JPH03107759A - Manufacturing method of column packing material - Google Patents

Abstract

PURPOSE:To improve durability and separability by modifying the inside and outside surfaces of the pores of a porous carrier respectively with the residual groups of silane coupling agents having a substituent exhibiting an aryl group or a chiral group and a hydrophilic property. CONSTITUTION:The residual group of the silane coupling agent with which the inside surface of the pores of the porous carrier is modified and which has the aryl group or the substituent exhibiting a chiral separating power acts to efficiently separate the various relatively small molecules, such as drugs, which can infilter the inside of the fine pores based on the difference in the degree of the hydrophobicity thereof or the difference in the stablizability of a clathrate complex or diastereomer complex. On the other hand, the residual group of the silane coupling agent with which the outside surface of the pores is modified and which has the hydrophilic group acts to prevent the adsorption of the macromoleculees of protein, etc., on the porous carrier and the protection of the carrier. The sepn. and analysis of the drugs, etc., with the high accuracy are executed over a long period of time by the combination of the above- mentioned effects. The direct sepn. of serum, etc., is possible as well.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a column packing material. For more details,
The present invention relates to a column packing material suitable for separation analysis and protein removal treatment of biological samples by liquid chromatography.

(b) Prior Art Conventionally, chromatography, particularly high performance liquid chromatography (hereinafter referred to as IPLc), has been widely used for analyzing drugs in biological samples such as serum. However, HPLC analysis of drugs in biological samples containing large amounts of proteins such as serum
Protein removal operations are indispensable in flowering, and such pretreatment operations have the drawbacks of requiring time and effort and impairing analytical precision or accuracy.

Therefore, in recent years, serum samples have been directly injected without protein removal, and various drugs contained in these samples have been separated and
Fillers for chromatography have been developed and are now in use because they allow quantitative determination. These fillers use porous glass or silica gel as a carrier and provide different properties inside and outside the pores. In such a packing material, proteins in serum (albumin and globulin) are macromolecules, so they do not enter the pores and pass through the column without being adsorbed on the hydrophilic outer surface (outer surface of the pores). Relatively small molecules will be adsorbed on the hydrophobic inner surface (inner surface of the pore) and separated.

Specific examples of such fillers include Japanese Patent Application Laid-Open No. 60-5625
An example is the invention described in Publication No. 6. In this invention, a filler is used in which the outer surface of a silica filler to which octadecylsilyl (ODS) groups are chemically bonded is coated with protein. This packing material is obtained by adsorbing bovine serum albumin or rabbit plasma protein onto a column and denaturing it.

In addition, as a method for producing a filler similar to this, as described in JP-A-61-65159 (method of Pinkerton et al.) and JP-A-1-123145 (method of Haginaka et al.), 2) Next, only the hydrophobic groups on the outer surface are cleaved using an enzyme that is itself a macromolecule and cannot enter the pores. 3) After that , there is a method of introducing wood-philic groups to the outer surface. More specifically, someone's method uses porous silica into which glycerylpropyl groups have been introduced as a starting material,
An oligopeptide (glycyl-phenylalanyl-phenylalanine, etc.) is bonded to this via carbonyldiimidazole, and the phenylalanine side chain on the outer surface is cleaved by hydrolysis using carboxypeptidase A, a protein hydrolase. This is the way to do it.

As a result, the inner surface contains glycyl as a hydrophobic ligand.
Phenylalanyl-phenylalanine remains, and the outer surface becomes a hydrophilic glycyl-glycerylpropyl group. On the other hand, in the latter method, porous silica into which aminopropyl groups have been introduced is used as a starting material, and octanoyl chloride is reacted in the presence of triethylamine to introduce hydrophobic groups through amide bonds. In this method, acyl groups on the outer surface are hydrolyzed by acylase, and amino groups on the outer surface are reacted with glycidol to make them water-soluble.

(c) Problems to be Solved by the Invention However, among the above-mentioned packing materials, ODS silica packing coated with protein may cause elution of adsorbed and denatured proteins when used for a long period of time, and elution of denatured proteins may occur. It has drawbacks in terms of durability and separation ability, such as the inability to obtain columns with high separation efficiency.

In addition, since the methods of Pinkerton and Haginaka et al. utilize an enzymatic reaction, the process is complicated and the characteristics of the obtained packing material tend to vary, and the separation ability of small molecules such as drugs There was a limit to the number of species.

This invention was made under such circumstances, and aims to provide a packing material that has high protein removal ability and drug separation ability, is highly durable, and can be easily manufactured with good reproducibility. be.

(d) Means for Solving the Problems According to the present invention, the inner surfaces of the pores in the porous carrier are modified with a silane coupling agent residue having an aryl group or a substituent exhibiting chiral resolution. , and the outer surface of the pores is modified with a silane coupling agent residue having a hydrophilic group.

A water-insoluble substance having a hydroxyl group on the surface can be used as the porous carrier serving as the matrix of the column packing material of the present invention, and for example, so-called porous silica gel or porous glass can be used. However, other than this, a porous polymer made of a synthetic resin having hydroxyl groups can also be used. Suitable porous carriers are usually those having a particle size of 5 to 20 μm and a specific surface area of 200 to 50 (about 1 m''/9).

The column packing material of the present invention is obtained by modifying the inner and outer surfaces of the porous carrier with silane coupling agent residues having different substituents.

Here, the inner surface of the pore is modified with a silane coupling agent residue having an aryl group or a substituent exhibiting chiral resolution. Here, examples of the aryl group include a phenyl group and a naphthyl group, and examples of the substituent exhibiting chiral resolution include a cyclodextrin-containing group, a branched cyclodextrin-containing group, a chiral amide and urea derivative-containing group, and a crown ether List the containing groups. Here, modification with aryl group-containing silane coupling agent residues is mainly employed when separating drugs based on differences in the degree of hydrophobicity, and modification with silane coupling agent residues exhibiting chiral resolving ability is It is mainly employed when separating each enantiomer of an active drug, and is selected as appropriate.

On the other hand, the outer surface of the pore is modified with a silane coupling agent residue having a hydrophilic group. Here, the hydrophilic groups include hydroxyl group,
Examples include an amino group and a carboxyl group. This modification of the outer surface of the pores is mainly performed for the purpose of preventing adsorption and retention of proteins on the filler and facilitating protein removal treatment and protein separation and removal treatment.

The modification of the inner and outer surfaces of the pores can be achieved, for example, by condensing and adding a silane coupling agent having various substituents to the surface hydroxyl groups of a porous carrier such as porous silica gel or porous glass, and modifying the terminal substituents as necessary. This can be carried out by converting the substituent into another substituent. Suitable examples of such silane coupling agents include substituted tri(lower alkoxy)silanes, terminally substituted alkyltri(lower alkoxy)silanes, etc. Specifically, substituted trimethoxysilanes, substituted triethoxysilanes, γ- Substituted monoprobyltrimethoxysilane, γ-substituted monoprobyltriethoxysilane, and the like. Among these, preferred examples of silane coupling agents for modifying the pore inner surface include aryl group-substituted trimethoxy (or ethoxy) silane and 3-
Isocyanatopropyltrimethoxy (or ethoxy)
Examples include carbamic acid cyclodextrin ester-substituted propyltrimethoxy (or ethoxy) silane obtained by reacting silane and cyclodextrin. Further, one preferable example of the silane coupling agent after modifying the pore inner surface is 3-glycidoxypropyl trimethoxy (or ethoxy) silane.

The column packing material of the present invention can be efficiently produced by utilizing the difference in reactivity between the inner surface of the pores and the outer surface of the pores in the surface hydroxyl groups of the porous carrier. In other words, the porous carrier has hydroxyl groups on the inner surface of the pores (pore inner surface) and the outer surface of the pores (pore outer surface), but according to the findings of the present inventors, the chemical reactivity on the outer surface of the pores higher than that of

Therefore, taking advantage of this difference in reactivity, first, after modifying the pore inner surface, a silane cubupling agent is applied to the porous carrier under mild condensation reaction conditions where the reaction with the pore inner surface can be virtually ignored. The corresponding silane coupling agent residue is bonded only to the outer surface of the pores, and then the silane coupling agent for modifying the inner surface of the pores is applied to the porous carrier under strong condensation reaction conditions that allow the reaction with the inner surface of the pores to proceed smoothly. The corresponding silane coupling agent residues are bonded to the pore inner surface by condensation with the remaining hydroxyl groups, and then the substituents of each silane coupling agent residue can be modified or converted as necessary. It can be made into

Any condensation reaction herein can be carried out by contacting the porous support with a coupling agent in a substantially anhydrous non-polar solvent (e.g., toluene, benzene, xylene, etc.) under reflux; Non-catalytic conditions are selected as mild condensation reaction conditions for selective modification of the outer surface of the pores, and conditions in which a condensation catalyst is added are selected as strong condensation reaction conditions for modification of the inner surface of the pores. As the condensation catalyst used here, an organic base is suitable, and examples thereof include N,N-diisopropylethylamine, triethylamine, pyridine, and the like.

Note that the amounts of the porous carrier and the coupling agent used in each condensation reaction are not particularly limited, but usually 0.5 to 4.0 equivalents of the coupling agent to the surface silanol It (for example, 8.5 μmol/♂). It is appropriate to consider it as a degree.

Further, when using a condensation catalyst, an amount of about 2.5 equivalents relative to the coupling agent is sufficient.

Furthermore, when using a terminal glycidoxyalkyltrialkoxysilane to modify the outer surface of the pore, for example, it is necessary to modify the terminal glycidoxy group into a hydrophilic group after modifying the inner surface of the pore. This can be achieved by converting it into a diol. In particular, the use of a terminal glycidoxy group is preferable because the inner surface of the pore can be modified without protecting the active hydrogen of the silane coupling agent residue bonded to the outer surface of the pore during production! ! I am like 1.

The column packing material of the present invention thus obtained can be packed into a suitable column container and suitably used as a column for liquid chromatography or a column for protein removal treatment.

(E) Effect The silane coupling agent residues modified on the inner surface of the pores of the porous carrier and having an aryl group or a substituent exhibiting chiral resolving ability can be used for various relatively small molecules such as drugs that can enter the pores. It acts to efficiently separate molecules based on differences in their hydrophobicity or differences in stability of inclusion complexes or diastereomeric complexes. On the other hand, the silane coupling agent residues modified on the outer surface of the pores and having a hydrophilic group act to prevent the adsorption and protection of macromolecules such as proteins on the porous carrier.

Together, these effects make it possible to perform high-precision separation and measurement of drugs and the like over a long period of time, and also to directly separate biological samples such as serum.

(to) Examples Examples! (Step 1) Preparation of the outer surface of the pores having 3-glycidoxypropyl groups To 2 g of Develosil 6O-5 (particle size 5 gm, pore size 60, porous silica manufactured by Nomura Chemical Co., Ltd.) was added 120i12 of toluene, and azeotropic After removing water, 3.81 g of 3-glycidoxypropyltrimethoxysilane was added, and the mixture was stirred at 90 to 95° C. for 5 hours under non-catalytic conditions. Reacted silica was filtered and 603! & toluene and 6
After washing with 0 methanol, the outer surface looks like the one shown below.
- Obtain glycidoxypropylsilyl etherified silica.

(Step 2) Preparation of the inner surface of the pores having phenyl groups 80% of toluene was added to 29% of the 3-glycidoxypropyl silica obtained in step 1.
x(l) and stir.Furthermore, after adding 2 x Q of phenyltrimethoxysilane and 6 ml of N,N-diisopropylethylamine (condensation catalyst), reflux for 9 hours while stirring.Reacted silica is collected by filtration. , 60zQ of toluene and 601fluoromethanol to obtain silica with phenylsilyl etherified pore inner surfaces as shown below.

(Step 3) Preparation of silica having phenyl groups on the inner surface of the pores and diol groups on the outer surface of the pores The filler 29 prepared above is added to an aqueous solution of perchloric acid having a pH of 3.0, and refluxed for 4 hours with stirring. The obtained silica was washed with 60x Q of water and 60x (l) of methanol to produce the following silica whose pore inner surface was modified with a phenyl group-containing silane coupling agent residue and the pore outer surface was modified with a diol group-containing silane coupling agent residue. A filler of the present invention shown in Figure 1 was obtained.

Example 2 (Step 1) Preparation of pore outer surface with 3-glycidoxypropyl group Chemcosorb 5SI (particle size 5 μm, pore size 8
0 people, porous silica (manufactured by Kemco) 2g to toluene 80
After adding 11Q and removing water by azeotropy, 0.8zQ of 3-glycidoxypropyltrimethoxysilane was added, and the mixture was stirred at 90 to 95°C for 5 hours under non-catalytic conditions. The reacted silica is collected by filtration and washed with 60xQ toluene and 60ml methanol to obtain silica in which the outer surface of the pores has been converted to 3-glycidoxypropylsilyl ether.

(Step 2) Preparation of the inner surface of the pore that exhibits optical resolution (preparation of a filler in which β-cyclodextrin is chemically bonded to the inner surface of the pore). 4.59 and stir. Under a nitrogen stream, gradually add 3-isocyanatopropyltriethoxysilane 2.649, and for about 5 hours (infrared absorption vector of isocyanate at 2200-2300 cm-' disappears) Silica 29 prepared above (Step I) is added to this solution and refluxed for 20 hours with stirring.The reacted silica is filtered and treated with 60zQ each of pyridine, acetone, methanol and water. The modified silica shown below was obtained by washing.

(Step 3) Preparation of a filler having β-cyclodesquitrin on the inner surface of the pores and diol groups on the outer surface of the pores Approximately 29 grams of the silica prepared in the above (Step 2) was added to a 5% acetonitrile aqueous solution and kept at room temperature for 24 hours. By stirring, the filler of the present invention shown below was obtained, in which the inner surface of the pores was modified with a silane coupling agent residue containing β-cyclodesquitrin, and the outer surface of the pores was modified with a diol group-containing silane coupling agent residue.

Example 3 The filler prepared in Example 1 had an inner diameter of 4631111. A stainless steel tube for HPLC with a length of 5 cm was filled. Using this column, phenobarbital (20u9) was added to human control serum (sample A) and human control serum.
/ aL), 7 z 2) I: / (40u9/s
The separation of sample B) with standard addition of carbamazepine (10μ?/sL) was investigated. The mobile phase was 10
0mM NaH*PO* -too @M NaJPO
*-CHaCN (4,5-4,5-1) at 0.8 m
The liquid was delivered at L/sin, and detection was performed at 254 ns. Further, the injection amount was 10μ. The obtained chromatogram is shown in FIG. A in FIG. 1 shows sample A, in which the human serum protein peak eluted immediately after injection. B in FIG. 1 shows sample B, in which the peaks of phenobarbital (2), phenytoin (2), and carbamazepine (2) were eluted after the human serum protein, and were well separated from the serum components.

Example 4 The filler prepared in Example 2 had an inner diameter of 4 cm and a length of 100 cm.
It was filled into a single shoulder HPLC stainless steel tube. Using this column, human control serum (sample A) and human control serum were treated with hexobarbital (100μ9/
The separation characteristics of sample C to which ++L) was added as standard were investigated. Mobile phase was 1100I NaH*POa-100
111! If NatHPO4-CHaCN(2,5-
2,5-1) was delivered at a rate of 0.8 mL/ll1n, and detection was performed at 254 nm.

Further, the injection amount was 20μ. The obtained chromatogram is shown in FIG. A in FIG. 2 shows sample A, in which the human serum protein peak eluted immediately after injection. C in FIG. 2 shows sample C, in which a peak of hexobarbital (d-unit and 4-unit) elutes after human serum protein and is well separated from serum components.

The recovery rate of serum proteins in Examples 3 and 4 was approximately 100%. This indicates that the diol group-containing silane coupling agent residue is uniformly bonded to the outer surface of the pores of the silica, but is not substantially bonded to the inner surface of the pores.

Example 5 The packing material obtained in Example 2 was filled into a stainless steel tube with an inner diameter of 4.631111 mm and a length of 30 zm, and used as a column for protein removal from serum samples. Phenytoin (5 μ9/surname) and carbamazepine (1 μ
? /iL) was added as standard and 50 μL was injected. A liquid chromatograph flow path diagram for column switching is shown in FIG. In the figure, l is the analytical mobile phase, 2 is the pump,
3 is a protein removal column, 4 is a switching valve (the valve switching time is 20 to 5 minutes for the dotted line, 5 to 8 minutes for the solid line, and 8 minutes for the dotted line), 5 is the analytical column, 6 is the detector, and 7 is the A recorder, 8 an injector, 9 a pump, and 10 a mobile phase for pretreatment. Further, in the figure, the dotted line of the switching valve indicates a state where the pretreatment column and the analysis column are separated, and the solid line indicates the state where the pretreatment column and the analysis column are connected.

Here, as the pretreatment mobile phase 10, 14mM NaHt
POa-6nM Natl [POa flow rate 0.8mL/
Inject 50μL of serum sample into injector 8.
The solution was injected from the column, pretreated for 5 minutes, and analyzed using analytical column 5.

The analysis conditions were as follows: 1 g of Nucleosil 5C (manufactured by Nagel) was used as the stationary phase with an inner diameter of 4.6 mm.
, filled in a stainless steel tube for HPLC with a length of 150R11, and 14 mM Na as the mobile phase for analysis.
HtPOa + 6mMNaJPOa-CH'5C
N-CH30 port (5,5/2.5/2) flow rate 0.8m
The liquid was fed at L/+in, and detection was performed by absorption of 230n*. FIG. 4 shows the obtained chromatogram.

In the figure, ■ indicates phenytoin, and ■ indicates carbamazepine. Thus, it can be seen that the packing material of the present invention functions effectively as a column for protein removal.

(g) Effects of the Invention The column packing material of the present invention has sufficient hydrophilicity on the outer surface of the pores, and sufficient hydrophobicity or chiral recognition ability on the inner surface of the pores, so that it is difficult to detect biological samples containing proteins such as serum. In drug analysis, it is possible to analyze drugs by direct injection,
It can also be used as a protein removal column.

Since it can be produced by a chemical reaction using a silane coupling agent without an enzyme reaction, it is easy to manufacture and has good reproducibility, and the chemical bond imparts different properties to the inner and outer surfaces. Therefore, it does not cause deterioration and has excellent durability.

Therefore, the column packing material of the present invention is extremely useful in the field of liquid chromatography.

[Brief explanation of drawings]

Figures 1, 2, and 4 are chromatograms illustrating the results of separating and analyzing biological samples using the column packing material of the present invention, and Figure 3 is a chromatogram showing the liquid used in the example. It is a chromatographic flow path diagram. Fig. 1 Fig. N (ben) kimon (minute) Fig. 4

Claims (1)

[Claims] 1. The inner surface of the pores in the porous carrier is modified with a silane coupling agent residue having an aryl group or a substituent exhibiting chiral resolution, and the outer surface of the pore is a silane coupling agent residue having a hydrophilic group. Column packing material modified with