JPH04346830A - Packing material for chromatography - Google Patents

Abstract

PURPOSE:To obtain a packing material having good separation performance and excellent physical, chemical and biological stability by using porous carbon fine particles which are substantially spheres with a specified particle size and have coated surfaces. CONSTITUTION:The porous carbon fine particles as the supporting body of the packing material for chromatography consist of substantial spheres with sphericity of <=1/2 of the max. diameter, preferably <=1/4, and more preferably <=1/8. The average particle size of the porous carbon fine particles is 0.5-100mum and preferably 1-30mum. The surface of the porous carbon fine particle is modified with a coating material such as protein A, polyethylene glycohol and the like. The obtd. packing material for chromatography has high mechanical strength and durability against high temp. and has excellent chemical stability and biological stability and good separation performance.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a novel filler for chromatography, more specifically, it has high mechanical strength, can withstand high temperatures, and has good organic solvent resistance, acid resistance, alkali resistance, etc. The present invention relates to a packing material for chromatography that has substantially perfect spherical porous carbon particles as a support, which has excellent chemical stability, excellent biological stability, and good separation performance.

0002

BACKGROUND OF THE INVENTION In recent years, high performance liquid chromatography and gas chromatography have been rapidly used as means for component analysis and separation/preparation of single substances. In particular, as the development of packing materials and columns progresses in high-performance liquid chromatography, the range of targets that can be separated has expanded from organic low-molecular compounds to high-molecular compounds such as proteins, and the range of applications is expanding year by year. High-performance liquid chromatography can quickly and accurately separate substances that are easily destroyed depending on temperature conditions, and has become an indispensable separation method for research into material production using biotechnology and the development of pharmaceuticals. . Furthermore, recently, large columns with column diameters of 300 mm and 800 mm have appeared, and are beginning to be used for industrial-scale separation and fractionation, for example, for industrial fractionation of pig serum, carotene, chlorophyll, and the like. Typical separation modes currently used in liquid chromatography include reversed phase chromatography (RPC), normal phase chromatography (NPC), adsorption chromatography (LSC), and hydrophobic chromatography. (HIC), optical resolution chromatography (ERC), ion exchange chromatography (
IEC), ion chromatography (IC), gel filtration chromatography (GFC), gel permeation chromatography (GPC), affinity chromatography (AFC), and the like. The performance required of the packing material used in such chromatography differs depending on the separation mode of the chromatography, but in general, it should be spherical in shape, appropriately fine, and uniform in particle size. The properties include high mechanical strength, good organic solvent resistance, acid resistance, and alkali resistance, excellent chemical stability, high temperature resistance, and excellent separation performance. Conventionally, various fillers for chromatography have been developed, and among them, organic polymer fillers (porous polymers) and silica-based fillers are known as representative ones. However, the organic polymer filler has poor solvent swelling resistance;
It has the disadvantage that the eluent used is limited and the range of application is inevitably limited. On the other hand, silica-based fillers can be produced by first preparing a core material such as glass beads that is strong and has a uniform particle size, and then melting and fixing fine particles such as silica gel or silica sol to the surface of the core material, or further depending on the separation mode. The filler is obtained by modifying the filler by coating or chemical bonding with an appropriate substance, and then subjecting it to surface treatment as necessary, and compares the required performance with that of the organic polymer filler. Although it satisfies the target, it is not necessarily fully satisfying.
It has been desired to develop a packing material for chromatography that has better physical and chemical stability. Incidentally, since carbon has excellent chemical stability such as solvent resistance, acid resistance, and alkali resistance, and can withstand high temperatures, research and development of carbon-based fillers has recently been actively conducted. For example, a high-strength carbon adsorbent consisting of pyrolytically modified carbon black obtained by depositing a pyrolysis product of benzene on carbon black has been reported [Journal.
of liquid chromatography (J.Liq.C
chromatogra. )” Volume 119, Page 41 (1976)]. However, this method has the drawbacks of low capacity and poor reproducibility. In addition, a room-temperature reduction product of polytetrafluoroethylene with lithium amalgam [Journal of Chromatography (
J. Chromatogra. )” Volume 119, No. 61
Page (1980)], K by lithium amalgam
Chemically modified surface of fluoropolymer derivative with carbon obtained by reduction of el-F300 and surface modification by Grignard reaction [Anal.Chem., Vol. 53, p. 812 (1981) )], commercially available graphitized carbon black [J. Chromatogra. Vol. 269, p. 47 (1983)], coke and activated carbon ["
Journal of Chromatography (J.Chro
matogra. )” Volume 202, Page 3 (198
0 years)], mesophase carbon microbeads obtained by heating pitch (Japanese Patent Application Laid-Open No. 56-44846,
Attempts have been made to consider methods such as Japanese Unexamined Patent Publication No. 58-41351). However, with these pulverized type fillers made by finely crushing porous carbon, surface modified type fillers where the gel surface is coated with carbon, or spherulite type fillers, the pore size cannot be adjusted arbitrarily, and the pore size varies from amorphous to graphite. It has drawbacks such as the inability to control the crystalline state of carbon, and cannot be said to be practical as a packing material for chromatography.

0003

[Problems to be Solved by the Invention] Under these circumstances, the present invention has high mechanical strength, can withstand high temperatures, and has good chemical resistance such as organic solvent resistance, acid resistance, and alkali resistance. The purpose of this invention is to provide a packing material for chromatography that has not only excellent physical stability but also excellent biological stability and good separation performance.

0004

[Means for Solving the Problems] The present inventors have previously developed carbon-based particles having a specific pore volume distribution, pore volume, and carbon content that have excellent performance as a packing material for liquid chromatography. I found it. In order to develop a chromatography packing material having the above-mentioned preferable properties, the present inventors conducted further intensive research on this carbon-based packing material, and found that it has a substantially true sphere and a specific particle size. We have discovered that this objective can be achieved by modifying the surface of porous carbon particles with a coating agent,
Based on this knowledge, we have completed the present invention.

That is, the present invention provides a method for chromatography in which the surface of porous carbon particles having a sphericity of 1/2 or less of the maximum radius and an average particle size of 0.5 to 100 μm is modified with a coating agent. It provides a filler. The present invention will be explained in detail below. In the chromatography packing material of the present invention, the porous carbon fine particles used as the support have a sphericity of 1/2 of the maximum radius.
Below, preferably 1/4 or less, more preferably 1/8
It is necessary to be a substantially perfect sphere. Generally, it is known that the separation performance of a chromatography filler improves as it approaches a perfect sphere, as its particle size decreases, and as its particle size distribution narrows. In the present invention, sphericity refers to the diameter of a large circle formed when a carbon particle is rotated about an axis passing through its center in a direction perpendicular to the axis, and the maximum and minimum values of the diameter are measured. This is the value expressed as a percentage of the maximum radius of the carbon fine particles. Note that the maximum diameter and the minimum diameter are determined from scanning electron micrographs as an average value of 10 values. At this time, a scanning electron microscope photograph is taken at a magnification such that the particle size of the carbon particles to be measured is 10 mm or more on the photograph.

[0006] When the sphericity exceeds 1/2 of the maximum radius, the separation performance deteriorates and the object of the present invention cannot be fully achieved. In the present invention, it is necessary that the average particle diameter of the porous carbon fine particles be in the range of 0.5 to 100 μm, preferably 1 to 30 μm. This average particle size is 0.5μm
If it is less than 100 μm, the pressure loss will be large and operation will be difficult when packed in a column, and if it exceeds 100 μm, separation performance tends to decrease. Further, the pore diameter of the porous carbon fine particles is usually selected in the range of 0.1 to 500 Å, and it is desirable that the substantial surface area is 5 times or more the apparent surface area calculated from the diameter of the fine particles, such as Average particle size 1
For fine particles of 0 μm, the specific surface area is 2 to 500 m2/g,
Preferably, it is in the range of 10 to 500 m2/g. If the pore diameter and specific surface area are outside the above range, the object of the present invention cannot be fully achieved.

On the other hand, regarding the pore volume of the porous carbon fine particles, the sum of the volumes of pores with a radius of 10 to 50 nm is [
10-50], the sum of the volumes of pores with a radius of 1 to 50 nm is [
1-50], the pore volume index [10-50]/
It is desirable that [1-50] is 0.5 or more, preferably 0.6 or more, more preferably 0.8 or more. If the pore volume index is less than 0.5, the separation performance will be insufficient, which is not preferable. It is also advantageous for the total pore volume to be at least 0.15 ml/g, preferably at least 0.2 ml/g. If the total pore volume is less than 0.15 ml/g, it is difficult to retain the solute appropriately, and the separation performance may not be sufficiently exhibited, which is not preferable. Furthermore, the carbon fine particles have pores with a radius larger than 50 nm whose sum of volumes is 0.
．． The amount is preferably 1 ml/g or less, preferably 0.05 ml/g or less. Particles with a radius larger than 50 nm and a total volume of more than 0.1 ml/g have poor mechanical strength, and when used as a packing material for chromatography operated under high pressure, the particles are easily destroyed, resulting in poor reproducibility. This is not preferable because it may result in poor chromatographic separation.

[0008] The pore volume can be determined by a nitrogen gas adsorption method. For example OMNISORP360
BJH (Barret-Joyner Ha
lenda) method. The porous carbon fine particles in the present invention preferably have a carbon atom content of 97% by weight or more, preferably 99% or more. If the carbon atom content is less than 97% by weight, impurities other than carbon such as S, O, H, N, metals, etc. may be contained, which is undesirable because the separation performance may deteriorate. The method for producing such porous carbon fine particles is not particularly limited as long as it satisfies the above conditions. It can be produced by firing a granulated material comprising a binder at an appropriate temperature in an oxidizing, reducing or inert gas atmosphere.

Next, an example of a preferred method for producing the porous carbon particles will be explained. First, an average molecular weight of 3
A mixture of pitch of 00 or more, preferably 400 or more, a polymerizable monomer, and a polymerization initiator is subjected to suspension polymerization to form beads made of pitch and an organic polymer. The organic polymer is not particularly limited as long as it can form a substantially spherical network gel, but aromatic vinyl polymers such as polydivinylbenzene and polytrivinylbenzene and polyethylene dimethacrylate are preferred. Can be mentioned. Furthermore, if the average molecular weight of the pitch used at this time is less than 300, it may be difficult to obtain particles having the above-mentioned pore distribution characteristics. The average molecular weight of the pitch can be measured, for example, by using chloroform as a solvent and using a vapor pressure equilibrium method, which is a common method. Furthermore, the pitch includes, for example, pitch obtained in a petroleum processing process, pitch obtained in a coal carbonization process, synthetic pitch obtained from naphthalene, polyvinyl chloride, etc.
Either can be used.

There are no particular restrictions on the conditions for suspension polymerization, and methods conventionally used in the production of synthetic resins can be used. For example, pitch, a polymerizable monomer, a polymerization initiator, and an optional diluent or suspending agent are added to an aqueous medium, and the mixture is stirred at high speed, preferably at a temperature of 20°C or lower, to form a homogeneous suspension. After forming, usually 50 to 9
A method is used in which polymerization is carried out at a temperature in the range of 0°C for about 4 to 10 hours, preferably in a temperature range of 60 to 80°C for about 5 to 8 hours. At this time, the content of polymerizable monomer and pitch in the aqueous medium is usually 2 to 2
0% by weight, preferably in the range of 4 to 10% by weight. In addition, as a polymerization initiator, those conventionally used in suspension polymerization, such as α,α'-azobisisobutyronitrile, benzoyl peroxide, 2,2'-azobis(
2,4-dimethylvaleronitrile) and the like can be used.

Diluents that may be used as desired include organic solvents such as toluene, xylene, benzene and benzonitrile, and suspension stabilizers include polyvinyl alcohol and methyl cellulose. In this way, substantially spherical crosslinked organic polymer reticulated gel beads with internal pitch are formed. After the beads are removed from the reaction solution by means such as filtration, they are heated in the air at a temperature of about 250 to 380° C. for about several hours to make them infusible. Next, the infusible beads thus obtained are heated to a temperature of usually 1,100°C or higher, preferably 1,100°C to 1,100°C.
Desired porous carbon particles can be obtained by firing in vacuum or in an inert gas atmosphere such as argon or nitrogen at a temperature in the range of 3000°C. If the firing temperature is lower than 1100° C., the carbon content in the obtained carbon particles may be less than 97% by weight, which is not preferable.

The chromatography packing material of the present invention includes:
The surface of the porous carbon fine particles thus formed is coated with a coating agent to modify the fine particles, and the coating agent is a separation mode of chromatography in which the filler of the present invention is used. be selected as appropriate. As a specific example, a coating agent when the filler is used for affinity chromatography will be described. Substances of biological origin include pairs of complementary substances that exhibit specific affinity for each other, such as enzymes and substrates, enzymes and specific inhibitors, antigens and antibodies, lectins and sugars, and hormones and hormone receptors. . Such complementary substances are called "affinity ligands" or "affinants" or simply "ligands." When this ligand is immobilized on an insoluble carrier to create a column and a sample solution containing the target substance is supplied to this column, substances that have no affinity for the ligand will not be adsorbed, and only complementary substances will be adsorbed. If the adsorbed target substance is then eluted using an appropriate eluent, it can be recovered as a highly pure product. Such a purification method is called affinity chromatography.

[0013] Therefore, various ligands are used as coating agents. Examples of such coating agents include the following. In addition(
) indicates the substance to be adsorbed and separated. That is, the ligands used as coating agents include protein A (immunoglobulin), concanavalin A lectin (saccharide and sugar complex), malt lectin (N-acetylglucosamine), poly-U (nucleic acid binding protein), poly -A (nucleic acid oligonucleotide, RNA-specific protein), polylysine (plasminogen, RNA), blue dextran (enzyme, serum albumin), 5'-adenosine monophosphate (enzyme, adenosine triphosphate-dependent kinase), 2 ',5'-adenosine diphosphate (enzyme with nicotinamide adenine dinucleotide phosphate as coenzyme)
, heparin (blood coagulation protein, plasma protein), p
-Aminobenzamidine (trypsin, thrombin, etc.), p-aminobenzyl phosphate (alkaline phosphatase, etc.), aminopeptidase M (hydrolysis of N-terminal amino acid), p-aminophenylphosphorylcholine (C-reactive protein, Others), avidin (biotin, etc.), D-biotin (avidin, avidin derivatives, etc.), carboxypeptidase Y (hydrolysis of C-terminal amino acid), p-chloromercuribenzoate (papaya peptidase, others), cibacron blue (vitamin D binding plasma proteins, etc.), dextran sulfate (
prothrombin conjugate, etc.), 17-β-estradiol-17-hemisuccinate (estradiol receptor, etc.), β-galactosidase (hydrolysis of sugars), gelatin (fibronectin, etc.), glycyl-L
-Tyrosyl-azobenzylbutyric acid (carboxypeptidases A, B, Y, etc.), iminobiotin (avidin, avidin analogs, etc.), trypsin inhibitors (trypsin, etc.), aprotinin (kallikrein), trypsin (aprotinin), D-tryptophan methyl ester ( α-chymotrypsin), pepstatin (renin), leupeptin (trypsin), galatosamine agarose (soybean lectin), lactose agarose (castor bean lectin, peanut lectin), maltose agarose (nata bean lectin), various monoclonal antibodies (corresponding Antigens), etc.

[0014] When the filler is used for normal phase or reverse phase chromatography, coating agents such as carbon wax, ethylene glycol, cyanoethyl silicone, hydrocarbon polymers, polyamide, and squalene are used. In addition, when the filler is used for optical resolution chromatography, as a coating agent,
For example, phenyl urea, naphthylurea, 3,5-dinitrobenzoyl methyl ester, N-3,5-dinitrobenzoyl-(R)-phenylglycine, N-3,5-
Dinitrophenylaminocarbonyl-L-valine, 1-
Fluoro-2,4-dinitrophenyl-5-L-alaninamide, O-(p-nitrobenzyl)-N,N'-(
diisopropyl) isourea, D-phenylglycine,
Cyclodextrin, polyacrylamide, albumin, α1-acid glycoprotein (orosomucoid), ovomucoid, etc. are used.

On the other hand, when the packing material is used for hydrophobic chromatography, polyethylene glycol is usually used as a coating agent, and when it is used for chelate formation chromatography, a complex-forming reaction reagent is used as the coating agent. or coordination exchange reagents are used. For example, a packing material using a phenylboron compound as a coating agent is used for the selective separation of catecholamines, and is also applicable to the separation of various general saccharides.

Furthermore, when the packing material of the present invention is used for gas chromatography, coating agents such as p,p'-azoxydiphenethol, BC-120
, N,N-bis(2-cyanoethyl)formamide, p
-Toluidine, bis(2-ethoxyethyl)adipate, butanediol succinate, carbowax 400
0 monostearate, cyclohexane dimethyl succinate, dibutyl maleate, diethylene glycol adipate, diethylene glycol succinate, di(2-ethylhexyl) sebacate, diglycerol, dinonyl phthalate, dioctyl sebacate, ethyl-N,N-
Dimethyl oxamate, ethylene glycol adipate, ethylene glycol phthalate, ethylene glycol succinate, ethylene glycol tetrachlorophthalate, 1,2,3,4,5,6-hexakis (2-cyanoethoxycyclohexane), N-lauroyl-N -L
-valine-t-butyramide, neopentyl glycol adipate, neopentyl glycol sebacate, neopentyl glycol succinate, β,β'-oxydipropionitrile, phenyldiethanolamine succinate, polyethyleneimine, polyphenyl ether (
5 rings) OS-124, polyphenyl ether (6 rings) OS-138, polypropylene glycol, polypropylene imine, propylene carbonate, quadrol, squalene, sucrose acetate isobutyrate (SAIB), tetracyanoethylated pentaerythritol, tetraethylene penta Min, 1, 2, 3
, 4-tetrakis (2-cyanoethoxybutane), tetrahydroxyethylenediamine (THED), tris(
2-cyanoethyl) nitromethane (TCENM), as well as emalfor, EPON1001, etofat 60/25, fluorad, Halcomide M-18, Halcomide M-18-OL, halocarbon 10-25, halocarbon K-352, halocarbon wax , Igepal C
O-880, Igepal CO-990, PPE-20, P
PE-21, SP-216-PS, SP-301, SP
-1000, SP-1200, SP-1220, SP-
1500, SP-1510, SP-1700, SP-2
510, Triton X-100, Triton X-305, U
CON LB-550-X, UCON50-HB-2
80-X, UCON50-HB-2000, UCON5
0-HB-5100, DC-11, DC-200, DC
-550, GE-SF-96, GE-XE-60, OV
-1, OV-3, OV-7, OV-11, OV-22,
OV-61, OV-73, OV-105, OV-202
, OV-215, OV-1701, Silar5CP,
Silar10CP, SP-400, SP-2250,
SP-2310, SP-2401, UC W982, etc. are used. Fillers treated with these coating agents can be used not only for gas chromatography but also for supercritical solvent chromatography.

[0017] In order to coat the surface of the porous carbon particles with the coating agent, the porous carbon particles are first thoroughly dried. On the other hand, a coating agent-containing liquid is prepared by dissolving, dispersing, or suspending a desired coating agent in an appropriate solvent, and (1) the carbon fine particles are added to the coating agent-containing liquid and mixed with stirring, and then the solvent is evaporated. (2) a solvent filtration method in which the carbon particles are added to the coating agent-containing liquid and stirred and mixed, and then filtered to remove excess coating agent together with the solvent; (3) the carbon particles are packed in a column. after,
The surface of the carbon particles may be coated with a desired coating agent by using a method such as a column flow method in which the coating agent-containing liquid is circulated, and if necessary, reinforced with a crosslinking agent. The coating amount of the coating agent is preferably 0.1 to 25% by weight based on the carbon fine particles. Since the carbon fine particles of the present invention have strong adsorption power, the coating agent can be firmly fixed to the particle surface.

[0018] When the coating agent is a biopolymer substance such as a protein or other biologically derived substance, the solvent used for preparing the coating agent-containing liquid is:
Generally, polar solvents such as water, alcohol, acetonitrile, acetone, and tetrahydrofuran are used, and when the coating agent is a modified product such as a protein modified with polyethylene glycol, a nonpolar solvent is usually used as the solvent. used.

[0019] By coating and modifying the surface of porous carbon fine particles with a coating agent in this way,
Although the packing material for chromatography of the present invention is obtained, in the present invention, the stationary phase (coating layer) can be further chemically modified as desired depending on the type of coating agent. For example, porous carbon particles whose surfaces are coated with silicone polymer, silicic acid, alumina, cellulose, dextran, etc. can be further chemically modified to impart desired functions. in particular,
By introducing ion exchange groups into the surface of porous carbon particles treated with silicone polymer, they can be used as physically stable ion exchangers, and by introducing octadecyl groups, they can be used as reverse phase fillers. It will also happen.

The thus obtained packing material for chromatography of the present invention has excellent physical, chemical and biological stability as well as good separation performance. High performance liquid chromatography such as chromatography, adsorption chromatography, hydrophobic chromatography, optical resolution chromatography, ion exchange chromatography, ion chromatography, gel filtration chromatography, gel permeation chromatography, affinity chromatography, chelate formation chromatography It is suitably used as a filler in gas chromatography, supercritical solvent chromatography, etc.

[0021]

EXAMPLES Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to these examples in any way. Production Example 1 5% by weight of vacuum distillation residue oil with an average molecular weight of 600, 5% by weight of divinylbenzene, 1% by weight of polyvinyl alcohol, 0.25% by weight of azobisisobutyronitrile, 5% by weight of toluene
A suspension was prepared by stirring a mixture consisting of the weight percent and the remainder of ion-exchanged water at a high speed at 15° C. using a laboratory diffuser, and then heated at 80° C. for 6 hours with stirring to perform polymerization. Next, the generated beads are collected by filtration and 1
After drying at 00°C, it was heated in air at 350°C for 3 hours to make it infusible, and then heated in a nitrogen gas atmosphere for 2 hours.
It was baked at 500°C for 1 hour. After firing, the product was subjected to ultrasonic treatment in benzene, washed with a methanol/ether mixture, dried at 100° C., and then classified to produce porous carbon particles. Table 1 shows the elemental analysis results and physical properties of the porous carbon particles.

Production Example 2 Porous carbon particles were produced in the same manner as Production Example 1 except that the firing temperature was 2700°C. Table 1 shows the elemental analysis results and physical properties of this product.

Production Example 3 Porous carbon particles were produced in the same manner as Production Example 1 except that the firing temperature was 1500°C. Table 1 shows the elemental analysis results and physical properties of this product.

[0024]

[Table 1]

Note 1) Sphericity is a value expressed as a ratio of the difference between the maximum diameter and the minimum diameter to the maximum radius. Example 1 Hydrophobic chromatography filler The porous carbon fine particles obtained in Production Example 1 were heated at 120°C for 3 hours.
After drying for a period of time, the surface of the fine particles was coated with polyethylene glycol by a column flow method to prepare a hydrophobic chromatography packing material. The amount of polyethylene glycol coated was 5% by weight based on the fine particles. Next, using this packing material, lysozyme was separated by hydrophobic chromatography under the conditions shown below, and the chromatogram shown in FIG. 1 was obtained. Conditions Column: 4.6 mm x 10 cm Mobile phase A: 4.
0M ammonium sulfate aqueous solution Mobile phase B: 0.05M phosphate buffer (pH 6.0) Gradient: A → B (30 minutes) Flow rate: 0.5 ml/min Detection: UV 280 nm

Example 2 Packing material for gas chromatography After drying the porous carbon fine particles obtained in Production Example 2 at 120° C. for 3 hours, the surface of the fine particles was coated with diethylene glycol succinate using a column flow method. , a packing material for gas chromatography was prepared. The amount of diethylene glycol succinate coated was 2% by weight based on the fine particles. Next, using this filler, fatty acids having 12 to 18 carbon atoms were separated by gas chromatography under the conditions shown below to obtain the chromatogram shown in FIG. 2. Conditions Column: 2 mm x 100 cm Column temperature: 200°C N2 flow rate: 20 ml/min Detection: FID Sample: 1 Caprylic acid, 2 Myristic acid, 3
Arachidonic acid, 4 Palmitic acid, 5 Partolenic acid, 6 Stearic acid, 7 Oleic acid, 8
Linoleic acid, 9 Arachidic acid, 10 Linolenic acid

Example 3 Filling material for optical resolution chromatography
After drying the porous carbon fine particles obtained in Production Example 3 at 120° C. for 3 hours, the surfaces of the fine particles were coated with cyclodextrin by a column flow method to produce a packing material for optical resolution chromatography. The amount of cyclodextrin coated was 5% by weight based on the fine particles. Next, using this filler, ibuprofen was resolved by optical resolution chromatography under the conditions shown below to obtain the chromatogram shown in FIG. 3. Conditions Column: 4.6mm x 10cm Mobile phase: 0.0
1wt% triethylamine acetate solution (pH 4.5)/
Acetonitrile mixed solution (volume ratio 50/50) flow rate: 0.
5ml/min detection: UV254nm

Example 4 Filler for affinity chromatography The porous carbon fine particles obtained in Production Example 1 were
After drying at 0° C. for 3 hours, the surface of the fine particles was coated with protein A using a column flow method to prepare a packing material for affinity chromatography. The amount of protein A coated was 4% by weight based on the fine particles. Next, using this packing material, plasma proteins in human serum and I was analyzed by affinity chromatography under the conditions shown below.
gG was separated and the chromatogram shown in FIG. 4 was obtained. Conditions Column: 4.6 mm x 10 cm Starting and washing solution: Isotonic phosphate buffer Eluent: 0.01 M hydrochloric acid (pH 2.0) Flow rate: 1 ml/
Minute detection: UV280nm

[0029]

Effects of the Invention The chromatography packing material of the present invention is obtained by modifying the surface of substantially spherical porous carbon particles by coating them with a coating agent, and has high mechanical strength, resistance to high temperatures, and organic solvent resistance, acid resistance,
It has excellent chemical stability such as good alkali resistance, excellent biological stability, and good separation performance. It is suitably used as a filler for chromatography, supercritical solvent chromatography, etc.

[0030]

[Brief explanation of the drawing]

FIG. 1 is a chromatogram showing separation of lysozyme by hydrophobic chromatography in Example 1.

[0031]

FIG. 2 is a chromatogram showing separation of fatty acids having 12 to 18 carbon atoms by gas chromatography in Example 2.

[0032]

FIG. 3 is a chromatogram showing the resolution of ibuprofen by optical resolution chromatography in Example 3.

[0033]

FIG. 4 shows plasma proteins and Ig in human serum obtained by affinity chromatography in Example 4.
FIG. 2 is a chromatogram showing the separation of G.

Claims (2)

[Claims] 1. A packing material for chromatography, which is obtained by modifying the surface of porous carbon particles having a sphericity of 1/2 or less of the maximum radius and an average particle size of 0.5 to 100 μm with a coating agent. Claim 2: The porous carbon fine particles have a pore volume index [
10-50]/[1-50]0.5 or more, total pore volume 0
．． 2. The chromatography packing material according to claim 1, which has a total volume of pores with a radius exceeding 50 nm of 15 ml/g or more, a total volume of 0.1 ml/g or less, and a carbon content of 97% by weight or more.