JP2902122B2 - Column packing and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a column packing and a method for producing the same. The column packing can be used, for example, for separating a biological sample or deproteinizing by liquid chromatography.

[0002]

2. Description of the Related Art It is well known to use chromatography, for example, high performance liquid chromatography (HPLC) technology, to analyze substances (eg, drugs) in biological samples. However, when analyzing a biological sample containing a large amount of protein such as serum, a procedure for deproteinization was required before the sample was subjected to chromatography. However, there are drawbacks in that the operation of deproteinization is complicated and adversely affects the analysis accuracy.

[0003] Therefore, there has been a demand for the development of a packing material for chromatography capable of directly processing a biological sample without performing a deproteinization operation. As such a filler, one that imparts different properties to the inner surface and outer surface of the pores of the porous carrier, allows small molecules to be adsorbed on the inner surface of the pores, and allows macromolecules such as proteins to pass through without being adsorbed. Proposed. The following belong to this, but all have drawbacks and have not been satisfactory yet.

(I) After a hydrophobic group is introduced into the inner and outer surfaces, the hydrophobic group on the outer surface is cut off using an enzyme that cannot enter the pores, and then a hydrophilic group is bonded to the outer surface. JP-A-61-65159 and JP-A-1-123145.
issue). This involves an enzymatic reaction, which complicates the process and tends to vary in quality.

[0005] (ii) and a method similar to the above (i), using a plasma instead of an enzyme (JP-A-62-158113). This has the same disadvantages as (i) due to the use of plasma.

(Iii) The modifying silane coupling agent is reacted under mild reaction conditions to bind the modifying group substantially only to the outer surface, and then reacted with another modifying silane coupling agent under strong reaction conditions. With a modifying group attached to the inner surface (Summary of the 38th Annual Meeting of the Japan Society for Analytical Chemistry, page 4 (19
1989), lecture number 1A04). This is because it is difficult to set conditions because there is little difference in reactivity between the inner and outer surfaces of the pores,
Products tend to vary.

(Iv) First, a 3-glycidoxypropyl group is introduced into the inner and outer surfaces, then a phenyl group is introduced, and then the glycidoxy group is hydrolyzed. (Chromatographia Vol. 29, pp. 223-227). This is because the reagents that can introduce phenyl groups without distinction on the inner and outer surfaces are limited, it is difficult to adjust the ratio of phenyl groups to diol groups, the product tends to vary, and the separation ability is not satisfactory.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a column packing material which does not have the above-mentioned drawbacks, has excellent resolving power, reproducibility and durability, and is easy to produce.

[0009]

That is, the present invention provides: (1) an alkylsilyl group (an aromatic group as a substituent or a group having a chiral resolving ability) on the inner and outer surfaces of pores of a porous carrier; ) Or an arylsilyl group and (ii) a 2,3-dihydroxypropoxy lower alkylsilyl group as a mixture, and (2) a porous carrier. Is reacted with (i) a lower alkylsilane coupling agent having a glycidoxy group, (b) then an alkyl group (which may contain an aromatic group as a substituent or a group having chiral resolving ability) or aryl (C) hydrolyzing the glycidoxy group to form a diol group, and (d) lowering the glycidoxy group. A method for producing a column packing material according to the above (1), wherein a silane coupling agent is reacted and a glycidoxy group is hydrolyzed at the same time or thereafter to form a diol group. (It may contain an aromatic group or a group having chiral resolving ability as a substituent) or a silane coupling agent having an aryl group. (D) A lower alkyl silane coupling agent having a glycidoxy group And producing a diol group by hydrolyzing a glycidoxy group at the same time or afterwards, to provide a method for producing a column packing material according to the above (1).

As the porous carrier, granules composed of an inorganic porous material having a hydroxy group, for example, silica,
Includes porous bodies made of porous glass, hydroxyapatite, diatomaceous earth and the like. The porous carrier usually has a particle size of 5
It has a specific surface area of about 200 to 500 m ² / g at −20 μm. Silica having a large pore diameter is preferable.

The alkyl in the above-mentioned alkylsilyl group is preferably one having 4 to 20 carbon atoms.
18 are more preferred, and 8-15 are most preferred.

The alkyl may be straight-chain and may have one or more, for example 1-2, branches. Also,
As the substituent, one or more, for example, one to two aromatic groups (preferably a monocyclic aromatic group having 6 to 12 carbon atoms, preferably 6 to 8 carbon atoms such as phenyl, tolyl, xylyl, cumenyl, etc.) Can have. Further, as a substituent,
One or more, for example, 1-2 chiral resolving groups such as (R) -N-3,5-dinitrobenzoylphenylglycine, 3,5-dinitrobenzoylnaphthylglycine or valine, t-leucine or proline A group derived from a urea derivative with naphthylethylamine).

Representative examples of alkyl include butyl, pentyl, hexyl, heptyl, isoheptyl, 2-ethylhexyl, octyl, isooctyl, nonyl, 2,4,5
Trimethylhexyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, 3-methyl-4-ethylundecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosanyl, and aromatic substituents thereof, such as benzyl, phenethyl,
Styryl, 2-phenylpropyl and the like, and these groups substituted with a group having chiral resolution.

As the aryl in the arylsilyl group, those described above as substituents on alkyl, that is, monocyclic aromatic groups having 6 to 12 carbon atoms (for example, phenyl, tolyl, xylyl, cumenyl, etc.) Phenyl optionally having 1-2 lower alkyl substituents).

The lower alkyl in the 2,3-dihydroxypropoxy lower alkyl group may be a lower alkyl having 2 to 2 carbon atoms.
6 are preferable, and 3-5 are more preferable.
It can also have branches. The 2,3-dihydroxypropoxy group can be attached at any position of the lower alkyl.

[0016]

[Production method] The column packing of the present invention can be produced, for example, by the methods shown in the above (2) and (3). These methods will be described in detail as follows.

Method (2): First, in step (a), a lower alkylsilane coupling agent having a glycidoxy group is reacted with a porous carrier. As the lower alkylsilane coupling agent having a glycidoxy group, a lower alkyl group having three or less, preferably one glycidoxy group is bonded to a silicon atom, and a leaving group (for example, halogen, A compound in which at least one lower alkoxy, 1- (tri-lower alkylsilyl) imino lower alkoxy, preferably lower alkoxy) is bonded, and if necessary, one or two lower alkyl groups are bonded is used. This reaction is preferably performed in a substantially anhydrous non-polar solvent (eg, toluene, xylene, etc.) under reflux.

Next, in the step (b), the product obtained above is reacted with a silane coupling agent having an alkyl group or an aryl group. As a silane coupling agent having an alkyl group or an aryl group, three
Or less, preferably one alkyl group or aryl group is bonded, and a leaving group (preferably halogen such as chlorine or bromine) is bonded to the remaining bond, and 1-2 lower alkyl groups are optionally bonded. A bound compound is used. This reaction is preferably performed in the presence of a condensing agent (for example, a base such as pyridi or triethylamine). As the solvent, a substantially anhydrous non-polar solvent is used. The reaction is preferably performed under reflux. The amounts of the porous carrier and the coupling agent in the above two reactions are not particularly limited, but usually, the amount of the surface silanol (for example, 8.5 μmol /
The amount of the coupling agent is suitably about 0.5 to 4.0 equivalents to m ² ). When the condensing agent is used, an amount of about 2.5 equivalents to the coupling agent is sufficient.

Next, in step (c), the glycidoxy group in the product obtained above is hydrolyzed. This reaction can be performed by a method commonly used for hydrolysis of a glycidoxy group, for example, by refluxing in an aqueous solution of an acid containing acetonitrile (for example, perchloric acid).

Finally, in step (d), the product obtained above is reacted with a lower alkylsilane coupling agent having a glycidoxy group, and the glycidoxy group is hydrolyzed. It is not impossible to carry out these two reactions in two steps in the same manner as described above.
It is convenient to carry out simultaneously in stages. In the case of one stage, when the reaction is carried out in an acidic aqueous solution (for example, an aqueous solution of perchloric acid), both reactions can be carried out simultaneously.

Method (3): First, in step (e), a silane coupling agent having an alkyl or aryl group is reacted with a porous carrier. This reaction can be carried out in the same manner as in the above step (b). Next, in step (f), the product obtained above is reacted with a lower alkylsilane coupling agent having a glycidoxy group, and the glycidoxy group is hydrolyzed. This reaction can be carried out in the same manner as in the reaction of the step (d).

In the above method, step (a),
(D) and (f) a lower alkylsilane coupling agent having a glycidoxy group and a silane coupling agent having an alkyl group or an aryl group in the steps (b) and (e) (particularly, By adjusting the number of moles of the coupling agent), the ratio of the alkyl or arylsilyl group and the 2,3-dihydroxypropoxy lower alkylsilyl group bonded to the column packing material can be arbitrarily adjusted. Further, by increasing the number of moles of the coupling agent in the steps (d) and (f), the ratio of the diol group on the outer surface to the inner surface of the pore can be increased.

[0023]

The column packing of the present invention can easily adjust the ratio of alkyl or aryl groups and diol groups to be introduced into the porous carrier to an appropriate ratio, so that macromolecules such as proteins are not adsorbed. A relatively small molecule such as a drug can be separated and adsorbed based on the difference in hydrophobicity. In addition, the production is easy and reproducible without using an enzyme or plasma for the production, a product having a constant quality can be obtained, and the durability is excellent because an unstable group is not bonded. Therefore, the column packing material of the present invention is useful and highly valuable for analyzing drugs and metabolites in biological samples containing proteins such as serum. In addition,
Among the above-mentioned production methods, the method (3) has a small number of steps, so that there is little variation in column characteristics between batches and excellent reproducibility.

[0024]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail based on embodiments.

Example 1 Preparation of silica having octylsilyl group and 2,3-dihydroxypropoxypropylsilyl group (1) Preparation of silica having 3-glycidoxypropylsilyl group 2 g of Develosil 60-5 silica After adding 120 ml of toluene and removing water by azeotropic distillation, 3-
3.8 ml of glycidoxypropyltrimethoxysilane was added and reacted at 90 to 95 ° C. for 6 hours. The reacted silica was collected by filtration and washed with 60 ml of toluene and 60 ml of methanol to obtain silica having a 3-glycidoxypropylsilyl group. (2) Production of silica having octylsilyl group and 3-glycidoxypropylsilyl group 2 g of silica having 3-glycidoxypropylsilyl group
After adding 80 ml of toluene and 2.4 ml of octyltrichlorosilane and 6 ml of pyridine,
Refluxed for 9 hours. The reacted silica derivative is filtered, and 60
Wash with ml toluene and 60 ml methanol,
Silica having an octylsilyl group and a 3-glycidoxypropylsilyl group was obtained. (3) Production of silica having octylsilyl group and 2,3-dihydroxypropoxypropylsilyl group 2 g of the silica derivative produced in the above (2) was treated with 100% aqueous solution of perchloric acid having a pH of 3.0 and containing 30% of acetonitrile.
and refluxed for 7 hours. 50 ml of acetonitrile
After washing with, a pH containing 10% acetonitrile was further added.
It was added to 100 ml of a 3.0 aqueous solution of perchloric acid and refluxed for 8 hours. The obtained silica derivative was washed with 60 ml of water and 60 ml of methanol to give an octylsilyl group and 2,2.
Silica having a 3-dihydroxypropoxypropylsilyl group was obtained. (4) Introduction of 2,3-dihydroxypropoxypropylsilyl group again 4.64 ml of 2 g of the silyl derivative obtained in the above (3)
Containing 3-glycidoxypropyltrimethoxysilane
The solution was added to a perchloric acid aqueous solution having a pH of 3.5 and reacted at 90 ° C. for 4 hours. The obtained silica derivative was washed with 60 ml of water and 60 ml of methanol to obtain a target filler.

Example 2 Preparation of silica having butylsilyl group and 2,3-dihydroxypropoxypropylsilyl group (1) Preparation of silica having 3-glycidoxypropyl group 3 was prepared in the same manner as in (1) of Example 1. -A silica having glycidoxypropyl groups was obtained. (2) Production of silica having butyl group and 3-glycidoxypropyl group 2 g of the silica derivative produced in the above (1) and toluene 80
After adding 1.73 ml of butyltrichlorosilane and 6 ml of pyridine, the mixture was refluxed for 9 hours. The reacted silica derivative was collected by filtration, and 60 ml of toluene and 6 ml of toluene were removed.
After washing with 0 ml of methanol, butylsilyl group and 3
-Silica having glycidoxypropylsilyl groups was obtained. (3) Production of silica having butylsilyl group and 2,3-dihydroxypropoxypropylsilyl group 2 g of the silica derivative produced in the above (2) was mixed with 100 ml of a perchloric acid aqueous solution containing 5% acetonitrile and having a pH of 3.0.
And refluxed for 7 hours. After washing with 50 ml of acetonitrile, the solution was further adjusted to pH 3 containing 5% acetonitrile.
0 perchloric acid aqueous solution (100 ml) and refluxed for 8 hours. The obtained silica derivative was washed with 60 ml of water and 60 ml of methanol to obtain a silica derivative having a butylsilyl group and a 2,3-dihydroxypropylpropoxysilyl group. (4) Introduction of 2,3-dihydroxypropoxypropylsilyl group again 1.16 ml of 2 g of the silica derivative obtained in the above (3)
Containing 3-glycidoxypropyltrimethoxysilane
The solution was added to a perchloric acid aqueous solution having a pH of 3.5 and reacted at 90 ° C. for 4 hours. The obtained silica derivative was washed with 60 ml of water and 60 ml of methanol to obtain a target filler.

Example 3 Analysis of Drug by Direct Injection of Serum Sample The filler prepared in Example 1 was used with an inner diameter of 4.6 mm and a length of 5 cm.
In a stainless steel tube for HPLC. Using this column, human control serum (A) and phenobarbital (20 μg / ml) added to human control serum (B)
Was examined for separation. As the mobile phase, 100 mM NaH ₂ P
O ₄ -100 mM Na ₂ HPO ₄ —CH ₃ CN (4.25-4.25-1.5)
Was sent at 0.6 ml / min, and detection was performed at 254 nm. The injection volume was 10 μl. The obtained chromatograms are shown in FIG. 1 and FIG. In FIG. 1, the peak of human serum protein eluted immediately after injection. FIG. 2 shows that the peak of febarbital eluted after the human serum protein, and was well separated from the serum component.

Example 4 Preparation of Hybrid Functional Phase Silica Having Phenyl Group and Diol Group (1) Preparation of Silica Having Phenyl Group To 3.8 g of Devosyl 90-5 silica, 200 m of toluene was added.
After water was removed from the azeotrope, 1.42 ml (5 μmol / m ² ) of phenyltrimethoxysilane and 1.51 ml of pyridine were added, and the mixture was reacted under reflux for 7 hours. The reacted silica was collected by filtration and washed with 60 ml of toluene and 60 ml of methanol to obtain silica having a phenyl group. (2) Production of silica having phenyl group and 2,3-dihydroxypropoxypropyl group 1.4 g of the silica derivative obtained in the above (1) was 2.1 m
l of 3-glycidoxypropyltrimethoxysilane in a pH 3.5 aqueous solution, and reacted under reflux for 5 hours. The obtained silica derivative was washed with 60 ml of water and 60 ml of methanol to obtain a target filler.

Example 5 Analysis of Drug by Direct Injection of Serum Sample The filler prepared in Example 4 was used with an inner diameter of 4.6 mm and a length of 5 mm.
cm HPLC stainless steel tube. Using this column, separation of human control serum (C) and human control serum to which phenytoin (25 μg / ml) and carbamazepine (5 μg / ml) were added as a standard (D) were examined. The mobile phase was 100 mM NaH ₂ PO ₄ -100 m
_{M Na 2 HPO 4 -CH 3 CN} (4.75-4.75-
0.5) at a flow rate of 0.6 ml / min.
Performed in The injection volume was 20 μl. The obtained chromatograms are shown in FIGS. In FIG. 3, the human serum protein peak eluted immediately after injection.
In FIG. 4, it can be seen that the peaks of phenytoin and carbamazepine elute after the peak of the human serum protein and are well separated from the serum components.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of analyzing human serum using the column packing material of the present invention.

FIG. 2 shows the results of analysis of human serum containing phenobarbital using the column packing material of the present invention.

FIG. 3 is a diagram showing the results of analyzing human serum using the column packing material of the present invention.

FIG. 4 is a view showing the results of analysis of human serum containing phenytoin and carbamazepine using the column packing material of the present invention.

[Explanation of symbols]

 1 serum protein 2 phenobarbital 3 serum protein 4 phenytoin 5 carbamazepine

Continuation of the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) G01N 30/48 B01D 15/08 B01J 20/22 CA (STN)

Claims (3)

(57) [Claims] 1. The method according to claim 1, wherein the inner and outer surfaces of the pores of the porous carrier are
(I) an alkylsilyl group (which may contain an aromatic group as a substituent or a group having chiral resolving ability) or an arylsilyl group; and (ii) a 2,3-dihydroxypropoxy lower alkylsilyl group. A column packing material characterized by being introduced. 2. A porous carrier is reacted with (i) a lower alkylsilane coupling agent having a glycidoxy group, and (b) then an alkyl group (an aromatic group as a substituent or a group having chiral resolving ability) Or a silane coupling agent having an aryl group; (c) hydrolyzing a glycidoxy group to form a diol group; and (d) reacting a lower alkyl silane coupling agent having a glycidoxy group. 2. The method for producing a column packing according to claim 1, wherein the glycidoxy group is hydrolyzed simultaneously or thereafter to form a diol group. 3. A silane coupling agent having an (e) alkyl group (which may contain an aromatic group as a substituent or a group having a chiral resolving ability) or an aryl group is reacted with the porous carrier; (F) Then, a lower alkylsilane coupling agent having a glycidoxy group is reacted, and the glycidoxy group is hydrolyzed at the same time or afterwards to produce a diol group. Production method.