WO1985000758A1

WO1985000758A1 - Improved silica-based chromatographic supports containing additives

Info

Publication number: WO1985000758A1
Application number: PCT/US1984/001227
Authority: WO
Inventors: Pedro Manoel Buarque De Macedo; Aaron Barkatt
Original assignee: Litovitz, Theodore, Aaron
Priority date: 1983-08-17
Filing date: 1984-08-03
Publication date: 1985-02-28
Also published as: JPS61500078A; EP0153937A1; EP0153937A4; CA1236075A

Abstract

Silica-based chromatographic and reactive materials with surfaces modified to contain or to be coated with oxides, hydrous oxides, hydroxides, carbonates or silicates of aluminum, iron, or other suitable metals such as zirconium or titanium. The materials exhibit good resistance to dissolution and resulting loss of activity or clogging. This good resistance is particularly evident even in the high pH region (above 8-9) where the dissolution rates and solubilities of aluminosilicates and of trivalent iron oxides are much smaller than those of silica.

Description

DESCRIPTION

IMPROVED SILICA-BASED CHROMATOGRAPHIC SUPPORTS CONTAINING ADDITIVES

TECHNICAL FIELD AND BACKGROUND ART Various forms of silica and silica-based chromato- graphic materials are of primary importance in separating fine organic and biochemical products from one another, from by-products and from excess reagents in reaction mixtures. For instance, silica of various particle sizes (e.g., 1 to 700 μm) and pore diameters (e.g., 1 nm to 1 ym) is used in a very large fraction of the high-pressure and the reverse- phase chromatographic separation techniques in use. For certain applications (e.g., affinity chromatography) active groups which exhibit different degrees of interaction with different components of a mixture to be separated are coated on or bonded to the silica surface, usually by means of using organo-silane reagents. A particular class of modified silica-based chromatographic supports are controlled pore glass . supports, which are coated with polar groups such as quarternary amine or sulfonic glycophases for use in ion- exchange chromatography. Controlled pore glass supports are also widely used in exclusion and affinity chromatograph . On the other hand, despite the availability of a large variety of silica supports coated with various functional groups and molecules, materials consisting of largely pure silica are used in a large number of separations. For instance, silica gel is the most widely used support in high pressure liquid chromatography. Molecules separated on pure and coated silica surfaces include amines, amino acids, purines, nucleosides, nucleotides, aminoglycosides, peptides, proteins, nucleic acids, enzymes and other organic and biochemical molecules as well as inorganic species such' as- metal ions and anions. In addition to their role in separations, silica- based supports are also used in carrying out chemical reactions using reagents which are immobilized on the grain and pore surfaces. Such reactive supports include, for instance, reducing supports to which reducing groups such as borohydride have been attached, supports which contain chelating groups to remove metal ions from solutions, and supports which contain immobilized enzymes and other bio¬ chemically active species. In spite of their wide usefulness, silica-based supports are subject to a number of limitations. These are evident in some cases more than in others. For instance, uncoated silica supports are more susceptible to deteriora¬ tion at high pH than supports coated with neutral molecules,- and coarse-grained, large-_pore glass supports are more durable than fine-grained, small pore gels. However, the problems encountered in the case of silica gels used for high-pressure liquid chromatography, for instance, also appear in the cases of other silica-based supports, although their extent may be smaller or they may appear after a longer period of service -

One serious problem which limits the application of silica supports is the poor durability of silica at the high pH range. Generally, mixtures of organic or biological molecules are separated by sorption on a support followed by fractional elution using eluants with varying combinations of pH and ionic strength. High pH separations are very useful in the case of proteins and other substrates. However, at pH values above 7.5 rapid silica dissolution takes place, leading to loss of activity of surface sites and to clogging due to reprecipitation at the lower parts of the column. Support deterioration becomes much more serious at elevated temperatures. Jackson and Fisher (Ind. Res. Dev. , Feb. 1983, pp. 130-33) state: "For a silica-based bonded phase, the packings are stable in the pH range of 2.0 to 7.5 Exposure to a mobile phase with a pH higher than 7.5 (less acid) causes dissolution of the silica and leads to voids in the column. The resulting chromatogram will exhibit peak broadening, and thus a loss of resolution, sensitivity (as the peak broadens, the peak height decreases, decreasing the sensitivity) , and quantitative accuracy. We need to note also that a sample of high pH can cause void formation at the column head as it is introduced onto the column. This effect can be observed regardless of the pH of the column. Of course, an occasional run outside the pH range, 2.0 to 7.5, ca be acceptable. But, frequent operation outside the range results in both poor.chromatography and greatly reduced column life". In addition to poor column performance and short lifetime, the enhanced silica dissolu¬ tion at pH values above 7.5 also causes considerable contam¬ ination of the separated products with silica. One way to overcome the problems associated with the use of silica columns at high pH environments, mentioned by Jackson and Fisher, is to replace such columns by resin-based, usually polystyrene/divinylbenzene, stationary phases. However, these materials have poor mechanical properties and are crushed at the high pressures required in typical liquid chromatography operations. Jackson and Fisher also emphasize that "even small changes in pH (e.g., by 0.5 of a pH unit) can have large effects on the shapes of chromatograms". Accordingly, even a small increase in the service range of silica-based stationary phases can result in considerable improvement of existing separation techniques and in making it possible to perform novel separations. Another problem associated with the use of silica- based supports is the fact that the silanol surface groups often form excessively strong bonding with the organic or biochemical species being purified and this requires the use of drastic conditions (in terms of solvent polarity, pH, ionic strength and temperature) to desorb the molecule of interest. In the case of many sensitive organic structures this leads to decomposition or loss of biological activity (for instance, denaturing of proteins) . A third problem associated with the use of silica supports is due to the existence of various configurations of silicon-oxygen on silica surfaces, including isolated hydroxyl groups, pairs of hydroxyl groups attached to the same Si atoms, pairs of hydroxyl groups attached to adjacent Si atoms, and siloxane (Si-O-Si) terminal groups. Since each of these configurations has a different affinity towards organic substrates, chromatographic peaks are often quite broad and the resolution of separating similar species is poor. It is well-known that hydrous oxides of various metal ions such as Al and Fe reduce the solubility of silica in water (U.S. Patent No. 2,267,831 issued and in aqueous solutions (U.S. Patent No. 4,332,031 issued May 25, 1982). This is due to the formation of a combined species (aluminosilicate, iron oxide-silicate, etc.) which has a very low solubility in water. The interaction involved in the formation of such species may consist of a chemical reaction, sorption, ion-exchange, colloid-colloid neutraliza¬ tion, etc. Furthermore-, the rise in solubility with in- creasing temperature is also smaller in the case of alumino- silicates than in the case of silica. However, according to studies of the chemical durability of silicate glasses in high pH media (Ohta and Suzuki, Am. Cera . Soc. Bull., 57, 602-604 (1978)), the addition of alumina or iron oxide to the glass results in increased corrosion rates. U.S. Patent No. 3,843,341 issued October 22, 1974 relates to thermally stable, mechanically strong microporous glass articles with large pore volumes, surface areas, and varying pore sizes, and methods for making such articles. In particle form, such as beads, the microporous glass articles are useful as catalyst supports in applications such as petroleum catalytic refiners and motor vehicle catalytic mufflers. The mechanical strength and the dimen¬ sional stability of the microporous glass articles at elevate temperatures can be improved if the articles are preshrunk, such as by brief exposure to high temperatures, before their intended use, and can be improved even further if treated with certain metal oxides. In particular, this improvement is achieved by treating the preshrunk porous glass article with a tin solution to deposit tin oxide thereon. The deposition is effected through soaking .in a tin chloride solution, drying and calcination at 700°C. The examples describe coating of the microporous glass articles with oxides of chromium, zirconium, copper and aluminum. However, tin oxide (Sn0₂) is a particularly desirable metal oxide to deposit on the porous glass articles of the invention. Tin oxide has been found to provide additional thermal stability to the porous glass bead. According to the patent, this is somewhat surprising in that other metal oxides such as Al-O,, ^Cr2°_ ^{and Zr0}2 ^{do not} P^rovi^de analogous results and do not show improved stability over the undoped glass.

The amount of tin oxide deposited within the pores according to this patent should be about 0.5 to 10 percent, preferably about 1 to 5 percent by weight. The metal oxide doping levels shown in this patent are 1.5% Sn0₂, 1% A^O,, 1.5% Cr₂0₃, 3.0% Zr0₂ and 3% CuO - Cr₂0-, respectively. In all cases, the metal oxide deposition treatment follows a high-temperature preshrinking step to produce a porous- glass article with improved stability in high-temperature applica— tions. U.S. Patent 3,923,688 issued December 2, 1975 is a continuation-in-part of the previous patent. It details the preparation of the microporous glass articles having a catalytic coating deposited thereon, where the catalyst is a metal oxide selected from the class consisting of Ti0₂,

V₂0-, Cr₂0₃, FeO, CoO, Ni0₂, CuO, Al₂0₃, Zr0₂ and SnO,. It also mentions the possible uses of the thermally stable porous glass of the invention as an ion exchange and desalin¬ ation medium, as a chromatographic support, a filter for gases and vapors and as a membrane providing an effective diffusion barrier for use in ultrafiltration and reverse osmosis. In these applications, the patent notes that porous glass offers expanded capabilities for use in systems employing high temperature and low pH which are generally not compatible with organic polymer membranes.

U.S. Patent 4,17a,270 issued December 11, 1979 describes an inorganic ion-exchanger supported on a carrier which is prepared by supporting an active component of inorganic ion exchanger, such as hydrous oxide of metal, for example, titanium, zirconium, etc. , on a porous carrier such as alumina, silica, or activated carbon, where the pH of a solution in contact with the active component and the carrier is adjusted so that the active component and the carrier can have zeta potentials of opposite polarities to each other, and an inorganic ion-exchanger having a larger amount of the supported active component and firmly supporting less bleed- able active component can be obtained by setting out a supporting condition on the basis of the polarity of zeta potential. This patent describes the preparation of supporte hydrous titanium and zirconium oxides, but the possible use of the hydrous oxide of titanium in admixture with manganese, zinc, tin, zirconium, silicon or rare earth element is also mentioned. According to the invention described in the patent, the carrier supports about 10% by weight of the hydrous metal oxide in the hydrolyzed state, on the basis of the carrier, and at least 5% by weight thereof, even after shaking.

U.S. Patent 4,333,847 issued June 5, 1982 relates to the immobilization of toxic, e.g., radioactive materials, internally in a silicate glass or silica gel matrix for extremely long periods of time. Toxic materials, such as radioactive wastes containing radioactive anions, and in some cases cations, which may be in the form of liquids, or solids dissolved or dispersed in liquids or gases, are internally incorporated into a glass matrix, having hydrous organofunctionalsiloxy groups, e.g., hydrous aminoalkylsiloxy or carboxyorganosiloxy, bonded to silicon atoms of said glass and/or hydrous polyvalent metals bonded to silicon atoms of said glass through divalent oxygen linkages or otherwise immobilized therein, by a process which involves the ion exchange of said toxic, radioactive anions with hydroxyl groups attached to said organofunctionalsiloxy groups or with hydroxyl groups attached to the hydrous polyvalent metal. In the case where hydrous polyvalent metals are used, said non-radioactive cationic polyvalent metals are selected from the group consisting of -Zr , -Pb' -Th and -Ti . The processes described in the patent for attaching said metals to the glass consist of cation exchange with protons or molecular stuffing, which lead to loading the glass with high concentrations of hydrous oxides of said metals.

U.S. Patent No. 3,677,938 issued July 18, 1972 relates to a chromatographic separation process using columns filled with porous silica. The porous silica used in the columns is prepared by calcination of silica gel at tempera¬ tures within the range of 400 to 1000°C. The silica gel used in the calcination is doped with foreign atoms including alkali metal cations (lithium, sodium, potassium and cesium) and acid anions (sulfate, phosphate, bromide, chloride and. iodide) . This formulation makes it possible to obtain yy-^x ^E substantially complete dehydration of the silica upon calcin¬ ation, leading to enhanced mechanical strength and stability of the silica gel particles.

U.S. Patent No. 3,722,181 issued March 27, 1983

^' 5 relates to a process for making a chromatographic packing having a polymeric stationary phase comprising repeating units of silicon. The organic stationary phase is bonded to a substrate which contains a metal or a metal oxide where the metal has a valence of 3-5, including non-alkaline metal

10 oxides, alumina, thoria, titania, zirconia and non-alkaline metals with an oxide skin. However, the preferred substrates are those which contain silica, such as diatomaceous earth, silica gel, glasses, sand, aluminosilicates, quartz, porous silica beads and clays. The purpose of the introduction of

15 Si or a polyvalent metal into the substrate surface is to provide for the formation of linkage with the polymolecular organosilicon stationary phase.

U.S. Patent No. 4,299,732 issued November 10, 1981 relates to amorphous. luminosilicates which are usef l as

20 catalysts. It describes the preparation of synthetic amorphous aluminosilicates by mixing under reaction conditions a source of silica such as an aqueous colloidal dispersion of silica particles, a source of alumina such as sodium aluminate prepared by dissolving alumina particles in excess

25 sodium hydroxide solution, a source of alkali metal such as sodium hydroxide, water and one or more polyamines other than a diamine. The reaction conditions are a temperature in the range 80° to 210°C, a pressure in the range of 70 to 400 psig and a reaction time in the range 20 to 100 hours.

30 The exact formulation of the aluminosilicate and the specific method of preparation have a large effect on the catalytic properties of the surface of aluminosilicate solids.

U.S. Patent 4,322,310 issued March 30, 1982 relates to a composition comprising a chiral organic amine covalently

35 linked via a carbamate, mercaptocarbamate, or urea linkage to a chain of atoms which in turn are covalently bound to a core support. The support described in the patent is used as a solid phase chromatographic medium in the separation of racemic mixtures. This patent illustrates, for example, the binding of a siϊyl group to an alumina support and having an alpha-methylbenzyl amine molecule bound through a carbamate linkage to said 3-propyl-silyl group. In general, supports mentioned in this patent include silica, alumina, glass and ceramic materials, and silylating agents are used to form a σovalent bonding to the chiral composition.

U.S. Patent No. 4,340,496 issued July 20, 1982 relates to an anion exchange composition that is useful in chromatographic separations comprising an inert porous particle having a tetra-substituted silane material fixedly attached by coyalent bonding, to the surface thereof. This patent demonstrates the use of the resulting weak anion exchange material in separating polar polyfunctional com¬ pounds such as proteins. The inert porous particle to which the silane material is attached is microparticulate silica in all the examples cited in the patent. The possible use of alumina, cross-linked dextran and cross-linked polystrene- divinylbenzene as inert porous particles is also mentioned.

U.S. Patent No. 4,359,389 issued November 16, 1982 relates to the purification of human fibroblast interferon using a two-stage purification method comprising (a) subject¬ ing an aqueous interferon solution to chromatography on porous glass beads and (b) subjecting the resulting aqueous interferon solution to chromatography on immobilized zinc chelate. The porous glass beads used in the first stage are of controlled (i.e., rather uniform) pore size between 170 and 1700 A (usually between 350 and 900 A) and a diameter which may be less uniform and may range in general between 50 and 500 μm. The beads are used to absorb the interferon from an aqueous solution having a neutral or slightly alkaline pH (around 7.4), and the interferon is eluted- using - lo ¬

an elution agent at acidic pH (6 to 4) and subjected to the second stage of purification by means of immobilized zinc chelate.

Accordingly,.it is an object of the present ^• 5 invention to provide an improved silica-based support.

A further object of the present invention is to provide* a silica-based chromatographic or reactive support which is relatively free from deterioration at high pH.

Yet a further object of the present invention is 10 to provide a silica-based support which has good durability of silica at high pH.

Another object of the present invention is to provide a silica-based chromatographic support for bonding organic or biochemical species in which the molecules can be 15 desorbed without substantial decomposition or loss of biological activity.

Another object of the present invention is to provide a silica-based chromatographic or reactive support which minimizes product contamination with silicate during 20 separation of mixtures or chemical reaction.

Yet another object of the present invention is to provide a silica-based chromatographic support which provides good resolution of separating similar species.

25 DISCLOSURE OF THE INVENTION

In accordance with the present invention, silica- based chromatographic materials are provided with surfaces modified to contain or to be coated with oxides, hydrous oxides, hydroxides, carbonates or silicates of aluminum,

³⁰ iron, or other suitable metals such as zirconium or titanium.

The materials exhibit good resistance to dissolution and resulting loss of activity or clogging. This is especially true in the high pH region (above 8-9) where the dissolution rates and solubilities of aluminosilicates and of trivalent

35 iron oxides are much smaller than those of silica. In addition, the modification of silica surfaces with metal species such as aluminum or iron is believed to lead to reduction of the polarity of surface groups and their binding strength with respect to organic species. Accordingly, it is believed that the separations of proteins, enzymes, etc. can be carried out under milder elution conditions without risking denaturing. Furthermore, such surface modification will reduce the non-uniformity of surface structures by preferential attachment to the more reactive silanol groups, resulting in narrower elution peaks and sharper resolution. Optimization on the choice of additives can lead to systematic tailoring of the reactivity and surface charge for specific purifications. The improve¬ ments resulting from the present invention should be most advantageous when combined with the use of siliceous materials with sharp pore size distribution such s controlled pore glasses. Modification of surface compositions with additives can be accomplished during the preparation of silica-based support materials or by means of subsequent treatments (e.g., contact with solutions which contain the additive).

Experiments have shown that treatment of siliceous support materials to incorporate alumina or iron oxide in the surface reduce the amount of corrosion and the occurrence of clogging during subsequent passage of high pH solutions without affecting the performance of the column in the separation of organic materials. As such, the present invention clearly represents a marked advance over the state-of-the-art discussed above.

BEST MODES FOR CARRYING OUT THE INVENTION

The silica-based material employed in accordance with the present invention can be any of the silica-based materials conventionally employed as chromatographic supports in liquid chromatography, particularly high-pressure liquid ' chromatography and ion-exchange chromatography. Suitable silica-based materials are porous glass and silica gel. Suitable porous glass compositions are described in U.S. Patent No. 3,549,524. The porous glass medium described in U.S. Patent No. 3,549,524 has a controllable pore size of narrow distribution and is prepared from a base glass having a composition which lies in a limited region of the ternary system RO*B₂0,*Si0₂ wherein the region comprises compositions which will separate by heat treatment into at least two phases, one of which is easily decomposable and the other substantially undecomposable. The term RO in U.S. Patent No. 3,549,524 is defined to mean any of the alkaline earth, alkali metal or heavy metal oxides wherein RO can be Li ⁰- Na₂0, ₂0, CaO, BaO, MgO, BeO, SrO, ZnO or PbO, or any combination thereof. The base glass composition can be of the type described in U.S. Patents 2,106,744 and 2,215,039. - Suitable mixtures of oxides include compositions wherein the base glass silica is present in amounts ranging from 50 to 83 weight percent, the RO (e.g., soda, potash, lithia) is present in amounts ranging from about 2 to 10 weight percent and the boric oxide is present in amounts from about 8 to 48 weight percent. The processing of the base glass is fully described in U.S. Patent No. 3,549,524, the disclosure of which is expressly incorporated herein by reference.

The literature also adequately describes the preparation of silica gel compositions which can be employed in this invention. These materials are commercially avail¬ able, for example, from Sigma Chemical Co., St. Louis, Missouri.

The silica-based support is modified to contain or be coated with oxides, hydrous oxides, hydroxides, carbonates or silicates of a suitable metal. The preferred metals are aluminum and iron. However, chromium, tin, lanthanum, rare earths, zinc, hafnium, thorium, gallium and, in particular, zirconium or titanium can also be used. Preferably, the . support is contacted with a suitable metal salt dissolved in an aqueous medium having a pH of above about 2. The pH of the aqueous medium is typically about 2 to 9 and preferably about 3 to 7.5. During the contact, the support is modified to contain or to be coated with the oxide, hydrous oxide, hydroxide, carbonate or silicate of the metal. The modifi¬ cation of or coating of the support can, in some cases, be controlled by changing the pH of the solution to -precipitate the metal. In other cases, the coating can be produced by sorption of the metal species from a solution at a constant pH.

In the case of iron, it is preferred to dissolve a ferrous salt in the aqueous medium rather than a ferric salt. However, the final coating material is a ferric ^' material, e.g., ferric oxide, due to the oxidatio^'n of the ferrous salt dμring or following modification or coating of the support.

The amount of aluminum, iron, zirconium, titanium, chromium, tin, lanthanum, rare earth, zinc, hafnium, thorium or gallium metal immobilized on the support is generally about 0.02 to 2 percent by dry weight, preferably about 0.02 to 0.5 percent, and more preferably about 0.05 to 0.2 percent, expressed as the metal oxide. The amount of metal immobilized on the support expressed as its oxide is deter¬ mined by drying the modified support, weighing the support, immersing the support in a strong (other than hydrofluoric) acid at room temperature for about 10 minutes, removing the sample from the acid, determining the metal content of the acid, drying the sample, and measuring the weight loss of the sample. The amount of metal immobilized on the support expressed as its oxide is computed by dividing the metal content of the acid by the dry weight of the sample prior to immersion in the acid. The sample should not have more than 5% weight loss on immersion in the acid.

The specific test which is preferred for deter- . mining the concentration of the metal expressed as its oxide in the modified support comprises washing a quantity of glcLSS or silica gel with a small amount of cold water; drying the sample overnight at about 110 to 120°C; weighing accurately about 0.1 ml of sample; stirring the sample with about 3 ml of about 6 molar nitric acid for about 10 minutes at room temperature; decanting the nitric acid and adding to the solid another about 3 ml of about 6 molar nitric acid; stirring for about 10 minutes at room temperature; decanting the nitric acid and mixing it with the first decanted portion; analyzing the sample for aluminum or other additives.

A preferred method of modifying the support is to treat a material such as porous glass, silica gel or other suitable silica-based support material with a solution of a metal species such as aluminum chloride or iron sulfate with or without subsequent changes in pH or oxidation conditions to coat the support material with the corresponding metal oxide, hydrous oxide, hydroxide, carbonate or silicate, and provide a support material modified with the desired concen¬ tration of metal. The support material can be subsequently packed into a column, made into a stationary or fluid bed or used in other suitable configurations to effect a chromato¬ graphic separation or a chemical, reaction.

While we do not intend to be bound by this des¬ cription, we believe that the support into which aluminum, iron, zirconium or titanium has been introduced is character¬ ized by a high concentration of metal oxide on the surface of the pores of the support. It is further believed that the concentration of the metal oxide gradually decreases from the surface of the pores so that a gradually decreasing concentration gradient is created. On the other hand, it must be recognized that leached glass may inherently contain aluminum. * However, the aluminum content is not at the surface of the pores, but rather in the interior of the glass matrix. The modification of the standard leached glass in accordance with the present invention provides a high concentration of aluminum at the surface of the pores but this is not believed to greatly effect the concentration in the interior of the matrix. This high surface aluminum content can be specifically determined by using the acid leaching test described above for determining the concentra¬ tion of metal oxide in the modified support. This acid leaching test does not substantially leach aluminum from the interior of the matrix which is inherently present in a standard leached glass. Thus, the acid leaching test only determines the quantity of aluminum or other additive added by way of the special processing techniques of the present invention.

The modified support of the present invention can be used as a chromatographic support to separate all of he various materials which have, previously been separated on conventional silica-based chromatographic materials. In particular, the modified support of the present invention can be used as a chromatographic support for separating amines, amino acids, purines, nucleosides, nucleotides, aminoglycosides, peptides, proteins, nucleic acids, enzymes and other organic and biochemical molecules. The silica- based material typically has a mesh size of about 10 to less than 325 mesh (U.S. Standard Sieve) and preferably a mesh size of about 50 to 200. The pore size typically ranges from about 1 nm to 1 μm and preferably about 2 nm to 200 nm. The silica-based supports of the present invention exhibit resistance to dissolution and clogging, especially in the high pH regions above 7.5. Resistance to dissolution and clogging has been found to exist at pH's as high as 10. In general, a chromatographic column should perform well for about one month or longer at a pH up to about 7.5 and a lifetime of one day is considered to be useful. A chromato¬ graphic column packed with the modified support of the present invention has been found to function for at least ^' about a month at pH's well above 7.5. Thus, the modified support of the. resent invention shows exceptional resistance to dissolution and clogging at high pH.

As mentioned above, both uncoated silica gel or glass and such gels or^*glasses coated with or bonded to organic functional groups are used as stationary phases in chromatographic separations. The latter are described, for instance, in U.S. Patent No. 4,340,496, which describes a microparticulate silica gel coated with N-2-aminoethyl-3- a inopropyl trimethoxysilane to produce N-2-aminoethyl-3- aminopropyl silyl groups covalently chemically bonded to the surface of the silica gel.. The present invention can be used to produce stationary phases consisting of silica gel or glass supports with as well without coating with organic functional groups. Furthermore, the modified supports of the present - invention are not restricted to stationary phases to be used in chromatographic separations. Reactive supports, such as materials to which reducing, chelating or enzymatically active species are attached can also be made using the present invention to have high stability, in particular in high pH environments. The use of such supports is expected to result in reduced dissolution, less contamination, improved reaction selectivity and higher thermal stability. It is believed to be particularly surprising that the modified supports of the present invention such as the aluminum oxide modified supports do not form colloids. It is also believed to be particularly surprising that a small amount of metal oxide such as aluminum oxide does not materially change the amount of silica leached out of the chromatographic support yet increases the stability of the column and its resistance to clogging by as much as more than 500%. It is also surprising, in view of reported studies that show enhanced corrosion in high pH media in the case of silicate glasses which contain alumina or iron oxide in their bulk composition (Ohta and Suzuki, Am. Ceram. Soc. Bull., 51_, 602-604 (1978)), that a surface treatment of silicate-based stationary phases with such oxides contribute to marked improvement in their resistance to high pH corros¬ ion. The following non-limiting examples further illustrate the invention.

Example 1 Two samples of chromatographic grade silica gel (Sigma Chemical Co., St. Louis, Missouri, #S-4133, 100 to

200 mesh) were treated with a solution of 16 mg/1 Al (present as aluminum chloride) at a pH of 5.5. In each case, a quantity of 8g of silica gel was shaken with a volume of 0.5 1 of the solution for 2 days. After washing in water and drying, the modified silica gel was found to contain 0.137% Al in one case and 0.094% Al in the other. Four chromato¬ graphic columns were prepared, each with a bed height of about 4.5 cm and a cross-section of 1 cm. Two of the columns were loaded with the original silica gel and two with the Al-modified gel. A 0.005 M pH 10 buffer solution made up with potassium carbonate, potassium borate and potassium hydroxide was used as an influent in all cases. Flow was gravitational under normal atmospheric pressure and the hydrostatic head was 2 m high. The initial flow rate in all cases was adjusted to approximately 0.3 ml/min by means of a stopcock. The two columns loaded with unmodified silica gel clogged up, one after 4.5 hours and the other after 10 hours, and the flow could not be restarted even when the stopcocks were fully opened. The Al-modified columns showed no clogging even after 20 hours. Silica concentrations in the effluent from the unmodified columns after the first 3 hours of flow were 42 and 52 mg/1 Si compared with 28 and 37 mg/1 Si in the case of the two Al- modified columns. Accordingly, the improvement in column. durability and performance which occurs upon treatment with a small amount of aluminum species is very noticeable. Further experiments have shown that high-silica porous glass can also be loaded with approximately 0.1% Al upon similar treatment.

Example 2 Three batches of silica gel were prepared: (A) a _ quantity of 7.669 g of 100-200 mesh silica gel (Sigma Chemical Co. S-4133) was slurried with a volume of 20 ml of de-ionized water; (B) a quantity of 7.669 g of the same type of silica gel used in (A) was stirred for three days with a volume of 500 ml of an aqueous solution which contained 20 ml of a solution of 1000 mg/1 aluminum in dilute hydrochloric acid, 1.7 ml of 50% w/w aqueous^"sodium hydroxide, and 24 ml of pH 7 phosphate buffer; (C) a quantity of 7.669 g of the same type of silica gel used in (A) was stirred for three days with a volume of 500 ml of an aqueous solution which contained 5 g of ferrous^"sulfate heptahydrate and 0.05 g of hydroxylamine hydrochloride. During this time the iron was slowly oxidized by air to the trivalent state as shown by the fact that the silica gel became yellowish brown.

A mixture of three dyes was prepared in a volume of 10 ml of pH 9 borate buffer solution similar to that used in Example 1. The quantities of dyes dissolved in this volume were 0.05 g of xylenol blue, 0.036 g of neutral red and 0.05 g of quinaldine red. The dyes were dissolved in the buffer solution by stirring for 10 minutes.

The three batches of silica gel designated (A) , (B) , (C) above were each placed in a chromatographic column with a cotton plug. The bed height of each column was 12.5 cm and the volume of each column was 11.8 ml. A volume of approximately 1500 ml of a solution of pH 9 borate buffer (diluted 1:10 in de-ionized water) at a flow rate of approxi mately 3 ml/min. was used. The hydrostatic head was 165 cm. At the end of this stage, the pH of the effluent of each of the three columns was observed to have stabilized at a value of 9.0. The excess influent above each column was drained off and 3 drops of the mixed dye solution described above were placed just above the top of the column and permitted to flow down into the uppermost part of the column. The flow of the dilute pH 9 buffer solution through the column was thereupon resumed in order to observe the separation of the dyes on the column.

In all three cases, the xylenol orange (colored blue) passed very quickly through the column (about 10 minutes for complete elution) . Neutral red (orange-brown) took approximately two hours to pass through the column. Quinaldine red (bright red-purple) remained at the top of the column and moved downwards at a rate of only about 1 mm/hr. The behavior of the dyes on the three columns was identical. However, the flow characteristics were quite different. The untreated silica gel column (A) was observed to clog, and the stopcock below the column had to be adjusted every few minutes in order to re-adjust the flow rate to its original level of 3 ml/hr. This column became completely clogged at the end of two hours and flow could not be continued even with the stopcock open all the way. The iron oxide-treated silica gel column (B) showed little clogging during the initial 10-hour period, but after being permitted to stand for 30 days in contact with the influent, the flow slowed down appreciably and it was necessary to open the stopcock all the way in order to maintain the flow. The alumina-treated silica gel column (C) did not exhibit any clogging or slowing down of flow even after 30 days. The test showed that the iron oxide treatment and, in particular, the alumina treatment greatly enhanced the chemical stability and flow characteristic of the silica gel columns in high pH environments without impairing the chromatographic resolution exhibited by the columns.

O PI

Claims

1. A. chromar-ographic or reactive support compris¬ ing a porous support formed from porous glass or silica gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxide, hafnium oxide, thorium oxide and gallium oxide, said modified porous support containing about 0.02 to 0.5% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

2. The support of claim 1 wherein said metal oxide is aluminum oxide.

3. The support of claim 1 wherein said metal oxide is ferric oxide.

4. The support of claim 1 wherein said porous support is silica glass.

5. The support of claim 1 wherein said porous support is silica gel.

6. The support of claim 1 wherein said acid leach is conducted using about 6 molar nitric acid at room tempera¬ ture for about ten minutes.

7. The support of claim 1 wherein the content of metal oxide in said modified support is about 0.05 to 0.5% by dry weight.

8. The support of claim 1 wherein the content of metal oxide in said modified support is about 0.02 to 0.2% by dry weight.

9. The chromatographic support of claim 1 wherein the porous glass or silica gel is coated with or bonded to organic, organometallic or enzymatically functional groups.

10. The reactive support of claim 1 wherein the porous glass or silica gel is coated with or bonded to reducing groups.

11. The reactive support of claim 1 wherein the porous glass or silica gel is coated with or bonded to chelating groups. •

12. The reactive support of claim 1 wherein the porous glass or silica gel is coated with or bonded to enzymatically active groups.

13. A chromatographic or reactive support com- _ prising a porous support formed- from silica gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxide, hafnium oxide, thorium oxide and gallium oxide, said modified porous support containing about 0.02 to 2% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

14. The support of claim.13 wherein said metal oxide is aluminum oxide.

15. The support of claim 13 wherein said metal oxide is ferric oxide.

16.. The support of claim 13 wherein said acid leach is conducted using about 6 molar nitric acid at room temperature for about ten minutes.

17. The support of claim 13 wherein the porous glass or silica gel is coated with or bonded to organic, organometallic or enzymatically functional groups.

18. The support of claim 13 wherein the porous glass or silica gel is coated with or bonded to enzymati¬ cally active groups.

19. A process for separating materials by liquid chromatography comprising passing said material through a chromatographic column packed with a chromatographic support composition and separating said materials on passage of one or more mobile phases having a pH above about 7.5 through said chromatographic column, said support being formed from porous glass or silica gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxide, hafnium oxide, thorium oxide and gallium oxide, said modi¬ fied porous support containing about 0.02 to 0.5% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

20. The process of claim 19 wherein said materials are selected from the group consisting of amines, amino acids, purines, nucleosides, nucleotides, aminoglycosides, ^" _ peptides, proteins, nucleic acids and enzymes. _/^DRE_

21. The process of claim 19 wherein any of said one or more mobile phases has a pH of about 8 to 10.

22. The process of claim 19 which is carried out under high pressure.

23. A process for carrying out a chemical reactio by passing one or more reactants in one or more mobile phases having a pH above about 7.5 through a column con- taining a reactive support composition and causing said reaction to take place, said support being formed from porous glass or silica. gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxide, hafnium oxide, thorium oxide and galli-um oxide, said modi¬ fied porous support containing about 0.02 to 0.5% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

24. The process of claim 23 wherein any of said one or more mobile phases has a pH of about 8 to 10.

25. A process for separating materials by liquid chromatography comprising passing said materials through a chromatographic column packed with a chromatographic support composition and separating said materials on passage of one or more mobile phases having a pH above about 7.5 through said chromatographic column, said support being formed from porous silica gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxide, hafnium - oxide, thorium oxide and gallium oxide, said modified porous

•' .' support containing about 0.02 to 2% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

26. The process of claim 25 wherein said materials are selected from the group consisting of amines, amino acids, purines, nucleosides, nucleotides, aminoglycosides, peptides, proteins, nucleic acids and enzymes.

27. The process of claim 25 wherein any of said one or more mobile phases has a pH of about 8 to 10.

28. The process of claim 25 which is carried out under high pressure.

29. A process for carrying out a chemical reactio by passing one or more reactants in one or more mobile phases having a pH above about 7.5 through a column con- taining a reactive support composition and causing said reaction to take place, said support being formed from porous silica gel and modified with a metal oxide selected from the group consisting of aluminum oxide, iron oxide, zirconium oxide, titanium oxide, chromium oxide, tin oxide, lanthanum oxide, rare earth oxides, zinc oxides, hafnium oxide, thorium oxide and gallium oxide, said modified porous support containing about 0.02 to 2% by dry weight of said metal oxide, said content of metal oxide being determined by the quantity of metal oxide which is acid leached from said modified support.

30. The process of claim 29 wherein any of said one or more mobile phases has a pH of about 8 to 10.

31. A process for modifying a porous support formed from porous glass or silica gel comprising treating said porous support with a solution of a metal selected from the group consisting of aluminum, iron, zirconium, titanium, chromium, tin, lanthanum, rare earth, zinc, hafnium, thorium and gallium to coat the porous support with an oxide, hydrous oxide, hydroxide, carbonate or silicate or said metal in a concentration of about 0.02 to 0.5% by dry weight of said metal oxide, said concentration being determined by acid leaching said modified support.

32. The process of claim 31 wherein a mobile phase containing materials to be separated by liquid chroma¬ tography is passed through said packed column after said porous support, is coated with said metal oxide.

33. A chromatographic or reactive support com¬ prising a porous support formed from silica glass or silica gel modified with a metal oxide selected from the group consisting of aluminum oxide and ferric oxide to a concen¬ tration of about 0.02 to 0.5% by dry weight, said concentra¬ tion of metal oxide being determined by acid leaching said modified support in an about 6 molar nitric acid solution at about room temperature for about ten minutes to determine the amount of metal oxide leached from said modified support and comparing this amount with the dry weight of said modified support before leaching, said support having good resistance to clogging and void formation at a pH above 7.5.

34. The support of claim 33 wherein said support is resistant to clogging and void formation at a pH of above about 7.5 for at least about one month.

35. A process for modifying a porous support formed from silica gel comprising treating said porous support with a solution of a metal selected from the group consisting of aluminum^*, iron, zirconium, titanium, chromium, tin, lanthanum, rare earth, zinc, hafnium, thorium and gallium to coat the porous support with an oxide, hydrous oxide, hydroxide, carbonate or silicate or said metal in a concentration of about 0.02 to 2% by dry weight of said metal oxide, said concentration being determined by acid leaching said modified support.

36. The process of claim 35 wherein a mobile phase containing materials to be separated by liquid chroma¬ tography is passed through said packed column after said porous support is coated with said metal oxide.

37. A chromatographic or reactive support com¬ prising a porous support formed from silica gel modified with a metal oxide selected from the group consisting of aluminum oxide and ferric oxide to a concentration of about 0.02 to 2% by dry weight, said concentration of metal oxide being determined by acid leaching said modified support in an about 6 molar nitric acid solution at about room tempera¬ ture for about ten minutes to determine the amount of metal oxide leached from said modified support and comparing this amount with the dry weight of said modified support before leaching, said support having good resistance to clogging and void formation at a pH above 7.5.

38. The chromatographic support of claim 37 wherein said support is resistant to clogging and void formation at a pH above about 7.5 for at least about one month.