that / 80 (V / V%) was used, and a chromatogram of FIG.

[0016]

According to the chromatographic method using the chromatographic packing material of the present invention, nucleic acids such as adenosine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-triphosphate, adenosine and adenine can be used. It is possible to separate substances with high resolution and in a short time.

[Brief description of the drawings]

FIG. 1 shows the chromatogram obtained in Example 3.

FIG. 2 shows a chromatogram obtained in Example 4.

[Explanation of symbols]

 1. Adenosine 5 'monophosphate, 2. Adenosine 5' diphosphate, 3. Adenosine 5 'triphosphate, 4. Adenosine, 5. Adenine

──────────────────────────────────────────────────続 き Continued on the front page (56) References Shuzo Akiyama, Development of 3- (N-substituted) aminopropylsilyl silica gel with π-electron system, Analytical Chemistry, Japan Society for Analytical Chemistry 1993 Dec. 42, No. 12, 817-823 Stwart, Development of an Enzyme-Linked Immunosorbent Assay for C.I. I. Reactive Blue 2, Journal of Chromatography, 1992, Vol. 633, 1-14 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 30/48 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A compound of the general formula (I) Wherein R represents a group of the following formula (II) or (III): Embedded image A chromatographic filler characterized in that the amino derivative represented by the formula is grafted on an inorganic carrier having a hydroxyl group on its surface. 2. The method according to claim 1, wherein the inorganic carrier is silica gel.
A chromatographic filler as described.