WO1992015385A1

WO1992015385A1 - Butadiene acrylonitrile polymeric coating and chromatographic packing material

Info

Publication number: WO1992015385A1
Application number: PCT/US1992/001822
Authority: WO
Inventors: Ethan S. Simon; Kevin B. Holland; Christopher Mcclanahan
Original assignee: Simon Ethan S; Holland Kevin B; Christopher Mcclanahan
Priority date: 1991-03-04
Filing date: 1992-03-04
Publication date: 1992-09-17
Also published as: CA2102425A1

Abstract

A chromatographic packing material includes a coated support material which is a chromatographically suitable substrate. An immobilized butadiene acrylonitrile polymer coating is provided on the substrate. The copolymer can be crosslinked by gamma radiation, or by means of a crosslinking agent such as dicumyl peroxide. The support material can be silica, alumina, diatomaceous earth, zeolite, porous glass or carbon, but preferably is spherical lamellar shaped crystals of aluminum hydroxide. The aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core with a particle density ranging from 0.3 to 2.5 g/cm3 and a diameter of 2 to 150 microns. The organic materials are separated by providing a bed of packing material selected from the group consisting of silica and alumina, diatomaceous earth, zeolite and porous glass with a polymeric coating being bonded thereto. Organic materials are introduced to the bed, and an eluting fluid is added. The fluid and one of the organic materials are removed from bed, and the materials are then separated and removed from the fluid. A chromatographic packing material is disclosed comprising a coated support material which is a chromatographically suitable substrate, with an uniform immobilized functionalized coating. A chromatographic column is disclosed having a stationary phase with a suitable substrate coated support material and an uniform immobilized functionalized coating. A stationary phase for reversed-phase liquid chromatography consisting of a crosslinked, polybutadiene-coated, macroporous, alumina substrate is disclosed wherein the micropores have a diameter predominantely in the range of 50 to 1000 Angstroms, and the alumina substrate is so occludingly coated with polybutadiene that the integrity of the stationary phase is not adversely affected by extended exposure to liquid environments having a pH of 12. The method for preparing a stationary phase for liquid chromatography by occludingly coating onto a macroporous alumina substrate having micropores of a diameter predominantely in the range of 90 to 500 Angstroms, an unsaturated polybutadiene oligomer having pendant vinyl groups and a molecular weight less than 50,000 Daltons. The polybutadiene oligomer is partially crosslinked to the substrate to provide a polybutadiene coating having pendant vinyl groups. The hydrophobicity of the polybutadiene coating is increased by providing alkyl groups onto said polybutadiene coating, so that the retention time of o-xylene is increased by at least 30 % over retention time of a stationary phase coated with polybutadiene having 18 mole percent vinyl groups per butadiene unit.

Description

BUTADIENE ACRYLONITRILE POLYMERIC COATING AND CHROMATOGRAPHIC PACKING MATERIAL BACKGROUND OF THE INVENTION Field of the Invention In one embodiment, this invention relates to the immobi- lization of hydrocarbonaceous polymers on inorganic support materials for subsequent use as chromatographic stationary phases. More specifically, this invention relates to the coating of a polymer onto a metal oxide followed by in situ crosslin ing of the polymer thereby producing stationary phases that exhibit unique chromatographic selectivities and excellent pH and chemi- cal stability. In a second embodiment the invention relates to the i mobi- lization and subsequent functionalization of a hydrocarbonaceous polymers on inorganic support materials for subsequent use as chromatographic stationary phases. More specifically, this inven- tion relates to the coating of a polymer onto a metal oxide fol- lowed by in situ crosslinking of the polymer and chemical functionalization thereby producing stationary phases that ex- hibit unique chromatographic selectivities and excellent pH and chemical stability. Prior Art Chemically modified silica supports are currently the most widely used stationary phases for reversed-phase liquid chro atog- raphy. By reversed-phase chromatography it is meant that the ad- sorbent is less polar than the eluting solvent, and in normal phase chromatography the adsorbent is more polar than the eluting solvent. That is, in reversed-phase chromatography, the more non-polar sample components interact more with the relatively non-polar stationary phase and thus elute later than polar sample components. Typical mobile phases for reversed phased chromatog- raphy are aqueous buffers, water, methanol, acetonitrile, tetrahydrofuran, and mixtures of water or buffer with these or- ganic solvents. However, these alkyl-bonded silica-based materials suffer from two major limitations: first, residual silanol groups fre- quently have adverse effects on chromatographic performance, and second, silica-based materials are stable only over a pH range of 2 - 8.5. In particular, of the various commercially available silica-based stationary phases, cyano bonded phases are con- sidered to be the least rugged. Polymeric supports exhibit enhanced pH stability but are often limited by their lack of structural rigidity and low efficiencies due to the poor dif- fusional properties of solutes in these materials. As a result of the aforementioned difficulties, attention has been given to an approach that involves the deposition of a hydrophobic, chemically stable polymer onto the surface of an in- organic carrier followed by a radical-initiated cross-linking reaction which serves to immobilize a thin layer of polymer on the surface of the support. This general approach has been taken by various researchers (e.g. Schomburg, Regnier, etc.) but, to date, no material has been reported which exhibits the combina- tion of acid and base stability, high efficiency and good chromatographic selectivities, especially for macrocyclic an- tiobiotics. Macrocyclic antibiotics, such as erythromycin are widely used and improved methods for their analysis and separa- tion are highly desirable for macrocyclic antibiotics. Methods for coating inorganic supports with polymers to create supports for chromatography are known. Schnecko and Bieber (Schnecko, H. and Bieber, O. Die Anσewandte Mackromolekulare Chemie 1971, 20, 111-119) describe coating Chromosorb P with polymers including polybutadiene and hydroxy-terminated polybutadienes and nitrile rubber, as well as dibromo polybutadiene after amine treatment. The use of dicumyl peroxide is disclosed as an agent for subse- quently crosslinking the polymers to create stationary phases for gas chromatography. This disclosure in inapplicable to the in- stant invention because of the differences between gas and liquid often developed with the chromatography columns. The high pres- sure liquid within the column subjects the base material to condi- tions which are much more severe, or substantially different from those experienced in gas chromatography. Unlike liquid chromatog- raphy, gas chromatography can not be applied to the separation of solids, such as proteins and peptides, because they can not be entrained in the mobile gas phase. Schomburg et al. (Schomburg, G. ; Kohler, J; Figge, H. ; Deege, A.; Bien-Vogelsang, U. , Chromatoσraphia 1984, .18., 265-274) report the immobilization of polymers on particles of silica and alumina using Co irradiation to prepare stationary phases for liquid chromatography and describe further improvements in subse- quent publications (including Bien-Vogelsang, U. ; Deege, A. ; Figge, H. ; Khler, J.; Schomburg, G., Chromato raphia 1984, 19, 170-179; Figge, H. ; Deege, A.; Kohler, J.; Schomburg, G. _____ Chromatography 1986, 351. 393-408; Kolla, P, Kohler, J. Schom- burg, G. Chromatoσraphia 1987, 2_3, 465-472). Kosaka et al. (U.S. Patent 4,054,353, Aug. 30, 1977) describe the radiation-induced crosslinking of monomers on the surface of inorganic substrates and simple chemical modification of some polymers, such as sulfonation of styrene. Berezkin et al. (Berezkin, V.G. ; Kolbanovskii, Yu. A.; Kyazimov, E.A. Zh. Fiz. Khim. 1966, 4JD, 1921) also describe modifying supports by depositing monomers and crosslinking by irradiation. Regnier et al. in a series of publications and in a patent (U.S. Patent 4,245,005, Jan. 13, 1981) describe the adsorbtion of coatings, such as amines, to inorganic supports and then the crosslinking of the coatings by chemical means to create εtation- ary phases suitable for ion-exchange chromatography. A disadvantage of these methods is that they produce station- ary phases which exhibit chromatographic behavior different froc that of commonly-used, commercially-available materials. There is a great reluctance among those who practice chromatography to use stationary phases that exhibit unfamiliar behavior because of certain stationary phases is not applicable. Accordingly, it would be very desirable to have a stationary phase that exhibits chromatographic behavior similar to commonly-used, commercially- available materials, but which also exhibits improved chemical and mechanical stability to alleviate the deficiencies of inor- ganic supports coated with organosilanes and of polymeric sup- ports. The materials described by this invention may be shown to exhibit excellent stability under acidic and basic conditions, high efficiency and good chromatographic selectivities especially for proteins and peptides. SUMMARY OF THE INVENTION In accordance with the present invention, chromatographic stationary phases are provided which consist of a thin layer of crosslinked polymer on an inorganic support. These materials are shown to overcome many of the disadvantages associated with chemi- cally bonded metal oxides and polymeric materials. The com- posites herein described exhibit a high degree of pH and chemical stability while providing a surface chemistry that is ideally suited for the separation of classes of compounds such as an- tibiotics and complex carbohydrates. The process for preparing such chromatographic stationary phases can involve the in situ chemical modification of the coated/crosslinked polymer in order to produce a surface chemistry that is tailored to a particular chromatographic separa- tion. The chromatographic packing material includes a coated sup- port material. The coated support material is a chromatographi- cally suitable substrate, having a uniform immobilized coating. The coating is a butadiene acrylonitrile copolymer. The packing material is preferably employed in reversed-phase chromatography. The copolymer is crosslinked, preferably through gamma radia- tion, or through the use of a photoinitiator or thermally. The copolymer can contain a thermal initiator, as for example dicumyl The support material can be any of the particles used for this purport, as well known in the art, as for example, silica, alumina diatomaceous earth, zeolite or porous glass. The preferred support material is aluminum hydroxide particles. The aluminum hydroxide particles are preferably spherical lamellar shaped crystals. The crystals are preferably bonded together at a central core and extend radially outwardly from a central core. The particle density can range from 0.3 to 2.5 g/cm³, and the par- ticle diameter can be in the range from 2 to 150 microns. The copolymer can be derived from a liquid copolymer which contains pendent reactive groups and can be carboxyl terminated. The carboxyl terminated copolymers may be considered to be long chain dicarboxlyic acids having functionalities between about 1.8 and 2.4. The copolymer can be derived from a liquid copolymer which is vinyl terminated and have reactive acrylate vinyl groups. Preferably the copolymer is predominantly butadiene, with the butadiene to acrylonitrile ratio being on the order of 5 to 1. The ratio, however can be from about 1:1 to about 10:1. Also in accordance with the present invention, chromatographic stationary phases are provided which consist of a functionalized thin layer of crosslinked polymer on an inorganic support. These materials are shown to overcome many of the disad- vantages associated with chemically bonded metal oxides and polymeric materials. The composites herein described exhibit a high degree of pH and chemical stability while providing a sur- face chemistry that is ideally suited for the separation of classes of compounds such as proteins and peptides. The process for preparing such chromatographic stationary phases involves the chemical modification of a polymer-coated in- organic support in order to produce a surface chemistry that is tailored to a particular chromatographic separation. The chromatographic packing material is a coated chromato- graphically suitable substrate, and immobilized functionalized coating on said substrate, said coating being a polymer having employed in reversed-phase chromatography. The polymer can be butadiene or a butadiene acrylonitrile copolymer, or other polymers, as well known in the art. The grafted moieties or monomeric groups can be octadecene or octene. The support material can be any of the well known materials used for this purpose, as for example, silica, alumina, diatomaceous earth, zeolite or porous glass. Preferably, the sup- port material is aluminum hydroxide particles, which are spheri- cal lamellar shaped crystals. Preferably, the aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core. The particle density can range from 0.3 to 2.5 and the particle diameter is from about^' 2 to 150 microns. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows chromatograms obtained before and after ex- posure to pH 10,5 mobile phase. Figure 2 shows the separation of several macrolide an- tibiotics at pH 11.0. Figure 3 shows the separation of several penicillin an- tibiotics at pH 3.0. DESCRIPTION OF THE PREFERRED EMBODIMENTS The chromatographic stationary phases comprise an inorganic carrier onto which has been coated and crosslinked a layer of or- ganic polymer. The inorganic carriers that may be used in the present invention include, but are not necessarily limited to silica, silica gels, glass, carbon, bentonite, hydroxyapatite, zirconia, titania and alumina. The preferred carrier is alumina having a known average pore size, known particle size and known surface area. It is preferred that the alumina has an average pore size of 50 - 1000 Angstroms, a surface area of 5 - 250 m²/g, preferably 40-100 m /g, and a particle size of 3 - 25 microns. The primary requirement for the inorganic carriers is that they be essentially water insoluble and have sufficient surface area ² The polymeric coating may be applied to the inorganic sup- port by known methods referred to above. The organic polymers employed in the present invention include, but are not neces- sarily limited to poly(butadiene) , poly(butadiene-acrylonitrile) and others. The primary requirements for the polymers are that they be easily solubilized to facilitate the coating process and that they possess chemical functionalities, such as unsaturated carbon-carbon bonds, which allow crosslinking and the subsequent grafting of monomers to the polymer. The preferred polymeric coat- ing consists of crosslinked polybutadiene. Further details of carrier materials are found in the prior art, as for example U.S. Patents 4,786,628, 4,822,593, and 4,045,353, and the disclosures of which are incorporated herein by reference or in the book, "Packing and Stationary Phases in Chromatographic Techniques" edited by K. K. Unger (Marcel Dekker, 1990) . The organic polymers employed in the present invention in- elude, but are not necessarily limited to the range of Hycar Reac- tive Liquid Polymers available from B.F. Goodrich, Inc. (e.g. Hycar 1300X40, Hycar 1300X43, etc.). The primary practical re- quirements for the polymers are that they be easily solubilized to facilitate the coating process and that they possess chemical functionalities which allow crosslinking and/or chemical grafting reactions. In order to produce the stationary phases described here, a solution is prepared containing typically 5-50% (w/w relative to the weight of support being used) of polymer in a suitable sol- vent (e.g. tetrahydrofuran, ethyl acetate) . To the solution is also added any necessary radical initiators or stabilizers at a level of 0-13% w/w. Inorganic carrier, such as alumina, is added to the solution in a round-bottomed flask and shaken for several minutes. The solvent is then removed by evaporation at reduced pressure using a rotary evaporator until the material is free- flowing. The polymer-coated support is then subjected to a crosslink- ing reaction using a free radical initiator (e.g. dicumyl peroxide) at elevated temperatures or by use of gamma irradiation from a ^oυCo source. After crosslinking, the materials are typi- cally washed with 1% glacial acetic in hexane or ethyl acetate followed by a wash with hexane. The washed material is then dried and packed into columns. Specific details for particular stationary phases are included in Typically, a monomer and additive, if used, are coated onto a polymer-coated inorganic support followed by irradiation using a ⁶⁰Co source which serves to graft the monomer onto the polymer coating. The monomers, such as 1-octadecene, preferably contain un- saturation and do not contain chemical functionalities which are unstable in the pH range of 1-13. The amount of monomer used is 5-50% (w/w) relative to the amount of support used. To create a reversed-phase stationary phase suitable for the separation of peptides and proteins, the preferred monomer is 1-octadecene. The additives are free radical initiators such as peroxides (e.g. dicumyl peroxide or benzoyl peroxide), or free radical sta- bilizers (such as allyl methacrylate or N-allyl acrylamide) . The amount of additive used is 1-15% (w/w) relative to the amount of monomer used. The grafting process is preferably carried out by mixing the monomer, additive and support for 5-15 minutes in a solvent that dissolves the monomer and additive, and then by removing the sol- vent by rotary evaporation at reduced pressure until the material is a relatively free-flowing powder. This step coats the support with the monomer and additive. The grafting reaction can be carried out either at elevated temperatures, or by using gamma-irradiation either in the presence of air or nitrogen. If irradiation from a ⁶⁰Co source is used, the total dose of radiation is preferably in the range Following the grafting step, the supports should be washed with a suitable solvent, such as hexane, to remove unbound monomer and additives so that the material is suitable for chromatographic purposes. In the case of polymer-coated alumina materials, a solution of 1% glacial acetic acid in hexane is ef- fective in removing unbound material and preventing subsequent leaching of unbound material during chromatography. Examples 1 - 7 below. Specific details for the preparation of materials and their use in chromatography are given in Ex- amples 8 - 14 below. EXAMPLE 1 This example describes a process for coating of alumina with poly(butadiene-acrylonitrile) by irradiation. Poly(butadiene-acrylonitrile) (25 g, Hycar VTBNX (1300X43), B.F. Goodrich Co., Cleveland, OH) and dicumyl peroxide (2.5 g, Polysciences, Inc., arrington, PA) were dissolved in 5.00 L of ethyl acetate. Sonication aided the dissolution of the polymer. The slightly cloudy solution was added to a 1-L round-bottomed flask containing 250 g of alumina powder (8 micron Unisphere^R alumina, Biotage, Inc., Charlottesville, VA) and the suspension was shaken for 15 min. Removal of solvent by rotary evaporation at reduced pressure yielded a free-flowing powder. The flask containing the coated powder was placed near a ⁶⁰Co array for 24 h. at a dosage rate of 2 x 10⁵ Rad/h. Following irradiation, the sample was washed with -3 mL of a solution of hexane containing 1% of acetic acid per gram of alumina and then with -3 mL of hexane per gram of alumina. The product was dried either at reduced pressure to give a free- flowing powder which was used to effect a range of separations by high-performance liquid chromatography as described in Examples 2-4. The ethyl acetate of this example can be replaced by other solvents, such as tetrahydrofuran, methyl ethyl ketone, or aromatic solvents, as well known to those skilled in the art. In this example, the exact total radiation dosage is not critical. Increasing the total time of irradiation up to 7 days did not affect the subsequent chromatographic performance of the product. Other °Co sources could also be used advantageously. In this example, the solvents used to wash the product can be advantageously replaced by other solvents, such as ethyl acetate, and other solvents suitable for solubilizing poly(butadiene-acrylonitrile) or dicumyl peroxide and their degradation products. It should be understood that additives other than dicumyl peroxide or no additives may be added to affect the crosslinking step. Examples 6 and 8 illustrate that this generalization is pos- sible. Crosslinking can be achieved by means other than irradia- tion, such as thermal or photochemical treatment. Example 5 il- lustrates the use of thermal crosslinking. EXAMPLE 2 Chromatographic performance of the alumina-based cyano sta- tionary phase. A 3.5 g quantity of pol (butadiene-acrylonitrile)-coated alumina prepared as in Example 1 above was packed into a 4.6 mm i.d. x 250 mm stainless steel column using methanol at a pressure of 6000 psi. A test mixture consisting of theophylline, p-nitroaniline, methyl benzoate, phenetole, and o-xylene (1 mg/mL each in 50% aqueous acetonitrile) was prepared and injected onto the column. The test mixture components were eluted using a mobile phase of 45% water and 55% acetonitrile at a flow rate of 0.5 mL/min. The efficiency of the column was found to be 35,500 plates/meter with an o-xylene retention of 13.5 minutes. The pH of the water portion of the mobile phase was raised to 10.5 with aqueous ammonia and after operation at this pH for 24 hours the efficiency was found to be 35,200 plates/meter and the o-xylene retention 13.4 minutes. This represents <1% loss of retention and efficiency. These chromatographic measurements showing the high level of performance of the material at high pH are shown in Figure 1. EXAMPLE 3 The chromatographic performance of the material described in Example 2 is further illustrated in Figure 2. This chromatogram shows the separation of a mixture of macrolide antiobiotics and was generated using a mobile phase of 80% 0.02M KH₂P0₄ 20% acetonitrile, apparent pH 10.9 and a flow rate of 1.2 mL/min. The samples chromatographed are indicated in the figure caption. After operation for 72 hours at pH 10.9, the measured loss of column efficiency was found to be less than 1%. Example 4 The same column as was used in Example 3 was used for the separa- tion of a set of penicillins (Figure 3) using a mobile phase of 72% .015-M phosphate buffer (pH 3.0) and 28% acetonitrile. Thus the invention exhibits good stability over a wide pH range with good separation efficiency. Example 5. This example describes a process for coating of alumina with poly (butadiene-acrylonitrile) by thermal treatment. 1 g of Hycar VTBNX (1300X43) and 0.1 g of dicumyl peroxide were dissolved in 30 mL of ethyl acetate. The resulting solution was added to 10 g of 8 micron Unisphere alumina in a 100-mL round-bottomed flask and the suspension was shaken for 15 min. Removal of solvent by rotary evaporation at reduced pressure yielded a free flowing powder. The powder was heated in an atmosphere of nitrogen at 110°C for 30 minutes an then at 140°C for 3 h. The reaction flask was allowed to cool to room temperature under a positive pressure of nitrogen and then washed and used to effect separations following Example 2. The retention time of o-xylene was 11 min. and the efficiency was 17,000 plates/meter. When the same alumina was coated with polybutadiene the retention time of o-xylene was 9.6 Example 6 The procedure described in Example 1 was carried out, except the reactants were 0.5 g of poly (butadiene-acrylonitrile) ,0.5 g of allyl methacrylate and 5 g of alumina. The retention time of o-xylene was 14.1 min, and the efficiency was 25,000 plates/meter. This example showed that dicumyl peroxide may be replaced by other additives. Example 7 A solution of 2.5 g of poly(butadiene-acrylonitrile) , 0.25 g of dicumyl peroxide and 0.25 g of divinyl benzene in 175 mL of ethyl acetate was shaken with 25 g of 8 micron alumina powder for 10 min. Following removal of the solvent by rotary evaporation at reduced pressure, the material was irradiated using a °Co source at 1.65 x 10⁶ Rad/h for 24 hr. The material was washed as follows: The sample was slurried in 200 mL of ethyl acetate and soni- cated for 10 min. The solvent was removed by filtration using a Bchuer funnel. This procedure was repeated and the material was then washed with 150 L of hexane. The material was packed ac- cording to Example 2. The retention time of o-xylene was 16 min. This Example 7 shows that the addition of other crosslinking agents can alter the characteristics of the material by altering retention times. Hycar is a registered trademark of B. F. Goodrich for butadiene homopolymers and butadiene/acrylonitrile copolymers. The isomer content is largely cis/trans with vinyl (1,2 addition of butadiene) being 25 % or less. They have reactive groups in both terminal positions of the polymer chain and may have addi- tional reactive groups pendent on the chain. Some do not contain solvents or other unreactive components. The designator letter C indicates a carboxyl group, V indicates a vinyl group, A indi- cates an amine group, T indicates terminal reactive groups, B in- dicates butadiene, N indicates Acrylonitrile and X indicates the presence of pendent reactive groups. The CT series of carboxyl terminated liquid polymers may be considered long chain dicarbox- lyic acids having functionalities between 1.8 and 2.4. The typi- cal properties are as follows:

1300X13 26

32

.057

570,000

9.14

.960 1.8

3,200 The HYCAR Vinyl terminated (VT) liquid polymers have reac¬ tive acrylate vinyl groups and can be reacted into systems involv¬ ing cures by free radical mechanisms. The reactive vinyl group is separate from the cis/trans/vinyl unsaturation contributed by the polymerized butadiene of the polymer backbone. Typical properties for the methacrylated polymer is as follows:

Hycar polymer acrylonitrile % bound Acid number Brookfield Viscosity mPa.s or cP, 27° Specific gravity 25°/25°C Solubility Parameter

EXAMPLE 8 This example describes the attachment of octadecene to polybutadiene-coated alumina by irradiation. Octadecene (3.0 g) and allyl methacrylate (0.24 g) were dis- solved in 50 mL of hexane. The solution was added to a 100-mL round-bottomed flask containing 10.0 g of alumina powder coated with polybutadiene (8 micron Unisphere^R-PBD, Biotage, Inc. , Char- lottesville, VA) and a magnetic stirring bar. The solution was stirred for 15 min and the solvent was removed by rotary evapora- tion at reduced pressure. The flask was evacuated, refilled with nitrogen, and capped. The flask containing the coated powder was placed near a ⁶⁰Co array and received a total dosage of 1.6 x 10⁷ Rad during a period of 3 days. Following irradiation, the sample was washed with 100 mL of a solution of hexane containing 1% of acetic acid and then with 100 mL or hexane. The product was dried at reduced pressure to give a powder which was packed into columns and used to effect the chromatographic separations described in Examples 2 and 3. EXAMPLE 9 Description of chromatographic performance of the alumina- based C18 stationary phase. A 3.5 g quantity of polybutadiene-coated alumina prepared as in Example 1 above was packed into a 4.6 mm i.d. x 250 mm stain- less steel column using methanol at a pressure of 6000 psi. A test mixture consisting of theophylline, p-nitroaniline, methyl benzoate, phenetole, and o-xylene (1 mg/mL each in 50% aqueous acetonitrile) was prepared and injected onto the column. The test mixture components were eluted using a mobile phase of 45% water and 55% acetonitrile at a flow rate of 0.5 mL/min. The sta- tionary phase was then subjected to alternate cycles of isocratic and gradient elution separations for 72 hours using tri- flouracetic acid (TFA)/water/acetontrile mobile phases. This was TFA in 50/50 water/acetonitrile and 0.1-M NaoH in 50/50 water/ethanol. This treatment resulted in losses of less than 1% in both retention and efficiency. The data is shown in Figure 1. EXAMPLE 10 The chromatographic performance of the material described in Example 8 is further illustrated in Figures 2 and 3. The condi- tions for the separation are described in the figure captions. As can be seen from the figures, the chromatographic performance on the invention compares quite favorably with that of a widely- used, commercially available silica-based column. The combina- tion of chromatographic performance and pH stability makes the present invention far superior to any comparable material cur- rently available. EXAMPLE 11 1-Octadecene (4.5 g) and allyl acrylamide (0.75 g) were dis- solved in 50 mL of ethyl acetate. 15.0 g of alumina powder coated with polybutadiene (8 micron Unisphere®-PBD, Biotage, Inc., Charlottesville, VA) was added to the solution, which was then shaken for 10 minutes. Following evaporation of the solvent by rotary evaporation at reduced pressure, the flask was evacuated and refilled with nitrogen. The evacuation/refilling procedure was repeated twice and the flask was capped. The flask containing the coated powder was placed near a Co array and received a total dosage of 1.5 x 10 Rad during a period of 76 h. Following irradiation, the sample was washed with 50 mL of hexane, then 100 mL of hexane containing 1% of glacial acetic acid, then with 50 mL of hexane. The product was dried at reduced pressure to give a powder which was packed into columns and used to effect chromatographic separations described in Ex- ample 2. The retention time of o-xylene was 13.6 min. EXAMPLE 12 The procedure according to Example 8 is carried out, with exception that 1-octene is used in place of 1-octadecene. The material is used to effect separations and the retention times of o-xylene and angiαtensin II are less than that obtained using material from Example 8. EXAMPLE 13 The procedure according to Example 8 was carried out, but the reaction was not carried out under an atmosphere of nitrogen. The retention time of o-xylene was 13.5 min. and that of aπgioten- sin II was 24 min. EXAMPLE 14 The procedure according to Example 8 was carried out but the amount of allylmethacrylate was reduced to 5% and the reaction was not carried out under an atmosphere of nitrogen. The reten- tion time of o-xylene was 13.1 and that of angiotensin II was 22 min.

Claims

What is claimed is:

CLAIM 1. A chromatographic packing material comprising a coated support material, said coated support material being a chromatographically suitable substrate, and a immobilized coating on said substrate, said coating being a butadiene acrylonitrile copolymer.

CLAIM 2. The chromatographic packing material of claim 1, wherein said copolymer is crosslinked.

CLAIM 3. The chromatographic packing material of claim 1, wherein said copolymer is crosslinked by gamma radiation.

CLAIM 4. The chromatographic packing material of claim 1, wherein said copolymer contains a crosslinking agent.

CLAIM 5. The chromatographic packing material of claim 4, wherein said crosslinking agent is dicumyl peroxide.

CLAIM 6. A chromatographic column having a stationary phase, said stationary phase comprising a coated support material, said coated support material being a chromatographically suitable sub- strate, and immobilized polymeric coating on said substrate, said coating being a butadiene acrylonitrile copolymer.

CLAIM 7. The chromatographic column of claim 6, wherein said sup- port material is selected from the group consisting of silica, alumina, diatomaceous earth, zeolite, porous glass and carbon.

CLAIM 8. The chromatographic column of claim 7, wherein said sup¬ port material is aluminum hydroxide particles. CLAIM 9. The chromatographic column of claim 8, wherein said sup- port material is spherical lamellar shaped crystals of aluminum hydroxide.

CLAIM 10. The chromatographic column of claim 9, wherein said aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core.

CLAIM 11. The chromatographic column of claim 10, wherein the par- tide density ranges from 0.3 to 2.5 g/cm³.

CLAIM 12. The chromatographic column of claim 10, wherein said particles have a diameter of 2 to 150 microns.

CLAIM 13. Method of separating organic materials, said method com- prising the steps of a) providing a bed of packing material selected from the group consisting of silica and alumina, diatomaceous earth, zeolite and porous glass, said packing material having bonded thereto, a polymeric coating, said coating having immobilized coating, said coating being a crosslinked butadiene acrylonitrile copolymer, b) introducing organic materials to said bed, c) adding an eluting fluid to said bed, d) removing said fluid and one of said organic materials from said bed, and e) separating said material removed in step (d) from the fluid.

CLAIM 14, The method of claim 13, wherein said packing material is spherical aluminum hydroxide particles. CLAIM 16. The method of claim 15, wher^ein said aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core.

CLAIM 17. The method of cl;.ιm 16, wherein the particle density ranges from 0.3 to 2.5 g/cm³.

CLAIM 18. The met.iod of claim 17, wherein said particles have a diameter of 2 to 150 microns.

CLAIM 19. The meth α of claim 13, wherein said copolymer is derived^' from a -iquid copolymer which contains carboxyl group.

CLAIM 20. The method of claim 13, wherein said copolymer is derived from a liquid copolymer which contains a vinyl "group.

CLAIM 21- The method of claim 13, derived from a liquid copolyr.er which contains terminal reactive groups.

CLAIM 22. The method of claim 13, wherein said copolymer is derived from a liquid copolymer which contains pendent reactive groups.

CLAIM 23. The method of claim 13, wherein said copolymer is derived from a liquid copolymer which is carboxyl terminated.

CLAIM 24. The method of claim 23 wherein said carboxyl ter- minated copolymer be considered long chain dicarboxlyic acids having functionalities between about 1.8 and 2.4.

CLAIM 25. The method of claim 13, wherein said copolymer is predominantly butadiene. CLAIM 26. The method of claim 25, wherein the ratio of butadiene to acrylonitrile is from about 1:1 to about 10:1.

CLAIM 27. The method of claim 13, wherein copolymer is derived from a liquid copolymer which is vinyl te^rminated and have reac- tive acrylate vinyl groups.

CLAIM 28. The chromatographic packing of Claim 1, wherein said copolymer is crosslinked thermally.

CLAIM 29. A chromatographic packing material comprisinσ a coated support material, said coated support material being a chromatographically suitable substrate, and an uniform immori- lized functionalized coating on said substrate, said coating being a polymer having terminal vinyl groups.

CLAIM 30. The chromatographic packing material of claim 29, wherein said polymer is a butadiene acrylonitrile copolymer.

CLAIM 31. The chromatographic packing material of claim 29, wherein said terminal vinyl groups are octadecene.

CLAIM 32. The chromatographic packing material of claim 29, wherein said terminal vinyl groups are octene.

CLAIM 33. The liquid chromatography packing material of Claim 29, wherein said polymer is a butadiene homopolymer.

CLAIM 34. A chromatographic column having a stationary phase, said stationary phase comprising a coated support material, said coated support material being a chromatographically suitable sub- strate, and an uniform immobilized functionalized coating on said substrate, said coating being a polymer having terminal vinyl CLAIM 35. The chromatographic column of claim 34, wherein said support material is selected from the group consisting of silica, alumina, diatomaceous earth, zeolite and porous glass.

CLAIM 36. The chromatographic column of Claim 35, wherein said support material is aluminum hydroxide particles.

CLAIM 37. The chromatographic column of Claim 36 wherein said sup- port material is spherical lamellar shaped crystals of aluminum hydroxide.

CLAIM 38. The chromatographic column of Claim 37 wherein said aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core.

CLAIM 39. The chromatographic column of Claim 38 wherein the par- tide density ranges from 0.3 to 2.5.

CLAIM 40. The chromatographic column of Claim 38 wherein said par- tides have a diameter of 2 to 150 microns.

CLAIM 41. Method of separating organic materials, said method com- prising the steps of providing a bed of packing material selected from the group consisting of silica, alumina, diatomaceous earth, "zeolite and porous glass, said packing material having bonded thereto, a polymeric coating, said coating having an uniform immo- bilized functionalized coating, said coating being a polymer having terminal vinyl groups.

CLAIM 42. The method of claim 41, wherein said packing material is spherical aluminum hydroxide particles.

CLAIM 43. The method of claim 42, wherein said support material is s herical lamellar sha ed cr stals of aluminum h droxide. CLAIM 44. The method of claim 43, wherein said aluminum hydroxide crystals are bonded together at a central core and extend radially outwardly from a central core.

CLAIM 45. The method of claim 44, wherein the particle density ranges from 0.3 to 2.5.

CLAIM 46. The method of claim 45, wherein said particles have a diameter of 2 to 150 microns.

CLAIM 47. The method of claim 46, wherein said polymer is a butadiene acrylonitrile copolymer.

CLAIM 48. The method of claim 45, wherein said terminal vinyl groups are octadecene.

CLAIM 49. The method of claim 45, wherein said terminal vinyl groups are octene.

Claim 50. In a stationary phase for reversed-phase liquid chromatography consisting of a crosslinked, polybutadiene-coated, macroporous, alumina substrate having micropores of a diameter predominately in the range of 50 to 1000 Angstroms, and wherein the alumina substrate is so occludingly coated with said polybutadiene that the integrity of the stationary phase is not adversely affected by extended exposure to liquid environments having a pH of 12; the improvement wherein the polybutadiene coat- ing has attached thereto sufficient alkyl groups whereby the sta- tionary phase exhibits increased hydrophobicity so that the reten- tion time of o-xylene is increased by at least 30% over retention time for a stationary phase coated with polybutadiene having at least about 18 mole percent vinyl groups per butadiene unit. Claim 52. A stationary phase according to claim 51 wherein sub- stantially all of said alkyl groups have 18 carbons atoms.

Claim 53. A stationary phase according to claim 50 wherein said alumina substrate comprises a plurality of microporous platelets bonded together to form a macroporous, substantially spherical particle having a nominal diameter of 8 micrometers.

Claim 54. A stationary phase according to claim 50 comprising at lease 0.05 alkyl side chains per olefin monomer unit of said polymer coating.

Claim 55. In a stationary phase for reversed-phased liquid chromatography consisting of a crosslinked, polybutadiene-coated, macroporous, alumina substrate having micropores of a diameter predominately in the range of 90 to 500 Angstroms, and wherein the alumina substrate is so occludingly coated with said polybutadiene that the integrity of the stationary phase is not adversely affected by extended exposure to liquid environments having a pH of 12, and wherein the polybutadiene contains pendant vinyl groups; the improvement wherein the polybutadiene coating has grafted thereto sufficient alkyl groups whereby the station- ary phase exhibits increased hydrophobicity so that the retention time of o-xylene is increased by at least 30% over retention time for a stationary phase coated with polybutadiene having 18 mole percent vinyl groups per butadiene unit.

Claim 56. A stationary phase according to claim 55 wherein said alumina substrate comprises a plurality of microporous platelets bonded together to form a macroporous, substantially spherical particle having a nominal diameter of 8 micrometers. Claim 57. A stationary phase according to claim 56 comprising at least 0.1 chromatographically-functional saturated aliphatic side chains per olefin monomer unit of said polymer.

Claim 58. A stationary phase according to claim 57 wherein said alkyl group comprises 14 to 20 carbon atoms.

Claim 59. A method for preparing a stationary phase for liquid chromatography comprising: a. occludingly coating onto a macroporous alumina substrate having micropores of a diameter predominately in the range of 90 to 500 Angstroms, an unsaturated polybutadiene oligomer having pendant vinyl groups and a molecular weight less than 50,000 Dal- tons, b. partially crosslinking said polybutadiene oligomer to provide on said substrate a polybutadiene coating having pendant vinyl groups; c. increasing the hydrophobicity of said polybutadiene coat- ing by providing thereon alkyl groups onto said polybutadiene coating, whereby the stationary phase exhibits increased hydrophobicity so that the retention time of o-xylene is in- creased by at least 30% over retention time or a stationary phase coated with polybutadiene having 18 mole percent vinyl groups per butadiene unit.

Claim 60. A method according to claim 59 wherein said alkyl groups are provided by graftlinking of alpha olefins, wherein said graftlinking is initiated by use of chemical grafting agents comprising peroxides or Lewis acids.

Claim 61. A method according to claim 59 wherein said alkyl groups are provided by graftlinking, wherein said graftlinking is initiated by use of electromagnetic radiation.