GB2113226A

GB2113226A - Porous substrate for analyzing hydrophilic substances having low molecular weight

Info

Publication number: GB2113226A
Application number: GB08300650A
Authority: GB
Inventors: Teruo Sakagami; Masuhiro Shoji; Noriyuki Arakawa; Naohiro Murayama
Original assignee: Kureha Corp
Current assignee: Kureha Corp
Priority date: 1982-01-13
Filing date: 1983-01-11
Publication date: 1983-08-03
Also published as: GB8300650D0; SE457797B; FR2519762B1; GB2113226B; DE3300366C2; SE8207467L; SE8207467D0; DE3300366A1; FR2519762A1

Abstract

A porous substrate useful for analyzing hydrophilic substances having low molecular weight, for example by liquid chromatography, comprises a cross-linked copolymer having methylol groups, the copolymer being made of styrene or substituted styrene monomer and a cross-linking agent copolymerizable with the monomer, the outer surface thereof being hydrophilic and the inner surface of pores of the substrate being less hydrophilic, and is prepared by subjecting the styrene monomer and the cross-linking agent to suspension polymerisation in water in the presence of a pore regulator and a water-soluble polymer and providing the resultant cross-linked copolymer with methylol groups, e.g. by chloroalkylation followed by hydrolysis.

Description

SPECIFICATION Porous substrate for analyzing hydrophilic substances having low molecular weight The present invention relates to a porous substrate for analyzing hydrophilic substances having low molecular weight and a process for preparing the same. More particularly, the invention relates to a porous substrate for analyzing hydrophilic substances having low molecular weight comprising a cross-linked copolymer having methylol groups which is made of a monomer of styrene-series and a cross linking agent copolymerizable with the monomer.

The outer surface of the substrate is hydrophilic due to a water-soluble polymer attached thereto and the inner surface of pores in the substrate is hydrophobic or less hydrophilic than the outer surface of the substrate.

Recently, a substrate capable of interacting specifically with a substance in a living body has been developed, for example, in a high-speed liquid chromatography which is frequently used to analyze the substances of living bodies, in particular, urine, serum or the like. On the other hand, a substrate which may be dispersed into urine, serum or the like and may interact with a specific substance contained therein may be applied to affinity-separation of the substances and/or antigen-antibody reaction.

It is well known that a porous cross-linked polymer of styrene may be prepared by suspension polymerization in water of styrene and a cross-linking agent copolymerizable with styrene in the presence of a pore regulator. For example, styrene-divinylbenzene copolymer is described in detail in J. Polymer. Sci. Part A-2, 835 (1964). In this process, although minute beads can be obtained by using a water-soluble polymer as a main suspending agent, the watersoluble polymer is adhered to the surface of the minute beads and the polymer is difficult to be completely removed even if washing repeatedly.

The minute beads has not been used in a gel permeation chromatography using an organic solvent since the pressure is increased due to viscosity of the adhered polymer and certain undesired interaction is caused to take place.

On the other hand, relatively pure cross-linked polymer of styrene may be obtained when as a suspending agent is used phosphate which is difficultly soluble in water such as calcium phosphate and magnesium phosphate, since the phosphate can be easily removed by washing the resulting minute beads with acid or the like.

However, the obtained polymer can not be dispersed well in water, various buffers, serum, urine and the like due to hydrophilic surface thereof.

It is an object of the invention to provide a substrate for analyzing hydrophilic substances having low molecular weight.

Another object of the invention is to provide a substrate of which the outer surface is highly hydrophilic, the inner surface of pores of which is less hydrophilic than the outer surface.

Still another object of the invention is to provide a process for preparing such a substrate.

The porous substrate of the present invention comprises a cross-linked copolymer having methylol groups. The copolymer is made of a monomer of styrene-series and a cross-linking agent copolymerizable with the monomer. The outer surface of the substrate is hydrophilic because of a water-soluble polymer attached thereto. The inner surface of pores in the substrate is hydrophobic or less hydrophilic than the outer surface of the substrate.

The process of the invention comprises subjecting a mixture of a monomer of styreneseries and a cross-linking agent copolymerizable with the monomer to suspension polymerization in water in the presence of a pore regulator and a water-soluble polymer, drying the resultant cross-linked copolymer and providing the copolymer with methylol groups.

The invention will be described in detail hereinafter.

The porous cross-linked copolymer is prepared by suspension polymerization of a monomer of styrene-series and a cross-linking agent copolymerizable with the monomer in a solution of a water-soluble polymer in the presence of a pore regulator. The monomer of styrene-series herein includes styrene, a-methylstyrene chloromethylstyrene and a mixture thereof. For the cross-linking agent copolymerizable with the monomer, divinylbenzene, trivinylbenzene, triallyl isocyanurate, a dimethyacrylate of a polyhydric alcohol, a diacrylate of a polyhydric alcohol or diallyl phthalate may be used. Among these agents, divinylbenzene is most preferable since methylol groups are easily introduced in this case.

The amount of the cross-linking agent is preferably in the range of 30 to 70% by weight of the total amount of the monomer and the crosslinking agent. The most preferable copolymer is a styrene-divinylbenzene copolymer in the invention.

The monomer and the cross-linking agent are radically polymerized in the presence of a watersoluble polymer of high concentration in the invention. An example of the water-soluble polymer is polyethylene oxide, polyvinyl alcohol, saponified polyvinyl acetates with various saponification degrees, polyvinyl pyrrolidone, methyl cellulose or the like. The amount of the water-soluble polymer is preferably in the range of 5 to 60 parts by weight, more preferably 10 to 40 parts by weight to 100 parts by weight of the mixture of the monomer and the cross-linking agent in order to provide the resulting copolymer with good hydrophilicity.With less than 5 parts by weight of the water-soluble polymer sufficient hydrophilicity is not obtained, and with more than 60 parts by weight of the water-soluble polymer a completely spherical minute bead is not obtained and there are some troubles in the introduction of methylol groups to the resulting bead since the viscosity of the system is increased. Into the aqueous solution of the water-soluble polymer may be added a small amount of a suspending agent of phosphate such as hydroxyapatite and the like or an anionic surfactant.

The diameter of the copolymer can be regulated in the invention by the kinds and amounts of the water-soluble polymer, the proportion of the amounts of the monomer and water, stirring strength, the amount of the surfactant and the like. The preferable substrate in the invention is a spherical bead having a diameter in the range of 1 to 308.

The pore regulator is used for controlling the pore size of the substrate and may be an inert organic solvent which is soluble in the monomer.

An example of the pore regulator is an aromatic hydrocarbon such as benzene, toluene, xylene and the like, a chlorohydrocarbon such as trichloroethylene, chloroform, carbon tetrachloride and the like, an aliphatic hydrocarbon such as n-hexane, n-heptane, n-octane, n-dodecane and the like, or a mixture thereof. In the invention, the pore size may be easily controlled by varying the kinds and/or amounts of the pore regulator. Usually, 20 to 300 parts by weight of the pore regulator is preferably used to 100 parts by weight of the mixture of the monomer and the cross-linking agent.

The pore size of the substrate of the invention may be varied according to the object for use. In order to analyze hydrophilic substances having low molecular weight without adsorbing protein having high molecular weight, the exclusive molecular weight of the substrate of the invention is preferably less than 30,000, that is, the molecular weight of serum protein. On the other hand, in order to adsorb protein having the molecular weight of 30,000 or more while analyzing the hydrophilic substances having low molecular weight it is preferable to be at least 30,000.

An initiator of polymerization may be the conventional initiator used in the radical suspension polymerization of vinyl monomer, for example, an organic peroxide such as benzoyl peroxide and butyl perbenzoate, an azocompound such as azobisisobutyronitrile, and the like. Benzoyl peroxide is preferably used since the water-soluble polymer may graft easily.

When the obtained porous cross-linked copolymer on the surface of which the watersoluble polymer is attached or grafted is used for a substrate, a hydrophilic substance is difficult to diffuse into the pores since hydrophilicity is not yet sufficiently high, although the copolymer may be dispersed into water. Furthermore, the copolymer is highly viscous due to the watersoluble polymer present on the surface thereof, and the pressure increases on elution resulting in the low flow rate of eluent and low reproducibility of elution when packed in a column.

Accordingly, in the invention methylol groups are introduced in the copolymer in order to solve such a defect.

A method of introduction of methylol groups is known, for example, a method of chloromethylating the copolymer followed by hydrolysis. An example of the method of chloromethylation is a method using formaldehyde or a derivative thereof and hydrochloric acid and zinc chloride as a catalyst, a method using chloromethyl methyl ether in the presence of aluminium chloride or tin tetrachloride as a catalyst, or the like. In the chloromethylating reaction, the watersoluble polymer on the surface of the copolymer may also react and the copolymer colors more than that in the case without any water-soluble polymer resulting in the brown surface of the substrate. The water-soluble polymer reacted chemically is partially released from the substrate resulting in low viscosity of the substrate.

When chloromethyl methyl ether, for example, is used, 2 to 20 parts by weight of chloromethyl methyl ether is preferably used per one part by weight of the cross-linked copolymer. 3 to 10 parts by weight of chloromethyl methyl ether is more preferably used in view of homogenious stirring and the loss of chloromethyl methyl ether due to evaporation in the course of reaction. The most preferable weight ratio of chloromethyl methyl ether to the cross-linked copolymer is about 5:1. The preferable catalyst is anhydrous SnOl4 in view of catalyzing ability and easy treatment after the chloromethylation. The amount of the catalyst is usually in the range of 0.5 to 30 parts by weight per 100 parts by weight of chloromethyl methyl ether and may be selected suitably in view of the degree of chloromethylation.The reaction is usually carried out at a temperature in the range of O to 580C (the boiling point of chloromethyl methyl ether).

The degree of chloromethylation of the copolymer is preferably 0.2 chloromethyl groups or more per one aromatic moiety.

After chloromethylation, chloromethyl groups are converted to methylol groups (-CH2OH) by hydrolyzing in an alkaline condition at room temperature or more. In the H2O-NaOH system, the reaction rate is low and the addition of methanol accelerates the reaction rate. The concentration of an alkali the amount of methanol, the temperature and the reaction time may be selected according to the desired introduction of methylol groups.

The degree of hydrolysis of chloromethyl groups is preferably about 50% or more.

Accordingly the number of methylol groups introduced in the substrate is preferably 0.1 to 0.5 per one aromatic moiety.

The obtained substrate of the invention has the outer surface which is highly hydrophilic and accordingly it can be well dispersed in an aqueous medium. The inner surface of pores in the substrate is also hydrophilic through less than the outer surface, and accordingly, low molecular weight hydrophilic substances may enter freely into the pores. The inner surface of pores is only provided with methylol groups but adhered by no water-soluble polymer, and it is more hydrophobic than the outer surface. The viscosity of the substrate of the invention is so low that a high flow rate may be obtained in a column packed with the substrate in a high-speed liquid chromatography. Furthermore, in a high-speed liquid chromatography the reproducibility is good and low molecular weight hydrophilic substances are well fractionated.

The substrate of the invention having an exclusive molecular weight of less than 30,000 and a diameter of 1 to 30 #u may be used preferably as a substrate for analyzing hydrophilic substances of low molecular weight, in particular, those contained in a sample which contains also high molecular weight substances, for example, serum protein, since it may not adsorb such high molecular weight substances, and accordingly, it may be used for continuous analysis of such a sample.

On the other hand, the substrate of the invention having exclusive molecular weight of at least 30,000 may be used in various fields, for example, a substrate for separating hydrophilic low molecular weight substances, for precolumn removing protein, for an antigen-antibody reaction or the like, since it may adsorb high molecular weight substances such as hydrophilic protein completely. In this case, the diameter of this substrate may be in the range of 1 to 30,u, although it is preferably in the range of 1 to 30 # when it is to be desired for use in a column for analyzing low molecular weight hydrophilic substances.

The invention will be illustrated in more detail with referring to the following non-limiting examples.

Example 1 In an autoclave, a solution of 12.5 g of methyl cellulose in 625 g of water containing 0.125 g of sodium lauryl sulfate was introduced, and after adding 21.8 g of styrene, 18.2 g of divinylbenzene and 60 g of toluene as a pore regulator into the autoclave, polymerization was initiated by addition of benzoyl peroxide as an initiator and carried out at 600C for 17 hours while stirring vigorously. The obtained polymer was washed thoroughly with water and then with acetone. The washed polymer was dried at 400C under reduced pressure. The diameter of the dried polymer is in the range of 3 to 20 y. By subjecting the polymer to sifting, beads of 10 to 20 y in diameter were collected.The exclusive molecular weight of the polymer was about 7000 which was determined by using polystyrene of known molecular weight and tetrahydrofuran as an eluent.

In 35 ml of chloromethyl methyl ether, 5 g of the beads and 1.5 ml of anhydrous tin tetrachloride were added, and the mixture was heated under a reflux condenser for 6 hours at about 50 to 600 C. The color of the beads became darker into blackish brown with the progress of reaction.

After the reaction was over, the beads were washed several times in methanol containing hydrochloric acid and then with acetone to be yellow ocher in color. The degree of chloromethylation of the beads was about 0.62 chloromethyl group per one aromatic moiety, the degree being determined by comparing the infrared absorption peaks at 1600 cm-' due to aromatic moiety and at 1260 cm~' due to chloromethyl group with a calibration curve obtained from mixtures of cumene and p-isopropylbenzyl chloride of different mixing ratios.

The chloromethylated beads were heated in an aqueous 10% solution of sodium hydroxide at 600C for 9 hours to carry out hydrolysis. In the hydrolyzed beads, the strength of infrared absorption peak at 1260 cm~' due to chloromethyl group was reduced, and on the other hand, an absorption peak appeared at 1090 to 1100 cm-' due to C--O group. From the extent of reduction of the strength of peak 1260 cm-1, it was found that about 55% of chloromethyl groups had been converted to methylol groups, that is, the introduction of methylol groups into the beads was about 0.34 per one aromatic moiety.

The obtained beads of the invention was dispersed extremely well in water and various buffer solutions without recognizable coagulation or agglomeration.

After packing a stainless steel tube of 4 mm in diameter and 500 mm in length with the substrate of the invention, 20 ,uI of a mixture of paminobenzoic acid, creatinine and uric acid was subjected to chromatography while using the column and an aqueous 1/20 M phosphoric buffer solution as an eluent. The conditions of chromatography were as follows; detector of ultraviolet at 250 nm, elution rate of 1 ml/min and a pressure of 20 kg/cm2. The elution time was 10.4 min to p-aminobenzoic acid, 7.5 min to creatinine and 5.5 min to uric acid.

On the other hand, a mixture of bovine serum albumin and the three components mentioned above was subjected to chromatography on the same column. Albumin was eluted at first and the four components were completely separated from each other (refer to Fig. 1 which shows the elution curve). On repeating the chromatographic operation, the adsorption of albumin was observed only a little in the first and second repetition, however, not observed thereafter at all.

Consequently, almost the same result was obtained thereafter.

Namely, it was found that the substrate of the invention was extremely suitable for analyzing water-soluble substances of low molecular weight and did not adsorb a protein of high molecular weight.

Comparative example 1 After packing the same stainless steel tube with the polymer prepared in Example 1 but not yet chloromethylated, the same specimen as in Example 1 was subjected to chromatography. The pressure showed a value higher than 80 kg/cm2 at a flow rate of 1 ml/min, showing the large effect of the viscousness of methyl cellulose adhered on the inner surface of pores in the polymer. The four components of the specimen were almost not separated from each other and eluted very rapidly.

Comparative example 2 Five grams of the polymer prepared in Example 1 was chloromethylated in the same method as in Example 1 except for the reaction time of 24 hours instead of 6 hours in Example 1 to obtain the beads of about 64% of the degree of chloromethylation. The beads were hydrolyzed in a solution of 25 g of sodium hydroxide in 100 g of methanol at 600C for 15 hours to obtain the substrate with about 0.58 (mean) methylol group per one aromatic moiety.

After packing the same stainless steel tube as in Example 1 with the substrate, a mixture of bovine serum albumin, p-aminobenzoic acid, creatinine and uric acid was subjected to chromatography while using the column at a flow rate of the same eluent of 1 mI/min, the elution times were as follows p-aminobenzoic acid of 6.4 min, creatinine of 5.9 min and uric acid of 4.8 min.

Namely, the elution time was shorter than in Example 1, and p-aminobenzoic acid and creatinine could not be separated from each other.

The elution curve of the chromatography mentioned above is shown in Fig. 2. The results show the interaction between the hydrophilic substances of low molecular weight and the inner surface of pores in the substrate was larger in the substrate of the invention than in the substrate of this Comparative Example 2.

Example 2 In a similar manner to that in Example 1, 21.8 g of styrene and 18.2 of divinylbenzene were polymerized in the presence of 56.4 g of toluene and 3.6 g of n-dodecane as the pore regulator to obtain a styrene-divinylbenzene copolymer of an exclusive molecular weight of about 16,000. The copolymer was subjected to chloromethylation in the same conditions as in Example 1 except for 2 hours of reaction time instead of 6 hours in Example 1 to obtain a chloromethylated copolymer having an extent of chloromethylation of about 0.42 chloromethyl groups per one aromatic moiety. By subjecting the copolymer to hydrolysis in an aqueous 10% solution of sodium hydroxide at 600C for about 5 hours, a substrate containing methylol groups of about 0.2 (mean) per one aromatic moiety was obtained.

The column prepared by packing the same stainless steel tube as in Example 1 with the obtained substrate of the invention showed about 25 kg/cm2 of pressure at a flow rate of 1 ml/min of an aqueous 1/20 M phosphoric buffer solution.

The same amount, 20 yl, of a mixture of uric acid, creatinine and p-aminobenzoic acid was subjected to chromatography while using the column. The three components were completely separated. Test on a mixture of bovine serum albumin and the three components mentioned above showed that albumin was not adsorbed onto the column at all.

Example 3 Two grams of methyl cellulose was dissolved in 125 g of water, and 0.025 g of sodium lauryl sulfate was added. After the mixture was charged into an ampoule of 300 ml, into the ampoule were added 3.27 g of styrene, 2.73 g of divinylbenzene and 6.75 g of toluene and 2.25 9 of ndodecane as pore regulators, the mixture was then stirred at 600C for 1 7 hours to react while using benzoyl peroxide as an initiator. The resultant beads were washed with water and acetone and then dried at room temperature under reduced pressure. The diameter of the spherical beads was substantially uniform and about 20 y.

The exclusive molecular weight of the bead was about 100,000, this value being measured by eluting polystyrene having various predetermined molecular weights on a column packed with the beads.

In a reaction vessel 5 g of beads was placed followed by adding 35 ml of chloromethyl methyl ether and 1.5 ml of anhydrous tin tetrachloride. The mixture was then refluxed at 50 to 600C for 6 hours to chioromethylate the beads.

The beads colored progressively to dark brown with the reaction. The beads were washed after reaction with methanol containing hydrochloric acid repeatedly and finally with acetone. The color of the beads was finally yellow brown. The new absorption peaks appeared at 1260 and 670 cm-' in the infrared absorption spectrum of the beads after chloromethylation. The degree of chloromethylation was about 0.5 chloromethyl groups per one aromatic moiety.

The chloromethylated beads were added into a solution of 25 g of sodium hydroxide in 100 g of methanol, and the solution was maintained at 600C for 15 hours to react. The new I.R.

absorption peak was appeared at 1090 cm~' due to the introduction of-CH20H groups. The number of methylol groups was 0.3 per one aromatic moiety.

The obtained beads were dispersed very well in water various buffer solutions, urine or the like without aggregation.

The beads were packed into a stainless steel column of 4 mm in diameter and 500 mm in length for a high-speed liquid chromatography. A solution of bovine serum albumin in 1/20 M phosphoric buffer was subjected to the highspeed liquid chromatography using the phosphoric buffer as an eluent at a flow rate of 1 ml/min. No alubumin was eluted. Further, no albumin was eluted even after passing of 50 ,uI of 10% bovine serum albumin 50 times repeatedly.

Twenty 410f a sample of a solution of uric acid, creatinine, p-aminobenzoic acid and albumin in 1/20 M phosphoric buffer was passed though the column. Uric acid, creatinine and p-aminobenzoic acid were completely separated from one another while no albumin was eluted. Refer to Fig. 3 which shows a chart recording elution peaks at a chart speed of 1 cm/3 min and measured by wave length of 250 nm. The pressure was 25 kg/cm2 at the flow rate of 1 ml/min.

Example 4 A styrene-divinylbenzene copolymer was prepared in the same manner as in Example 1 except for using 45 g of toluene and 1 5 g of n dodecane as the pore regulators instead of 60 g of toluene. The exclusive molecular weight of the obtained polymer was about 100,000. Then methylol groups were introduced into the polymer in the same manner as in Example 1 to an extent of about 0.4 methylol groups per one aromatic moiety.

After packing the same tube as in Example 1 with the obtained substrate, albumin was subjected to chromatography, however, albumin was adsorbed onto the column and did not eluted.

Comparative example 3 A styrene-divinylbenzene copolymer was prepared in the same manner as Example 3 while using 13.9 g of calcium phosphate and 4.07 mg of sodium n-dodecylbenzenesu Ifonate instead of methyl cellulose and sodium lauryl sulfate of Example 3. After washing with water the copolymer was sieved to obtain beads having diameter of 10 to 20 y. However, the distribution of the bead diameter is broad. The exclusive molecular weight was about 100,000.

The obtained beads were washed thoroughly with hydrochloric acid to remove calcium phosphate. The beads were not dispersed in water at all and were agglutinated.

After drying the beads, chloromethylation was carried out as in Example 3. The beads colored only slightly to light yellow finally. Chloromethyl groups were converted to methylol groups as in Example 3. The obtained beads were less hydrophilic than those of Example 3 and the dispersion into water or urine was not good. In a column packed with the beads, albumin was not completely adsorbed and a very broad peak of albumin was obtained. The reason is considered that the beads is less hydrophilic than those of Example 3 and protein can not enter into the pores.

Example 5 Porous minute beads of styrene-divinylbenzene copolymer having the exclusive molecular weight of 300,000 were prepared in the same manner as in Example 3 while using 4.5 g of toluene and 4.5 g of n-dodecane as pore regulators. The beads were treated in the same manner as in Example 3 to obtain substrate having methylol groups. The obtained substrate was dispersed well in serum and urine. In the column packed with the substrate, albumin was not eluted even after passing 50 yl of 10% bovine serum albumin solution through the column 50 times repeatedly.

Claims

1. A porous substrate suitable for analyzing hydrophilic substances having low molecular weight, which substrate comprises a cross-linked copolymer having methylol groups, the copolymer being derived from a styrene monomer and a cross-linking agent copolymerizable with the monomer, the outer surface of the substrate being hydrophilic and the inner surface of pores of the substrate being less hydrophilic than the outer surface of the substrate.

2. A substrate according to claim 1, in which the styrene monomer is styrene, a-methylstyrene, chloromethylstyrene or a mixture of two or more thereof.

3. A substrate according to claim 1 or 2, in which the cross-linking agent is selected from divinylbenzene trivinylbenzene, triallyl isocyanurate, dimethacrylates and diacrylates of polyhydric alcohols and diallyl phthalate.

4. A substrate according to any one of the preceding claims, in which the copolymer is a styrenedivinylbenzene copolymer.

5. A substrate according to any one of the preceding claims, which is in the form of a spherical bead having a diameter of from 1 to 30 y.

6. A substrate according to any one of the preceding claims in which the cross-linked copolymer has an exclusive molecular weight of less than 30,000.

7. A substrate according to any one of claims 1 to 5, in which the cross-linked copolymer has an exclusive molecular weight of at least 30,000 and is capable of adsorbing protein.

8. A substrate according to any one of the preceding claims in which the cross-linked copolymer has 0.1 to 0.5 methylol groups per aromatic moiety.

9. A substrate according to any one of the preceding claims, which is for use in a liquid chromatography.

10. A porous substrate suitable for analyzing hydrophilic substances having low molecular weight and being substantially as hereinbefore described in any one of Examples 1 to 5.

11. A process for preparing a porous substrate as claimed in any one of the preceding claims, which process comprises subjecting the styrene monomer and the copolymerizable cross-linking agent to suspension polymerization in water in the presence of a pore regulator and a watersoluble polymer and providing the resultant crosslinked copolymer with methylol groups.

12. A process according to claim 11, wherein the amount of the cross-linking agent is from 30 to 70% by weight of total weight of the styrene monomer and the cross-linking agent.

13. A process according to claim 11 or 12, wherein the water-soluble polymer is selected from polyethylene oxide, saponified polyvinyl acetates, polyvinyl alcohol, polyvinyl pyrrolidone and methyl cellulose.

14. A process according to any one of claims 11 to 13, wherein the amount of water-soluble polymer is from 5 to 60 parts by weight per 100 parts by weight of the total amount of the styrene monomer and the cross-linking agent.

15. A process according to claim 14, wherein the amount of the water-soluble polymer is from 10 to 40 parts by weight per 100 parts by weight of the total amount of the styrene monomer and the cross-linking agent.

16. A process according to any one of claims 11 to 15, wherein the pore regulator is an inert organic solvent soluble in the monomer.

17. A process according to claim 16, wherein the pore regulator is an aromatic hydrocarbon, a chlorohydrocarbon, an aliphatic hydrocarbon or a mixture of two or more thereof.

18. A process according to claim 17, wherein the pore regulator is benzene, toluene, xylene, trichloroethylene, chloroform, carbon tetrachloride, n-hexane,n-heptane, n-octane or ndodecane or a mixture of two or more thereof.

19. A process according to any one of claims 11 to 1 8, wherein the amount of the pore regulator is from 20 to 300 parts by weight per 100 parts by weight of the total amount of the styrene monomer and the cross-linking agent.

20. A process according to any one of claims 11 to 19, wherein the resultant cross-linked copolymer is provided with methylol groups by chloromethylating the copolymer followed by hydrolysis.

21. A process for the preparation of a porous substrate suitable for analyzing hydrophilic substances having low molecular weight, said process being substantially as hereinbefore described in any one of Examples 1 to 5.

22. A method for analyzing hydrophilic substances having low molecular weight, which method comprises contacting a porous substrate as claimed in any one of claims 1 to 10 or which has been produced by a process as claimed in any one of claims 11 to 21 with the hydrophilic substances under analysis.

23. A method according to claim 22 which is a liquid chromatography method.

24. A method according to claim 22 or 23 in which the substances under anlysis are separated.

25. A method of separating substances by liquic chromatography substantially as hereinbefore described in any one of Examples 1 to 3.