A ph stable chromatographic material and method of producing the same

Abstract

pH stable chromatographic materials for use in chromatographic separations and methods of producing pH stable chromatographic materials for use in chromatographic separations are provided. In addition, attenuated silica particle materials for use in chromatographic separations and methods of attenuating the pore size of a silica particle material for use in chromatographic separations are provided.

Description

[0001] A pH Stable Chromatographic Material and Method of Producing The Same

[0002] Field

[0003] The present disclosure is directed, in part, to pH stable chromatographic materials, methods of producing pH stable chromatographic materials, methods of attenuating the pore size of a silica particle, and pH stable attenuated silica particle materials.

[0004] Background

[0005] Silica is the most common chromatographic stationary material used in liquid chromatography. Generally, porous silica is modified with an organic functional group(s) and the resulting modified silica is employed as a chromatographic stationary phase separation material achieving desired separation characteristics for particular analytes of interest. Although various organic materials used to modify silica can be chemically stable against strongly alkaline mobile phases, the resulting modified silica generally results in chromatographic materials lacking in mechanical strength that can suffer from dissolution and compaction, and attendant loss of separation performance. Previous efforts have been made to produce chemically and mechanically stable chromatographic materials that perform better under highly alkaline conditions. U.S. Patent No. 6,686,035 to Waters et al. discloses a method to prepare a porous inorganic / organic hybrid material as a stationary phase for chromatography. This continuous hybrid silica material is modified by hydrothermal treatment in an autoclave to enlarge the surface pore structure and form reactive silanol groups. The limited available reactive surface silanols on such hybrid inorganic / organic particles can be improved by subsequent modifications, as disclosed in U.S. Patent No. 6,528,167. Improved surface reactive silica silanols can be obtained by known reactions of silica particles with various catalysts during hydrolysis, including fluoride ions and the like, as described by Kirkland and Kohler (U.S. Patent No. 5,108,595). U.S. Patent No. 8,277,883 includes the use of fluoride or ammonium fluoride in the reaction of organosilanes with silica particles to produce a hybrid inorganic / organic silica particle for chromatographic application. U.S. Patent No. 8,658,038 describes singular, or repetitive polycondensation reactions, of silica surfaces with multifunctional organosilanes, with intervening hydrolysis of reactive groups, and particle dehydration to improve silica stationary phase stability. U.S. Patent No. 9,308,520 discloses a method of altering the silanol groups of silica stationary phase separation materials after functionalization in order to increase the chemical stability of the stationary phase separation material by distributing organic groups from the surface into the silica-based material in a gradually decreasing concentration. U.S. Patent No. 11,642,653 discloses inorganic / organic hybrid materials wherein a surrounding material is condensed on a superficially porous hybrid core material. In many cases, hybrid inorganic / organic particles have thick surfaces, and / or are continuously functionalized throughout the particle structure. In other cited examples, the processes require complex manipulations of the particles, increasing cost and difficulties in reproducible manufacturing. Thus, there is a need for a stable chromatographic material, both chemically and mechanically, which is minimally modified on the surface. A suitable hybrid inorganic / organic chromatographic column packing material is desired to be of high surface area, to promote high retention of analytes and resistance to mass overloading, while also not possessing very small pore sizes, which will exhibit resistance to mass transport, and poor column efficiencies at higher linear velocity, via restricted diffusion in the stationary phase (see discussion in Synder, Kirkland and Dolan, Introduction to Modem Liquid Chromatography. 3rd Ed., Godinho, et al., (2020) J. Chromatogr. 1634, 461678). Thus, it is desired that the surface modification will not form thick layers, which occlude the porous structure, thereby reducing the effective pore size to negatively affect column performance. Ideally, a surface modification approach that yields a hybrid inorganic / organic material will also be applicable to a variety of silica particle types, including superficially porous particles, fully porous particles, and particles of a variety of discrete pore sizes. More specifically, there is a need for stable chromatographic material in which the surface silica incorporated carbon is no more than about 5%. Furthermore, there is an ongoing need for suitable material that can be employed as a chromatographic stationary phase separation material achieving desired separation characteristics for particular analytes of interest under high pH conditions.

[0006] Summary

[0007] The present disclosure provides methods of producing a pH stable chromatographic material comprising the steps of: i) providing an ammonium acetate buffer of pH from about 8 to about 10: ii) cooling the buffer mixture to a temperature of about 2 to about 25°C; iii) adding a silica to produce a 5 to 20% slurry (w / v); iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 25°C; v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours; vi) cooling the reaction mixture to a temperature less than about 40°C; vii) adjusting the pH of the reaction mixture to about 8 to about 10; viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours; ix) cooling the reaction mixture to a temperature of about 40 to about 80°C; x) isolating a coated silica by centrifugation / filtration; xi) dispersing the coated silica in water; xii) heating the coated silica water slurry to about 40 to about 100°C for about 4 to about 24 hours; xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C; and xiv) isolating the coated silica by centrifugation / filtration. rinsing the coated silica with a solvent, and drying the coated silica.

[0008] The present disclosure also provides pH stable chromatographic materials produced by the foregoing method.

[0009] The present disclosure also provides methods of attenuating the pore size of a silica particle wherein the pore size is changed by at least about 10% and no more than about 50%, the method comprising the steps of: i) providing an ammonium acetate buffer of pH from about 8 to about 10; ii) cooling the buffer mixture to a temperature of about 2 to about 25°C; iii) adding a silica to produce a 5 to 20% slurry (w / v); iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 25°C; v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours; vi) cooling the reaction mixture to a temperature less than about 40°C; vii) adjusting the pH of the reaction mixture to about 8 to about 10; viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours; ix) cooling the reaction mixture to a temperature of about 40 to about 80°C; x) isolating a coated silica by centrifugation / filtration; xi) dispersing the coated silica in water; xii) heating the coated silica water slurry to about 40 to about 100°C for about 4 to about 24 hours; xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C; and xiv) isolating the coated silica by centrifugation / filtration. rinsing the coated silica with a solvent, and drying the coated silica.

[0010] The present disclosure also provides attenuated silica particle materials produced by the foregoing method.

[0011] The present disclosure also provides any one or more of the foregoing pH stable chromatographic materials, or methods of producing a pH stable chromatographic material, or attenuated silica particle materials, or methods of attenuating the pore size of a silica particle, substantially as described with reference to the accompanying examples and / or figures.

[0012] Brief Description Of The Drawings

[0013] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure.

[0014] Figure 1 panel A shows a standard C18 silica product (HALO Cl 8) chromatograms, mobile phase: A, 10 mM ammonium bicarbonate / ammonia hydroxide. pH 10.0; B, acetonitrile; 50%B; Flow rate: 0.8 mL / min; Temperature: 60°C; Wavelength: 254 nm; Injection Volume: 0.5 pL; Sample: uracil, acenaphthylene, amitripty line.

[0015] Figure 1 panel B shows high pH stability' exhibited by the present disclosure (Elevate Cl 8) chromatograms, Mobile Phase: A, 10 mM ammonium bicarbonate / ammonia hydroxide, pH 10.0; B, acetonitrile; 50%B; Flow rate: 0.8 mL / min; Temperature: 60°C; Wavelength: 254 nm; Injection Volume: 0.5 pL; Sample: uracil, acenaphthylene, amitriptyline.

[0016] Figure 2 shows high pH stability of the present disclosure (Elevate Cl 8) compared to a standard C18 silica product (HALO-C18), using superficially prous silica particles of about 90 A pore size (HALO-C18) and 120 A pore size (Elevate C18) . Mobile Phase: A, 10 mM ammonium bicarbonate / ammonium hydroxide, pH 10.0; B, acetonitrile; 50%B; Flow rate: 0.8 mL / min; Temperature: 60°C; Wavelength: 254 nm; Injection Volume: 0.5 pL; Sample: uracil, acenaphthylene, amitripty line. Further testing of the HALO-C18 was discontinued due to excessive pressure. Figure 3 shows the separation of a mixture of synthetic oligonucleotides of 10 to 60 nucleotide bases in length using ion pairing reversed-phase. The separation used a 350 A pore size hybrid inorganic / organic superficially porous particle of the present disclosure, bonded with C18 on the surface, and loaded into a column of 2.1 mm I.D. x 50 mm. The separation was produced by gradient elution with flow rate of 0.5 mL / min, and a column temperature of 60°C, and gradient program shown in the figure, with Mobile phase A of 100 mM Tri ethylamine, pH 8.51, Mobile Phase B is acetonitrile.

[0017] Figure 4 shows the high pH stability of the present disclosure (Elevate Cl 8) using a hybrid inorganic / organic fully porous particle of 5 pm diameter and 95 A pore size. The separations are those obtained initially (injection 1) and after 3895 column volumes of high pH mobile phase (injection 70). Column: 2.1 mm I D. x 50 mm. Mobile Phase: 30% methanol (v / v) in 10 mM ammonium bicarbonate, pH 10.0. Flow rate: 0.21 mL / min, 50 °C, Absorbance at 254 nm.

[0018] Description Of Embodiments

[0019] Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong.

[0020] As used herein, the terms ‘"a” or "‘an7’ mean ‘"at least one” or “one or more” unless the context clearly indicates otherwise.

[0021] As used herein, the term “about” means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0022] As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive and open-ended and include the options following the terms, and do not exclude additional, unrecited elements or method steps.

[0023] The present disclosure provides methods of producing a pH stable chromatographic material comprising steps i) to xiv). The method of the present invention includes the step of i) providing an ammonium acetate buffer of pH from about 8 to about 10. In some embodiments, the concentration of ammonium acetate in step i) is from about 5 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 20 mM to about 30 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 5 mM to about 50 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 10 mM to about 40 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 20 mM to about 30 mM. In some embodiments, the pH is adjusted in step i) by the addition of an inorganic base, an organic base, or a combination thereof. In some embodiments the inorganic base is selected from the group consisting of NH4OH. NaOH. LiOH, KOH, Ca(OH)2, Ba(OH)2, Mg(OH)2, or any combination thereof. In some embodiments, the organic base is selected from the group consisting of triethylamine, N,N- diisopropylethylamine, 4-dimethylaminopyridine, l,8-Diazabicyclo[5.4.0]undec-7-ene, imidazole, or any combination thereof. The adjustment of the pH can be by the addition of an organic base or an inorganic base. In some embodiments, the adjustment in step i) is by the addition of NH4OH. In some embodiments, the adjustment in step i) is by the addition of NaOH. In some embodiments, the adjustment in step i) is by the addition of LiOH. In some embodiments, the adjustment in step i) is by the addition of KOH. In some embodiments, the adjustment in step i) is by the addition of Ca(OH)2. In some embodiments, the adjustment in step i) is by the addition of Ba(OH)2. In some embodiments, the adjustment in step i) is by the addition of Mg(OH)2. In some embodiments, the adjustment in step i) is by the addition of triethylamine. In some embodiments, the adjustment in step i) is by the addition of N,N- diisopropylethylamine. In some embodiments, the adjustment in step i) is by the addition of 4-dimethylaminopyridine. In some embodiments, the adjustment in step i) is by the addition of l,8-Diazabicyclo[5.4.0]undec-7-ene. In some embodiments, the adjustment in step i) is by the addition of imidazole. In some embodiments, the adjusted pH in step i) is from 9. 1 to 9.2. In some embodiments, the adjusted pH in step i) is 9.1. In some embodiments, the adjusted pH in step 1) is 9.2.

[0024] The method of the present invention includes the step of ii) cooling the buffer mixture to a temperature of about 2 to about 20°C. In some embodiments, the buffer mixture of step ii) is cooled to from 1 to 6°C. In other embodiments, the buffer mixture of step ii) is cooled to from 2 to 6°C. In other embodiments, the buffer mixture of step ii) is cooled to from 4 to 6°C. In yet other embodiments, the buffer mixture of step ii) is cooled to 5°C. In some embodiments, the buffer mixture of step ii) is cooled to 4°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0025] The method of the present invention includes the step of iii) adding a silica to produce a 5 to 20% slurry (w / v). In some embodiments, the silica is selected from the group consisting of a porous silica, a superficially porous silica, a non-porous silica, or any combination thereof. In some embodiments, the silica is a porous silica. In some embodiments, the silica is a superficially porous silica. In some embodiments, the silica is a non-porous silica. In some embodiments, the silica has a pore size of about 85 A. In some embodiments, the silica has a pore size of about 95 A. In some embodiments, the silica has a pore size of about 100A. In some embodiments, the silica has a pore size of about 110A. In some embodiments, the silica has a pore size of about 120A. In some embodiments, the silica has a pore size of about 130A. In some embodiments, the silica has a pore size of about 140A. In some embodiments, the silica has a pore size of about 150A. In some embodiments, the silica has a pore size of about 160A. In some embodiments, the silica has a pore size of about 170A. In some embodiments, the silica has a pore size of about 180A. In some embodiments, the silica has a pore size of about 190A. In some embodiments, the silica has a pore size of about 200A. In some embodiments, the silica has a pore size of about 250A. In some embodiments, the silica has a pore size of about 300A. In some embodiments, the silica has a pore size of about 350A. In some embodiments, the silica has a pore size of about 400A. In some embodiments, the silica has a pore size of about 450A. In some embodiments, the silica has a pore size of about 500A. In some embodiments, the silica has a pore size of about 550A. In some embodiments, the silica has a pore size of about 600A. In some embodiments, the silica has a pore size of about 650A. In some embodiments, the silica has a pore size of about 700A. In some embodiments, the silica has a pore size of about 750A. In some embodiments, the silica has a pore size of about 800A. In some embodiments, the silica has a pore size of about 850A. In some embodiments, the silica has a pore size of about 900A. In some embodiments, the silica has a pore size of about 950A. In some embodiments, the silica has a pore size of about 1000 A. In some embodiments, the porous silica has a pore size of about 85 A. In some embodiments, the porous silica has a pore size of about 95 A. In some embodiments, the porous silica has a pore size of about 100 A. In some embodiments, the porous silica has a pore size of about 1 lOA. In some embodiments, the porous silica has a pore size of about 120A. In some embodiments, the porous silica has a pore size of about 130A. In some embodiments, the porous silica has a pore size of about 140 A. In some embodiments, the porous silica has a pore size of about 150A. In some embodiments, the porous silica has a pore size of about 160A. In some embodiments, the porous silica has a pore size of about 170A. In some embodiments, the porous silica has a pore size of about 180A. In some embodiments, the porous silica has a pore size of about 190 A. In some embodiments, the porous silica has a pore size of about 200 A. In some embodiments, the porous silica has a pore size of about 250A. In some embodiments, the porous silica has a pore size of about 350A. In some embodiments, the porous silica has a pore size of about 400A. In some embodiments, the porous silica has a pore size of about 450A. In some embodiments, the porous silica has a pore size of about 500 A. In some embodiments, the porous silica has a pore size of about 550A. In some embodiments, the porous silica has a pore size of about 600A. In some embodiments, the porous silica has a pore size of about 650A. In some embodiments, the porous silica has a pore size of about 700 A. In some embodiments, the porous silica has a pore size of about 750A. In some embodiments, the porous silica has a pore size of about 800A. In some embodiments, the porous silica has a pore size of about 850A. In some embodiments, the porous silica has a pore size of about 900A. In some embodiments, the porous silica has a pore size of about 950A. In some embodiments, the porous silica has a pore size of about lOOOA. In some embodiments, the superficially porous silica has a pore size of about 85A. In some embodiments, the superficially porous silica has a pore size of about 95A. In some embodiments, the superficially porous silica has a pore size of about 100 A. In some embodiments, the superficially porous silica has a pore size of about 110 A. In some embodiments, the superficially porous silica has a pore size of about 120 A. In some embodiments, the superficially porous silica has a pore size of about 130A. In some embodiments, the superficially porous silica has a pore size of about 140 A. In some embodiments, the superficially porous silica has a pore size of about 150A. In some embodiments, the superficially porous silica has a pore size of about 160 A. In some embodiments, the superficially porous silica has a pore size of about 170A. In some embodiments, the superficially porous silica has a pore size of about 180A. In some embodiments, the superficially porous silica has a pore size of about 190 A. In some embodiments, the superficially porous silica has a pore size of about 200 A. In some embodiments, the superficially porous silica has a pore size of about 250A. In some embodiments, the superficially porous silica has a pore size of about 300A. In some embodiments, the superficially porous silica has a pore size of about 350A. In some embodiments, the superficially porous silica has a pore size of about 400A. In some embodiments, the superficially porous silica has a pore size of about 450A. In some embodiments, the superficially porous silica has a pore size of about 500 A. In some embodiments, the superficially porous silica has a pore size of about 550 A. In some embodiments, the superficially porous silica has a pore size of about 600A. In some embodiments, the superficially porous silica has a pore size of about 650A. In some embodiments, the superficially porous silica has a pore size of about 700 A. In some embodiments, the superficially porous silica has a pore size of about 750 A. In some embodiments, the superficially porous silica has a pore size of about 800 A. In some embodiments, the superficially porous silica has a pore size of about 850A. In some embodiments, the superficially porous silica has a pore size of about 900 A. In some embodiments, the superficially porous silica has a pore size of about 950A. In some embodiments, the superficially porous silica has a pore size of about lOOOA. In some embodiments, the concentration of the silica is 5 to 10% (w / v) in step iii).

[0026] The method of the present invention includes the step of iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 25°C. In some embodiments, the silane added in step iv) is selected from the group consisting of l,2-bis(tri ethoxy silyl)ethane, 1.2-bis(trichlorosilyl)ethane, tris(dimethylamino)ethyl silane, ethyltriethoxysilane, ethyltrimethoxysilane, methyltrichlorosilane, ethyltrichlorosilane, bis(trichlorosilyl)methane, bis(methyldimethoxysilyl)methane. 1,2-Bis(methyldichlorosilyl)ethane, 1.2- dichlorotetramethyldisilane, or any combination thereof. In some embodiments, the silane added in step iv) is l,2-bis(triethoxysilyl)ethane. In some embodiments, the silane added in step iv) is 1 ,2-bis(trichlorosilyl)ethane. In some embodiments, the silane added in step iv) is tris(dimethylamino)ethyl silane. In some embodiments, the silane added in step iv) is ethyltriethoxysilane. In some embodiments, the silane added in step iv) is ethyltrimethoxysilane. In some embodiments, the silane added in step iv) is methyltrichlorosilane. In some embodiments, the silane added in step iv) is ethyltrichlorosilane. In some embodiments, the silane added in step iv) is bis(trichlorosilyl)methane. In some embodiments, the silane added in step iv) is bis(methyldimethoxysilyl)methane. In some embodiments, the silane added in step iv) is 1,2-

[0027] Bis(methyldichlorosilyl)ethane. In some embodiments, the silane added in step iv) is 1,2- dichlorotetramethyldisilane. In some embodiments, the reaction mixture of step iv) is stirred for 6 hours. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of from 5 to 6°C. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of 5°C. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of 6°C.

[0028] The method of the present invention includes the step of v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours. In some embodiments, the reaction mixture of step v) is heated to 100°C for 6 hours. In some embodiments, step v) further comprises applying a gaseous stream. In some embodiments, the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof. In some embodiments, the gaseous stream is nitrogen. In some embodiments, the gaseous stream is argon. In some embodiments, the gaseous stream is carbon dioxide. In some embodiments, the gaseous stream is helium. In some embodiments, the gaseous stream is atmospheric air. In some embodiments, the gaseous stream is applied for about 6 to about 18 hours. In some embodiments, the gaseous stream is applied for 6 hours. In some embodiments, the gaseous stream is applied for 7 hours. In some embodiments, the gaseous stream is applied for 8 hours. In some embodiments, the gaseous stream is applied for 9 hours. In some embodiments, the gaseous stream is applied for 10 hours. In some embodiments, the gaseous stream is applied for 11 hours. In some embodiments, the gaseous stream is applied for 12 hours. In some embodiments, the gaseous stream is applied for 13 hours. In some embodiments, the gaseous stream is applied for 14 hours. In some embodiments, the gaseous stream is applied for 15 hours. In some embodiments, the gaseous stream is applied for 16 hours. In some embodiments, the gaseous stream is applied for 17 hours. In some embodiments, the gaseous stream is applied for 18 hours.

[0029] The method of the present invention includes the step of vi) cooling the reaction mixture to a temperature less than about 40°C. In some embodiments, the temperature of step vi) is from 5 to 6°C. In some embodiments, the temperature of step vi) is 5°C. In some embodiments, the temperature of step vi) is 6°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling j acket or an ice bath.

[0030] The method of the present invention includes the step of vii) adjusting the pH of the reaction mixture to about 8 to about 10. In some embodiments, the adjustment in step vii) is by the addition of an inorganic base, an organic base, or a combination thereof. In some embodiments the inorganic base is selected from the group consisting of NH4OH. NaOH. LiOH, KOH, Ca(OH)2, Ba(OH)2, Mg(0H)2, or any combination thereof. In some embodiments, the organic base is selected from the group consisting of triethylamine, N,N- diisopropylethylamine, 4-dimethylaminopyridine, l,8-Diazabicyclo[5.4.0]undec-7-ene, imidazole, or any combination thereof. The adjustment of the pH can be by the addition of an organic base or an inorganic base. In some embodiments, the adjustment in step vii) is by the addition of NH4OH. In some embodiments, the adjustment in step vii) is by the addition of NaOH. In some embodiments, the adjustment in step vii) is by the addition of LiOH. In some embodiments, the adjustment in step vii) is by the addition of KOH. In some embodiments, the adjustment in step vii) is by the addition of Ca(OH)2. In some embodiments, the adjustment in step vii) is by the addition of Ba(OH)2. In some embodiments, the adjustment in step vii) is by the addition of Mg(0H)2. In some embodiments, the adjustment in step vii) is by the addition of tri ethylamine. In some embodiments, the adjustment in step vii) is by the addition of N,N-diisopropylethylamine. In some embodiments, the adjustment in step vii) is by the addition of 4-dimethylaminopyridine. In some embodiments, the adjustment in step vii) is by the addition of l,8-Diazabicyclo[5.4.0]undec-7-ene. In some embodiments, the adjustment in step vii) is by the addition of imidazole. In some embodiments, the adjusted pH in step vii) is from 9. 1 to 9.2. In some embodiments, the adjusted pH in step vii) is 9. 1. In some embodiments, the adjusted pH in step vii) is 9.2. In some embodiments, step vii) further comprises applying a gaseous stream. In some embodiments, the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof. In some embodiments, the gaseous stream is nitrogen. In some embodiments, the gaseous stream is argon. In some embodiments, the gaseous stream is carbon dioxide. In some embodiments, the gaseous stream is helium. In some embodiments, the gaseous stream is atmospheric air. In some embodiments, the gaseous stream is applied for about 6 to about 18 hours. In some embodiments, the gaseous stream is applied for 6 hours. In some embodiments, the gaseous stream is applied for 7 hours. In some embodiments, the gaseous stream is applied for 8 hours. In some embodiments, the gaseous stream is applied for 9 hours. In some embodiments, the gaseous stream is applied for 10 hours. In some embodiments, the gaseous stream is applied for 11 hours. In some embodiments, the gaseous stream is applied for 12 hours. In some embodiments, the gaseous stream is applied for 13 hours. In some embodiments, the gaseous stream is applied for 14 hours. In some embodiments, the gaseous stream is applied for 15 hours. In some embodiments, the gaseous stream is applied for 16 hours. In some embodiments, the gaseous stream is applied for 17 hours. In some embodiments, the gaseous stream is applied for 18 hours.

[0031] The method of the present invention includes the step of viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours. In some embodiments, the reaction mixture of step viii) is heated to 100°C for 24 hours. The heating can be accomplished by any means known in the art such as, for example, a heating jacket.

[0032] The method of the present invention includes the step of ix) cooling the reaction mixture to a temperature of about 40 to about 80°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0033] The method of the present invention includes the step of x) isolating coated silica by centrifugation / filtrati on.

[0034] The method of the present invention includes the step of xi) dispersing the coated silica in water. In some embodiments, the concentration of the coated silica is 5 to 20% (w / v) in step xi). The dispersing step can be accomplished by using any means known in the art such as, for example, a paddle mixter or magnetic stirrer.

[0035] The method of the present invention includes the step of xii) heating the coated silica water slurry to about 40 to about 100°C for about 4 to about 24 hours. In some embodiments, the coated silica water slurry of step xii) is heated to 100°C for 24 hours. The heating can be accomplished by any means known in the art such as, for example, a heating jacket.

[0036] The method of the present invention includes the step of xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0037] The method of the present invention includes the step of xiv) isolating the coated silica by centrifugation / filtration, rinsing the coated silica with a solvent, and drying the coated silica. In some embodiments, the solvent in step xiv) is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, acetone, or any combination thereof. In some embodiments, the solvent in step xiv) is acetonitrile. In some embodiments, the solvent in step xiv) is tetrahydrofuran. In some embodiments, the solvent in step xiv) is methanol. In some embodiments, the solvent in step xiv) is acetone.

[0038] The present disclosure also provides pH stable chromatographic materials produced by any of the foregoing methods. The present disclosure also provides methods of attenuating the pore size of a silica particle wherein the pore size is changed by at least about 10% and not more than about 50%, the method comprising steps i) to xiv).

[0039] The method of the present invention includes the step of i) providing an ammonium acetate buffer of pH from about 8 to about 10. In some embodiments, the concentration of ammonium acetate in step i) is from about 5 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 20 mM to about 30 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 5 mM to about 50 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 10 mM to about 40 mM. In some embodiments, the concentration of ammonium acetate in step i) is from about 20 mM to about 30 mM. In some embodiments, the pH is adjusted in step i) by the addition of an inorganic base, an organic base, or a combination thereof. In some embodiments the inorganic base is selected from the group consisting of NH4OH, NaOH, LiOH. KOH, Ca(OH)2, Ba(OH)2. Mg(OH)2, or any combination thereof. In some embodiments, the organic base is selected from the group consisting of triethylamine, N,N- diisopropylethylamine, 4-dimethylaminopyridine, l,8-Diazabicyclo[5.4.0]undec-7-ene, imidazole, or any combination thereof. The adjustment of the pH can be by the addition of an organic base or an inorganic base. In some embodiments, the adjustment in step i) is by the addition of NH4OH. In some embodiments, the adjustment in step i) is by the addition of NaOH. In some embodiments, the adjustment in step i) is by the addition of LiOH. In some embodiments, the adjustment in step i) is by the addition of KOH. In some embodiments, the adjustment in step i) is by the addition of Ca(OH)2. In some embodiments, the adjustment in step i) is by the addition of Ba(OH)2. In some embodiments, the adjustment in step i) is by the addition of Mg(OH)2. In some embodiments, the adjustment in step i) is by the addition of tri ethylamine. In some embodiments, the adjustment in step i) is by the addition of N,N- diisopropylethylamine. In some embodiments, the adjustment in step i) is by the addition of 4-dimethylaminopyridine. In some embodiments, the adjustment in step i) is by the addition of 1.8-Diazabicyclo|5.4.0]undec-7-ene. In some embodiments, the adjustment in step i) is by the addition of imidazole. In some embodiments, the adjusted pH in step i) is from 9. 1 to 9.2. In some embodiments, the adjusted pH in step i) is 9.1. In some embodiments, the adjusted pH in step i) is 9.2.

[0040] The method of the present invention includes the step of ii) cooling the buffer mixture to a temperature of about 2 to about 25°C. In some embodiments, the buffer mixture of step ii) is cooled to from 5 to 6°C. In some embodiments, the buffer mixture of step ii) is cooled to 5°C. In some embodiments, the buffer mixture of step ii) is cooled to 6°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0041] The method of the present invention includes the step of iii) adding a silica to produce a 5 to 20% slurry (w / v). In some embodiments, the silica is selected from the group consisting of a porous silica, a superficially porous silica, a non-porous silica, or any combination thereof. In some embodiments, the silica is a porous silica. In some embodiments, the silica is a superficially porous silica. In some embodiments, the silica is a non-porous silica. In some embodiments, the silica has a pore size of about 85 A. In some embodiments, the silica has a pore size of about 95 A. In some embodiments, the silica has a pore size of about lOOA. In some embodiments, the silica has a pore size of about 110A. In some embodiments, the silica has a pore size of about 120A. In some embodiments, the silica has a pore size of about 130A. In some embodiments, the silica has a pore size of about 140A. In some embodiments, the silica has a pore size of about 150A. In some embodiments, the silica has a pore size of about 160A. In some embodiments, the silica has a pore size of about 170A. In some embodiments, the silica has a pore size of about 180A. In some embodiments, the silica has a pore size of about 190A. In some embodiments, the silica has a pore size of about 200A. In some embodiments, the silica has a pore size of about 250A. In some embodiments, the silica has a pore size of about 300A. In some embodiments, the silica has a pore size of about 350A. In some embodiments, the silica has a pore size of about 400A. In some embodiments, the silica has a pore size of about 450A. In some embodiments, the silica has a pore size of about 500A. In some embodiments, the silica has a pore size of about 550A. In some embodiments, the silica has a pore size of about 600A. In some embodiments, the silica has a pore size of about 650A. In some embodiments, the silica has a pore size of about 700A. In some embodiments, the silica has a pore size of about 750A. In some embodiments, the silica has a pore size of about 800A. In some embodiments, the silica has a pore size of about 850A. In some embodiments, the silica has a pore size of about 900A. In some embodiments, the silica has a pore size of about 950A. In some embodiments, the silica has a pore size of about 1000 A. In some embodiments, the porous silica has a pore size of about 85 A. In some embodiments, the porous silica has a pore size of about 95 A. In some embodiments, the porous silica has a pore size of about lOOA. In some embodiments, the porous silica has a pore size of about 110A. In some embodiments, the porous silica has a pore size of about 120A. In some embodiments, the porous silica has a pore size of about 130A. In some embodiments, the porous silica has a pore size of about 140 A. In some embodiments, the porous silica has a pore size of about 150A. In some embodiments, the porous silica has a pore size of about 160 A. In some embodiments, the porous silica has a pore size of about 170A. In some embodiments, the porous silica has a pore size of about 180A. In some embodiments, the porous silica has a pore size of about 190 A. In some embodiments, the porous silica has a pore size of about 200 A. In some embodiments, the porous silica has a pore size of about 250A. In some embodiments, the porous silica has a pore size of about 350A. In some embodiments, the porous silica has a pore size of about 400A. In some embodiments, the porous silica has a pore size of about 450A. In some embodiments, the porous silica has a pore size of about 500 A. In some embodiments, the porous silica has a pore size of about 550A. In some embodiments, the porous silica has a pore size of about 600A. In some embodiments, the porous silica has a pore size of about 650A. In some embodiments, the porous silica has a pore size of about 700 A. In some embodiments, the porous silica has a pore size of about 750A. In some embodiments, the porous silica has a pore size of about 800A. In some embodiments, the porous silica has a pore size of about 850A. In some embodiments, the porous silica has a pore size of about 900A. In some embodiments, the porous silica has a pore size of about 950A. In some embodiments, the porous silica has a pore size of about lOOOA. In some embodiments, the superficially porous silica has a pore size of about 85A. In some embodiments, the superficially porous silica has a pore size of about 95A. In some embodiments, the superficially porous silica has a pore size of about 100 A. In some embodiments, the superficially porous silica has a pore size of about 110 A. In some embodiments, the superficially porous silica has a pore size of about 120A. In some embodiments, the superficially porous silica has a pore size of about 130A. In some embodiments, the superficially porous silica has a pore size of about 140 A. In some embodiments, the superficially porous silica has a pore size of about 150A. In some embodiments, the superficially porous silica has a pore size of about 160 A. In some embodiments, the superficially porous silica has a pore size of about 170A. In some embodiments, the superficially porous silica has a pore size of about 180A. In some embodiments, the superficially porous silica has a pore size of about 190 A. In some embodiments, the superficially porous silica has a pore size of about 200 A. In some embodiments, the superficially porous silica has a pore size of about 250A. In some embodiments, the superficially porous silica has a pore size of about 300 . In some embodiments, the superficially porous silica has a pore size of about 350A. In some embodiments, the superficially porous silica has a pore size of about 400 A. In some embodiments, the superficially porous silica has a pore size of about 450A. In some embodiments, the superficially porous silica has a pore size of about 500 A. In some embodiments, the superficially porous silica has a pore size of about 550A. In some embodiments, the superficially porous silica has a pore size of about 600A. In some embodiments, the superficially porous silica has a pore size of about 650A. In some embodiments, the superficially porous silica has a pore size of about 700 A. In some embodiments, the superficially porous silica has a pore size of about 750A. In some embodiments, the superficially porous silica has a pore size of about 800A. In some embodiments, the superficially porous silica has a pore size of about 850A. In some embodiments, the superficially porous silica has a pore size of about 900 A. In some embodiments, the superficially porous silica has a pore size of about 950A. In some embodiments, the superficially porous silica has a pore size of about 1000A. In some embodiments, the concentration of the silica is 5 to 10% (w / v) in step iii). In some embodiments, the pore size of the silica in step iii) is changed by at least about 10% and no more than about 50%. In some embodiments, the pore size of the silica in step iii) is changed by about 10%. In some embodiments, the pore size of the silica in step iii) is changed by about 20%. In some embodiments, the pore size of the silica in step iii) is changed by about 30%. In some embodiments, the pore size of the silica in step iii) is changed by about 40%. In some embodiments, the pore size of the silica in step iii) is changed by about 50%. In some embodiments, the specific surface area of the silica in step iii) is changed by less than about 15%. In some embodiments, the specific surface area of the silica in step iii) is changed by less than about 10%. In some embodiments, the specific surface area of the silica in step iii) is changed by less than about 5%. In some embodiments, the specific surface area of the silica in step iii) is changed by less than about 2.5%. In some embodiments, the the pore size of the silica in step iii) is changed by at least about 10% and no more than about 50% and the specific surface area of the silica in step iii) is changed by less than about 15%.

[0042] The method of the present invention includes the step of iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 25°C. In some embodiments, the silane added in step iv) is selected from the group consisting of 1 ,2-bis(tri ethoxy silyl)ethane, l,2-bis(trichlorosilyl)ethane, tris(dimethylamino)ethyl silane, ethyltriethoxysilane, ethyltrimethoxysilane. methyltrichlorosilane, ethyltrichlorosilane, bis(trichlorosilyl)methane, bis(methyldimethoxysilyl)methane, 1,2-Bis(methyldichlorosilyl)ethane, 1,2- dichlorotetramethyldisilane, or any combination thereof. In some embodiments, the silane added in step iv) is l,2-bis(triethoxysilyl)ethane. In some embodiments, the silane added in step iv) is l,2-bis(trichlorosilyl)ethane. In some embodiments, the silane added in step iv) is tris(dimethylamino)ethyl silane. In some embodiments, the silane added in step iv) is ethyltriethoxysilane. In some embodiments, the silane added in step iv) is ethyltrimethoxysilane. In some embodiments, the silane added in step iv) is methyltrichlorosilane. In some embodiments, the silane added in step iv) is ethyltrichlorosilane. In some embodiments, the silane added in step iv) is bis(trichlorosilyl)methane. In some embodiments, the silane added in step iv) is bis(methyldimethoxysilyl)methane. In some embodiments, the silane added in step iv) is 1,2- Bis(methyldichlorosilyl)ethane. In some embodiments, the silane added in step iv) is 1,2- dichlorotetramethyldisilane. In some embodiments, the reaction mixture of step iv) is stirred for 6 hours. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of from 5 to 6°C. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of 5°C. In some embodiments, the reaction mixture of step iv) is maintained at a temperature of 6°C.

[0043] The method of the present invention includes the step of v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours. In some embodiments, the reaction mixture of step v) is heated to 100°C for 6 hours. In some embodiments, step v) further comprises applying a gaseous stream. In some embodiments, the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof. In some embodiments, the gaseous stream is nitrogen. In some embodiments, the gaseous stream is argon. In some embodiments, the gaseous stream is carbon dioxide. In some embodiments, the gaseous stream is helium. In some embodiments, the gaseous stream is atmospheric air. In some embodiments, the gaseous stream is applied for about 6 to about 18 hours. In some embodiments, the gaseous stream is applied for 6 hours. In some embodiments, the gaseous stream is applied for 7 hours. In some embodiments, the gaseous stream is applied for 8 hours. In some embodiments, the gaseous stream is applied for 9 hours. In some embodiments, the gaseous stream is applied for 10 hours. In some embodiments, the gaseous stream is applied for 1 1 hours. In some embodiments, the gaseous stream is applied for 12 hours. In some embodiments, the gaseous stream is applied for 13 hours. In some embodiments, the gaseous stream is applied for 14 hours. In some embodiments, the gaseous stream is applied for 15 hours. In some embodiments, the gaseous stream is applied for 16 hours. In some embodiments, the gaseous stream is applied for 17 hours. In some embodiments, the gaseous stream is applied for 18 hours.

[0044] The method of the present invention includes the step of vi) cooling the reaction mixture to a temperature less than about 40°C. In some embodiments, the temperature of step vi) is from 5 to 6°C. In some embodiments, the temperature of step vi) is 5°C. In some embodiments, the temperature of step vi) is 6°C.

[0045] The method of the present invention includes the step of vii) adjusting the pH of the reaction mixture to about 8 to about 10. In some embodiments, the adjustment in step vii) is by the addition of an inorganic base, an organic base, or a combination thereof. In some embodiments the inorganic base is selected from the group consisting of NH4OH, NaOH, LiOH, KOH, Ca(OH)2, Ba(OH)2, Mg(OH)2, or any combination thereof. In some embodiments, the organic base is selected from the group consisting of triethylamine, N,N- diisopropylethylamine, 4-dimethylaminopyridine, l,8-Diazabicyclo[5.4.0]undec-7-ene, imidazole, or any combination thereof. The adjustment of the pH can be by the addition of an organic base or an inorganic base. In some embodiments, the adjustment in step vii) is by the addition of NH4OH. In some embodiments, the adjustment in step vii) is by the addition of NaOH. In some embodiments, the adjustment in step vii) is by the addition of LiOH. In some embodiments, the adjustment in step vii) is by the addition of KOH. In some embodiments, the adjustment in step vii) is by the addition of Ca(OH)2. In some embodiments, the adjustment in step vii) is by the addition of Ba(OH)2. In some embodiments, the adjustment in step vii) is by the addition of Mg(OH)2. In some embodiments, the adjustment in step vii) is by the addition of tri ethylamine. In some embodiments, the adjustment in step vii) is by the addition of N,N-diisopropylethylamine. In some embodiments, the adjustment in step vii) is by the addition of 4-dimethylaminopyridine. In some embodiments, the adjustment in step vii) is by the addition of l,8-Diazabicyclo[5.4.0]undec-7-ene. In some embodiments, the adjustment in step vii) is by the addition of imidazole. In some embodiments, the adjusted pH in step vii) is from 9. 1 to 9.2. In some embodiments, the adjusted pH in step vii) is 9. 1. In some embodiments, the adjusted pH in step vii) is 9.2. In some embodiments, step vii) further comprises applying a gaseous stream. In some embodiments, the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air. or any combination thereof. In some embodiments, the gaseous stream is nitrogen. In some embodiments, the gaseous stream is argon. In some embodiments, the gaseous stream is carbon dioxide. In some embodiments, the gaseous stream is helium. In some embodiments, the gaseous stream is atmospheric air. In some embodiments, the gaseous stream is applied for about 6 to about 18 hours. In some embodiments, the gaseous stream is applied for 6 hours. In some embodiments, the gaseous stream is applied for 7 hours. In some embodiments, the gaseous stream is applied for 8 hours. In some embodiments, the gaseous stream is applied for 9 hours. In some embodiments, the gaseous stream is applied for 10 hours. In some embodiments, the gaseous stream is applied for 11 hours. In some embodiments, the gaseous stream is applied for 12 hours. In some embodiments, the gaseous stream is applied for 13 hours. In some embodiments, the gaseous stream is applied for 14 hours. In some embodiments, the gaseous stream is applied for 15 hours. In some embodiments, the gaseous stream is applied for 16 hours. In some embodiments, the gaseous stream is applied for 17 hours. In some embodiments, the gaseous stream is applied for 18 hours.

[0046] The method of the present invention includes the step of viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours. In some embodiments, the reaction mixture of step viii) is heated to 100°C for 24 hours. The heating can be accomplished by any means known in the art such as, for example, a heating jacket.

[0047] The method of the present invention includes the step of ix) cooling the reaction mixture to a temperature of about 40 to about 80°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0048] The method of the present invention includes the step of x) isolating a coated silica by centrifugation / fdtration.

[0049] The method of the present invention includes the step of xi) dispersing the coated silica in water. In some embodiments, the concentration of the coated silica is 5 to 20% (w / v) in step xi). The dispersing step can be accomplished by using any means known in the art such as, for example, a paddle mixter or magnetic stirrer.

[0050] The method of the present invention includes the step of xii) heating the coated silica water slurry to about 40 to about 100°C for about 4 to about 24 hours. In some embodiments, the coated silica water slurry of step xii) is heated to 100°C for 24 hours. The heating can be accomplished by any means known in the art such as. for example, a heating jacket.

[0051] The method of the present invention includes the step of xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C. The mixture can be cooled by any means known to those skilled in the art such as, for example, a cooling jacket or an ice bath.

[0052] The method of the present invention includes the step of xiv) isolating the coated silica by centrifugation / filtration, rinsing the coated silica with a solvent, and drying the coated silica. In some embodiments, the solvent in step xiv) is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, acetone, or any combination thereof. In some embodiments, the solvent in step xiv) is acetonitrile. In some embodiments, the solvent in step xiv) is tetrahydrofuran. In some embodiments, the solvent in step xiv) is methanol. In some embodiments, the solvent in step xiv) is acetone. In some embodiments, the surface silica incorporated carbon content of the coated silica is from about 0. 15% to about 4.5%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.15%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.2%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.3%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.4%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.5%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.6%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.7%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.8%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 0.9%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.0%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.1%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.2%.In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.3%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.4%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.5%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.6%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.7%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.8%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 1.9%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.0%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.1%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.2%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.3%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.4%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.5%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.6%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.7%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.8%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 2.9%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.0%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.1%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.2%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.3%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.4%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.5%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.6%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.7%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.8%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 3.9%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4.0%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4. 1%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4.2%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4.3%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4.4%. In some embodiments, the surface silica incorporated carbon content of the coated silica is about 4.5%. In some embodiments, the pore size of the coated silica is from about 85 A to about 1000 A. In some embodiments, the pore size of the coated silica is about 85 A. In some embodiments, the pore size of the coated silica is about 95 A. In some embodiments, the pore size of the coated silica is about 100 A. In some embodiments, the pore size of the coated silica is about 110 A. In some embodiments, the pore size of the coated silica is about 120 A. In some embodiments, the pore size of the coated silica is about 130 A. In some embodiments, the pore size of the coated silica is about 140 A. In some embodiments, the pore size of the coated silica is about 150 A. In some embodiments, the pore size of the coated silica is about 160 A. In some embodiments, the pore size of the coated silica is about 170 A. In some embodiments, the pore size of the coated silica is about 180 A. In some embodiments, the pore size of the coated silica is about 190 A. In some embodiments, the pore size of the coated silica is about 200 A. In some embodiments, the pore size of the coated silica is about 210 A. In some embodiments, the pore size of the coated silica is about 220 A. In some embodiments, the pore size of the coated silica is about 230 A. In some embodiments, the pore size of the coated silica is about 240 A. In some embodiments, the pore size of the coated silica is about 250 A. In some embodiments, the pore size of the coated silica is about 260 A. In some embodiments, the pore size of the coated silica is about 270 A. In some embodiments, the pore size of the coated silica is about 280 A. In some embodiments, the pore size of the coated silica is about 290 A. In some embodiments, the pore size of the coated silica is about 300 A. In some embodiments, the pore size of the coated silica is about 310 A. In some embodiments, the pore size of the coated silica is about 320 A. In some embodiments, the pore size of the coated silica is about 330 A. In some embodiments, the pore size of the coated silica is about 340 A. In some embodiments, the pore size of the coated silica is about 350 A. In some embodiments, the pore size of the coated silica is about 360 A. In some embodiments, the pore size of the coated silica is about 370 A. In some embodiments, the pore size of the coated silica is about 380 A. In some embodiments, the pore size of the coated silica is about 390 A. In some embodiments, the pore size of the coated silica is about 400 A. In some embodiments, the pore size of the coated silica is about 410 A. In some embodiments, the pore size of the coated silica is about 420 A. In some embodiments, the pore size of the coated silica is about 430 A. In some embodiments, the pore size of the coated silica is about 440 A. In some embodiments, the pore size of the coated silica is about 450 A. In some embodiments, the pore size of the coated silica is about 460 A. In some embodiments, the pore size of the coated silica is about 470 A. In some embodiments, the pore size of the coated silica is about 480 A. In some embodiments, the pore size of the coated silica is about 490 A. In some embodiments, the pore size of the coated silica is about 500 A. In some embodiments, the pore size of the coated silica is about 510 A. In some embodiments, the pore size of the coated silica is about 520 A. In some embodiments, the pore size of the coated silica is about 530 A. In some embodiments, the pore size of the coated silica is about 540 A. In some embodiments, the pore size of the coated silica is about 550 A. In some embodiments, the pore size of the coated silica is about 560 A. In some embodiments, the pore size of the coated silica is about 570 A. In some embodiments, the pore size of the coated silica is about 580 A. In some embodiments, the pore size of the coated silica is about 590 A. In some embodiments, the pore size of the coated silica is about 600 A. In some embodiments, the pore size of the coated silica is about 610 A. In some embodiments, the pore size of the coated silica is about 620 A. In some embodiments, the pore size of the coated silica is about 630 A. In some embodiments, the pore size of the coated silica is about 640 A. In some embodiments, the pore size of the coated silica is about 650 A. In some embodiments, the pore size of the coated silica is about 660 A. In some embodiments, the pore size of the coated silica is about 670 A. In some embodiments, the pore size of the coated silica is about 680 A. In some embodiments, the pore size of the coated silica is about 690 A. In some embodiments, the pore size of the coated silica is about 700 A. In some embodiments, the pore size of the coated silica is about 710 A. In some embodiments, the pore size of the coated silica is about 720 A. In some embodiments, the pore size of the coated silica is about 730 A. In some embodiments, the pore size of the coated silica is about 740 A. In some embodiments, the pore size of the coated silica is about 750 A. In some embodiments, the pore size of the coated silica is about 760 A. In some embodiments, the pore size of the coated silica is about 770 A. In some embodiments, the pore size of the coated silica is about 780 A. In some embodiments, the pore size of the coated silica is about 790 A. In some embodiments, the pore size of the coated silica is about 800 A. In some embodiments, the pore size of the coated silica is about 810 A. In some embodiments, the pore size of the coated silica is about 820 A. In some embodiments, the pore size of the coated silica is about 830 A. In some embodiments, the pore size of the coated silica is about 840 A. In some embodiments, the pore size of the coated silica is about 850 A. In some embodiments, the pore size of the coated silica is about 860 A. In some embodiments, the pore size of the coated silica is about 870 A. In some embodiments, the pore size of the coated silica is about 880 A. In some embodiments, the pore size of the coated silica is about 890 A. In some embodiments, the pore size of the coated silica is about 900 A. In some embodiments, the pore size of the coated silica is about 910 A. In some embodiments, the pore size of the coated silica is about 920 A. In some embodiments, the pore size of the coated silica is about 930 A. In some embodiments, the pore size of the coated silica is about 940 A. In some embodiments, the pore size of the coated silica is about 950 A. In some embodiments, the pore size of the coated silica is about 960 A. In some embodiments, the pore size of the coated silica is about 970 A. In some embodiments, the pore size of the coated silica is about 980 A. In some embodiments, the pore size of the coated silica is about 990 A. In some embodiments, the pore size of the coated silica is about 1000 A. In some embodiments, the surface silica incorporated carbon content of the coated silica is from about 0. 15% to about 4.5% and the pore size of the coated silica is from about 85 A to about 1000 A.

[0053] The present disclosure also provides attenuated silica particle materials produced by the any of the foregoing methods.

[0054] The present disclosure also provides attenuated silica particle materials having an alkaline stability, wherein the alkaline stability is determined by resistance to degradation of separation performance assessed by column efficiency, peak shape, and peak retention time during continuous challenge with a buffer at pH of at least 10 mixed with an organic solvent, at 50°C, of acidic, basic, and / or neutral analytes, and wherein performance is maintained for at least 2 days of continuous operation.

[0055] The present disclosure also provides attenuated silica particle materials having an alkaline stability, wherein the alkaline stability is determined by resistance to degradation of separation performance assessed by column efficiency, peak shape, and peak retention time during continuous challenge with lOmM ammonium bicarbonate / ammonium hydroxide at pH of at least 10 mixed with acetonitrile in a 1 : 1 volume ratio, at 50°C, of acidic, basic, and / or neutral analytes, and wherein performance is maintained for at least 2 days of continuous operation.

[0056] The present disclosure also provides attenuated silica particle materials having an alkaline stability, wherein the alkaline stability is determined by resistance to degradation of separation performance assessed by column efficiency, peak shape, and peak retention time during continuous challenge with 10-50 mM ammonium bicarbonate / ammonium carbonate at pH of at least 10 mixed with acetonitrile in a 1 : 1 volume ratio, at 50°C, of acidic, basic, and / or neutral analytes, and wherein performance is maintained for at least 2 days of continuous operation. In some embodiments, resistance to degradation of separation performance is determined by assessing column efficiency, peak shape, and peak retention time during continuous challenge of acidic, basic, and / or neutral analytes wherein the column efficiency, peak shape, and peak retention time of the acidic, basic, and / or neutral analytes are compared to a reference column efficiency, peak shape, and peak retention time of the acidic, basic, and / or neutral analytes. In some embodiments, the column efficiency is from about 80% to about 99% of the reference. In some embodiments, the column efficiency is 80% of the reference. In some embodiments, the column efficiency is 85% of the reference. In some embodiments, the column efficiency is 90% of the reference. In some embodiments, the column efficiency is 95% of the reference. In some embodiments, the column efficiency is 99% of the reference. In some embodiments, the peak shape is from about 80% to about 99% of the reference. In some embodiments, the peak shape is 80% of the reference. In some embodiments, the peak shape is 85% of the reference. In some embodiments, the peak shape is 90% of the reference. In some embodiments, the peak shape is 95% of the reference. In some embodiments, the peak shape is 99% of the reference. In some embodiments, the peak retention time is from about 80% to about 99% of the reference. In some embodiments, the peak retention time is 80% of the reference. In some embodiments, the peak retention time is 85% of the reference. In some embodiments, the peak retention time is 90% of the reference. In some embodiments, the peak retention time is 95% of the reference. In some embodiments, the peak retention time is 99% of the reference. In some embodiments, the analyte is acidic, basic, neutral, or a combination thereof. In some embodiments, the analyte is acidic. In some embodiments, the analyte is basic. In some embodiments, the analyte is neutral.

[0057] In order that the subject matter disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner.

[0058] Examples

[0059] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the materials, methods, procedures, compounds, compositions, devices or articles, claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors claim as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g.. amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, or at ambient temperature, which is at or near 22°C, and pressure is at or near atmospheric.

[0060] The elemental analysis (%C) values were measured by combustion analysis (Robertson Microlit Laboratories, Ledgewood, NJ). These values were employed to establish ligand coverage measures based on known composition of compounds and Specific Surface Areas (m2 / g). The specific surface areas (SSA), specific pore volumes (SPY) and the average pore diameters (APD) of these materials were measured using the multi-point N2 sorption method (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, Ga.). The SSA was calculated using the BET method, the SPY was the single point value determined for P / Po>0.98 and the APD w as calculated from the desorption portion of the isotherm using the BJH method. Particle sizes were measured using a Beckman Coulter Multisizer 3 analyzer (30 pm aperture. 70.000 counts; Miami. Fla.). The particle diameter (dp) was measured as the 50% cumulative diameter of the volume-based particle size distribution. The width of the distribution was measured as the 90% cumulative volume diameter divided by the 10% cumulative volume diameter (denoted 90 / 10 ratio). Generally, values of surface coverage are expressed as normalized to the elemental composition and SSA of samples, to yield molar surface coverage of the silica surface with ligand in pmol / m2. Suitable corrections were applied to account for carbon incorporated into the silica particle surface and are conducted distinctly for coverage of the primary ligand, usually the C 18 ligand, and the end-cap ligand, usually the trimethyl silane reagent.

[0061] Bonded-phase silica particles were employed to load stainless steel HPLC columns of various lengths and diamaters, as described in the Figures and Examples. These materials were applied to chromatographic separations of various small and larger molecules, primarily using reversed-phase separations. As an example, Figure 1 panels A and B show' a separation of a small molecule mixture, uracil, acenapthalene. and amitriptyline using a reversed-phase system with alkaline acqueous solution (pH 10), modified with acetonitrile as the organic modifier. The separation was accomplished using the Shimadzu Nexera LC instrument, at a flow rate and column temperature as specifed for this example. Detection of the analytes used absorbance at 254 nm. Chromatographic retention indices in isocratic elution (k’) are calculated using the standard formulae, with k’= (RTx-RT0) / RT0, in which the RTx refers to the retention time of the compound of interest, and RT0 is the unretained compounds, also known as the void volume marker, which in the examples is measured using uracil. Thus, a constant k’, measured in response to challenge by an aggressive mobile phase, is a desirable property, as this reflects resistance to column packing degradation in the mobile phase environment. In gradient elution, retention is measured in absolute time, usually in minutes, for evaluation of the stability of the separation. Longevity of column lifetimes, or resistance to column aging effects, can also be evaluated in reference to measures of the volume of mobile phase passed through the column bed (Synder, Kirkland and Dolan, Introduction to Modem Liquid Chromatography, 3rd Ed.). In such cases, a convenient measure is the number of column volumes (void volumes) that are passed through the bed. In general practice, and used interchangeably in the scientific literature, retention is measured as a function of time for the operation of the column (at fixed flow rate), the volume of solvent passed through the column, or the number of column volumes passed through the column.

[0062] Example 1:

[0063] A 20 mM ammonium acetate solution with a pH of 9. 1 was cooled to 4°C overnight. Next, 30 g of superficially porous silica of 156 A pore size and 72.6 m2 / g specific surface area (SSA) was added to a 500 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and 300 mL of the 20 mM ammonium acetate was added and the mixture was stirred. Then, 7.6 g of l,2-bis(triethoxysilyl)ethane was added and the mixture was stirred for 6 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature, the pH measured was 5.4 and was adjusted to pH 9. 1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The mixture was then filtered, dispersed as a 10% slurry, and then boiled in water overnight. The slurry was cooled, filtered, washed with water and methanol, and dried under vacuum. The resulting silica particles had a surface silica incorporated carbon content of 1.50%, a pore size of 118 A. and SSA of 73.0 m2 / g.

[0064] Example 2:

[0065] A 20 mM ammonium acetate solution with a pH of 9.1 was cooled to 4°C overnight. Next, 10 g of superficially porous silica of 151 A pore size and 72.8 m2 / g SSA was added to a 250 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, 100 mL of the 20 mM ammonium acetate was added and the mixture stirred before addition of 2.5 grams of L2-bis(triethoxysilyl)ethane. The mixture was stirred for 6 hours at low temperature. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under a nitrogen. The mixture was then cooled to room temperature, the pH measured was 5.4 and was adjusted to pH 9. 1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The silica was recovered by filtration, then dispersed as a 10% slurry, and the mixture was then boiled in water overnight. The slurry was cooled and filtered. The resulting silica particles had a surface silica incorporated carbon content of 1.63%, a pore size of 114 A, and SSA of 73.5 m2 / g.

[0066] Example 3:

[0067] A 50 mM ammonium acetate solution with a pH of 9. 1 was cooled to 4°C overnight. Next, 10 g of superficially porous silica with a pore size of 151 A and SSA of 72.8 m2 / g was added to a 250 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and 100 mL of 50 mM ammonium acetate was added and the mixture was stirred. Then, 2.5 g of 1 ,2-bis(tri ethoxy silyljethane was added and the mixture was stirred for 6 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature and the pH was adjusted to pH 9.1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The silica particles were recovered by filtration and dispersed as a 10% slurry in water. The mixture was then boiled in water overnight. The slurry was cooled and filtered. The resulting silica particles had a surface silica incorporated carbon content of 1.45%, a pore size of 111 A, and SSA of 75.0 m2 / g.

[0068] Example 4:

[0069] A 20 mM ammonium acetate solution with a pH of 9.1 was cooled to 4°C overnight. Next, 10 g of superficially porous silica with a pore size of 151 A and SSA of 72.8 m2 / g was added to a 250 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and 100 mL of the ammonium acetate was added and the mixture was stirred. Then. 2.5 g of l,2-bis(triethoxysilyl)ethane was added and the mixture was stirred for 4 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature and the pH was adjusted to pH 9.1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The solids were recovered by filtration, then dispersed in water as a 10% slurry and boiled overnight. The slurry was cooled and filtered. The resulting silica particles had a surface silica incorporated carbon content of 1.45%, a pore size of 116 A, and SSA of 76.4 m2 / g.

[0070] Example 5:

[0071] A 20 mM ammonium acetate solution with a pH of 9. 1 was cooled to 4°C overnight. Next, 10 g of superficially porous silica with a pore size of 151 A and SSA of 72.8 m2 / g was added to a 250 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and 100 mL of the ammonium acetate was added and the mixture was stirred. Then. 2.5 g of 1.2-bis(triethoxysilyl)ethane was added and the mixture was stirred for 18 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 6 hours under nitrogen. The mixture was then cooled to room temperature and the pH was adjusted to pH 9. 1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The solids were recovered by filtration, dispersed to form a 10% slurry in water, and the mixture was then boiled in water overnight. The slurry was cooled and filtered. The resulting silica particles had a surface silica incorporated carbon content of 1.43%, a pore size of 113 A, and SSA of 76.4 m2 / g.

[0072] Example 6:

[0073] A 20 mM ammonium acetate solution with a pH of 9. 1 was cooled to 4°C overnight. Next, 10 g of superficially porous silica with a pore size of 151 A and SSA of 72.8 m2 / g was added to a 250 mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and 100 mL of the ammonium acetate was added and the mixture was stirred. Then. 1.9 g of l,2-bis(triethoxysilyl)ethane was added and the mixture was stirred for 6 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature and the pH was adjusted to pH 9.1 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The solids were recovered, redispersed in water as a 10% slurry, and the mixture was then boiled in water overnight. The slurry was cooled and filtered. The resulting silica particles had a surface silica incorporated carbon content of 0.83%, a pore size of 130 A, and SSA of 75.1 m2 / g.

[0074] Example 7: 10 g of silica from the process in examples 1-5 dried at 110°C overnight was transferred to a 250 mL flask with a condenser and nitrogen stream. Next, 120 mL of toluene was added and the mixture was stirred. The mixture was then heated to 110°C and 6.2 g of octadecyldimethyl(dimethylamino)silane was added and stirred for 18 hours. The mixture was then cooled and filtered. The CIS-bounded silica was dried at 110°C overnight and endcapped with (N,N-dimethylamino)trimethylsilane. The total silica carbon content was 5.82%.

[0075] Example 8:

[0076] A 20 mM ammonium acetate solution with a pH of 9. 1 was cooled to 4°C overnight. Next 100g of superficially porous silica of 435 A pore size and SSA of 25.0 m2 / g was added to a 3L round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and lOOOmL of ammonium acetate was added and the mixture was stirred. Then, 9.2 g of 1,2- bis(triethoxysilyl)ethane was added and the mixture was stirred for 6 hours. The chilling wrap was removed and replaced with a heater. The mixture was then heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature, the pH measured was 5.44 and was adjusted to pH 9. 12 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The mixture was then boiled in water overnight. The slurry was cooled, filtered, washed with water and methanol, and dried under vacuum. The resulting silica particles had a surface silica incorporated carbon content of 0.56%, a pore size of 354 A, and SSA of 27.2 m2 / g.

[0077] 15 g of the resulting modified silica was dried under vacuum at 110°C overnight, wi th transfer to a 250 mL flask fitted with a condenser and nitrogen stream. Next, 180 mL of xylenes was added and the mixture was dispersed by overhead stirring, under nitrogen. The resulting mixture was heated to 139°C and 31.52 g of octadecyldimethyl(dimethylamino) silane was added and stirred for 18 hours. The mixture was then cooled and filtered, washed with THF, water, and acetonitrile. The C 18-bonded and modified silica was dried at 110°C overnight, and subsequently end-capped with (N.N-dimethylamino)trimethylsilane. The carbon content of the finish column packing material was 2.28%. Columns of this material were prepared in the 2. 1 I.D. x 50 mm format, exhibiting expected chromatographic efficiencies for separations of organic compounds with standard RP conditions, employing 70% acetonitrile in water. Figure 3 shows the separation of a synthetic oligonucleotide mixture of 10 to 60 bases in length (IDT. Inc. Coralville. IA. USA), using this wider pore RP material in a gradient elution ion pairing mobile phase, with 100 mM tri ethylamine acetate (Fluka, Seelze, Germany) adjusted to pH 8.5 as the buffer system, and acetonitrile as the organic modifier. Elution of the various length oligonucleotides proceeded in a manner expected, increasing retention based on chain length, and excellent stability of retention time over several days of use of the column was observed.

[0078] Example 9:

[0079] A 20 mM ammonium acetate solution with a pH of 9.1 was cooled to 4°C overnight. 15 g of Nucleodur 100-5 totally porous silica of nominal 5 pm particle diameter, 107 A pore size, and SSA of 334 m2 / g (Macherey -Nagel GmbH & Co. Duren. Germany) was added to a 250mL round-bottom flask. The flask was wrapped with a chilling wrap, cooled to 5-7°C, and a volume of 150mL of the cold ammonium acetate was added to the silica with overhead stirring of the mixture. After dispersion, 19.01 g of 1 ,2-bis(tri ethoxy silyljethane was added, and the mixture was stirred for 6 hours. The chilling wrap was removed and replaced with a heater. The mixture was heated to 100°C for 18 hours under nitrogen. The mixture was then cooled to room temperature, the pH measured as 5.41, then was adjusted to pH 9.12 with ammonium hydroxide. The mixture was then heated to 100°C for 24 hours. The resulting slurry was cooled, then centrifuged to remove the buffered solution, which was replaced by water, then brought to a boil for an additional 24 hours. The slurry was cooled, filtered, washed with water and acetone, and dried under vacuum. The resulting silica particles had a carbon content of 3.13%, a pore size of 94.2 A, and SSA of 265 m2 / g.

[0080] 15 g of the resulting modified silica was dried under vacuum at 110°C overnight, with transfer to a 250 mL flask fitted with a condenser and nitrogen stream. Next, 180 mL of xylenes was added and the mixture was dispersed by overhead stirring, under nitrogen. The resulting mixture was heated to 139°C and 31.52 g of octadecyldimethyl(dimethylamino) silane was added and stirred for 18 hours. The mixture was then cooled and filtered, washed with THF. water, and acetonitrile. The C 18-bonded and modified silica was dried at 110°C overnight, and subsequently end-capped with (N.N-dimethylamino)trimethylsilane. The carbon content of the finish column packing material was 16.27%. Columns of this material were prepared in the 2. 1 I.D. x 50 mm format, exhibiting expected chromatographic efficiencies for separations of organic compounds using elevated pH operating conditions, as shown in Figure 4. The Figure shows that minimal changes in efficiencies, peak shapes, or resolution of test mixtures are observed on use of the column for many samples, or extended periods of time. Table 1 presents results with the materials in the present examples, illustrating the effects of modifications of the silica particles on pore sizes and specific surface areas. Superficially porous particles (SPP), fully porous particles (FP) and wider pore SPP (WP SPP) are shown. Moderate and controlled effects are observed for pore size reduction using the surface modification of the present disclosure, for both SPP and FPP particles. Similarly, modest effects are observed on specific surface areas, indicating that these chromatographic materials are expected to exhibit acceptable chromatographic capacity factors and load tolerance, for both SPP and FPP types of silica, including smaller and larger pore silica bodies.

[0081] TABLE 1: Properties of Silica and Hybrid Inorganic / Organic Silica Materials

[0082] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to. journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims

What Is Claimed Is:

1. A method of producing a pH stable chromatographic material comprising the steps of: i) providing an ammonium acetate buffer of pH from about 8 to about 10; ii) cooling the buffer mixture to a temperature of about 2 to about 25°C; iii) adding a silica to produce a 5 to 20% slurry (w / v); iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 20°C; v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours; vi) cooling the reaction mixture to a temperature less than about 40°C; vii) adjusting the pH of the reaction mixture to about 8 to about 10; viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours; ix) cooling the reaction mixture to a temperature of about 40 to about 80°C; x) isolating a coated silica by centrifugation / filtration; xi) dispersing the coated silica in water; xii) heating the coated silica water slurry’ to about 40 to about 100°C for about 4 to about 24 hours; xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C; and xiv) isolating the coated silica by centrifugation / fdtration, rinsing the coated silica with a solvent, and dry ing the coated silica.

2. The method of claim 1, wherein the concentration of ammonium acetate in step i) is from about 5 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 20 mM to about 30 mM.

3. The method of claim 1, wherein the concentration of the silica is 5 to 10% (w / v) in step iii).

4. The method of claim 1, wherein the silica of step iii) is selected from the group consisting of a porous silica, a superficially porous silica, a non-porous silica, or any combination thereof.

5. The method of claim 1, wherein the adjustment in step i) is by the addition of an inorganic base, an organic base, or a combination thereof.

6. The method of claim 1, wherein the adjusted pH in step i) is from 9. 1 to 9.2.

7. The method of claim 1, wherein the buffer mixture of step ii) is cooled to from 5 to 6°C.

8. The method of claim 1, wherein the silane added in step iv) is selected from the group consisting of l,2-bis(triethoxysilyl)ethane, 1 ,2-bis(trichlorosilyl)ethane, tris(dimethylamino)ethyl silane, ethyltriethoxysilane, ethyltrimethoxysilane, methyltrichlorosilane, ethyltrichlorosilane, bis(trichlorosilyl)methane, bis(methyldimethoxysilyl)methane. 1,2-Bis(methyldichlorosilyl)ethane, 1.2- dichlorotetramethyldisilane, or any combination thereof.

9. The method of claim 1, wherein the reaction mixture of step iv) is stirred for 6 hours.

10. The method of claim 1, wherein the reaction mixture of step iv) is maintained at a temperature of from 5 to 6°C.

11. The method of claim 1, wherein the reaction mixture of step v) is heated to 100°C for 6 hours.

12. The method of claim 1, wherein step v) further comprises apply ing a gaseous stream.

13. The method of claim 12, wherein the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof.

14. The method of claim 12. wherein the gaseous stream is applied for about 6 to about 18 hours.

15. The method of claim 1, wherein the temperature of step vi) is from 5 to 6°C.

16. The method of claim 1, wherein the adjustment in step vii) is by the addition of an inorganic base, an organic base, or a combination thereof.

17. The method of claim 1, wherein the adjusted pH in step vii) is from 9.1 to 9.2.

18. The method of claim 1, wherein step vii) further comprises applying a gaseous stream.

19. The method of claim 18. wherein the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof.

20. The method of claim 18, wherein the gaseous stream is applied for 6 to 18 hours.

21. The method of claim 1, wherein the reaction mixture of step viii) is heated to 100°C for 24 hours.

22. The method of claim 1, wherein the concentration of the coated silica is 5 to 20% (w / v) in step xi).

23. The method of claim 1, wherein the coated silica water slurry of step xii) is heated to 100°C for 24 hours.

24. The method of claim 1, wherein the solvent in step xiv) is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, acetone, or any combination thereof.

25. A pH stable chromatographic material produced by the method according to any one of claims 1-24.

26. A method of atenuating the pore size of a silica particle wherein the pore size is changed by at least about 10% and no more than about 50%, the method comprising the steps of: i) providing an ammonium acetate buffer of pH from about 8 to about 10; ii) cooling the buffer mixture to a temperature of about 2 to about 20°C; iii) adding a silica to produce a 5 to 20% slurry (w / v); iv) adding a silane and allowing the reaction mixture to stir for about 1 to about 10 hours at a temperature of about 2 to about 25°C; v) heating the reaction mixture to about 40 to about 100°C for about 6 to about 18 hours; vi) cooling the reaction mixture to a temperature less than about 40°C; vii) adjusting the pH of the reaction mixture to about 8 to about 10; viii) heating the reaction mixture to about 40 to about 100°C for about 4 to about 24 hours; ix) cooling the reaction mixture to a temperature of about 40 to about 80°C; x) isolating a coated silica by centrifugation / filtration; xi) dispersing the coated silica in water; xii) heating the coated silica water slurry to about 40 to about 100°C for about 4 to about 24 hours; xiii) cooling the coated silica slurry to a temperature of about 40 to about 80°C; and xiv) isolating the coated silica by centrifugation / filtration. rinsing the coated silica with a solvent, and drying the coated silica.

27. The method of claim 26, wherein the concentration of ammonium acetate in step i) is from about 5 mM to about 50 M, from about 10 mM to about 40 mM, or from about 20 mM to about 30 mM.

28. The method of claim 26, wherein the concentration of the silica is 5 to 10% (w / v) in step iii).

29. The method of claim 26. wherein the silica of step hi) is selected from the group consisting of a porous silica, a superficially porous silica, a non-porous silica, or any combination thereof.

30. The method of claim 26. wherein the adjustment in step i) is by the addition of an inorganic base, an organic base, or a combination thereof.

31. The method of claim 26, wherein the adjusted pH in step i) is from 9.1 to 9.2.

32. The method of claim 26. wherein the buffer mixture of step ii) is cooled to from 5 to 6°C.

33. The method of claim 26, wherein the silane added in step iv) is selected from the group consisting of 1.2-bis(triethoxysilyl)ethane. 1 ,2-bis(trichlorosilyl)ethane. tris(dimethylamino)ethyl silane, ethyltriethoxysilane, ethyltrimethoxysilane, methyltrichlorosilane, ethyltrichlorosilane, bis(trichlorosilyl)methane, bis(methyldimethoxysilyl)methane, 1,2-Bis(methyldichlorosilyl)ethane, 1,2- dichlorotetramethyldisilane, or any combination thereof.

34. The method of claim 26, wherein the reaction mixture of step iv) is stirred for 6 hours.

35. The method of claim 26. wherein the reaction mixture of step iv) is maintained at a temperature of from 5 to 6°C.

36. The method of claim 26, wherein the reaction mixture of step v) is heated to 100°C for 6 hours.

37. The method of claim 26, wherein step v) further comprises applying a gaseous stream.

38. The method of claim 37. wherein the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air, or any combination thereof.

39. The method of claim 37. wherein the gaseous stream is applied for about 6 to about 18 hours.

40. The method of claim 26, wherein the temperature of step vi) is from 5 to 6°C.

41. The method of claim 26. wherein the adjustment in step vii) is by the addition of an inorganic base, an organic base, or a combination thereof.

42. The method of claim 26, wherein the adjusted pH in step vii) is from 9.1 to 9.2.

43. The method of claim 26, wherein step vii) further comprises applying a gaseous stream.

44. The method of claim 43, wherein the gaseous stream is selected from the group consisting of nitrogen, argon, carbon dioxide, helium, atmospheric air. or any combination thereof.

45. The method of claim 43, wherein the gaseous stream is applied for 6 to 18 hours.

46. The method of claim 26, wherein the reaction mixture of step viii) is heated to 100°C for 24 hours.

47. The method of claim 26. wherein the concentration of the coated silica is 10 to 20% (w / v) in step xi).

48. The method of claim 26, wherein the coated silica water slurry of step xii) is heated to 100°C for 24 hours.

49. The method of claim 26. wherein the solvent in step xiv) is selected from the group consisting of acetonitrile, tetrahydrofuran, methanol, acetone, or any combination thereof.

50. An attenuated silica particle material produced by the method according to any one of claims 26-49.

51. An attenuated silica particle material produced by the method according to any one of claims 26-49, wherein the pore size of the silica in step iii) is changed by at least about 10% and no more than about 30%.

52. An attenuated silica particle material produced by the method according to any one of claims 26-49, wherein the specific surface area of the silica in step iii) is changed by less than about 25%.

53. An attenuated silica particle material produced by the method according to any one of claims 26-49, wherein the pore size of the silica in step iii) is changed by at least about 10% and no more than about 50% and the specific surface area of the silica in step iii) is changed by less than about 25%.

54. An attenuated silica particle material according to any one of claims 50-53, wherein surface silica incorporated carbon content of the coated silica is from about 0. 15% to about 4.5%.

55. An attenuated silica particle material according to any one of claims 50-53, wherein the pore size of the coated silica is from about 85 A to about 1000 A.

56. An attenuated silica particle material according to any one of claims 50-53. wherein the surface silica incorporated carbon content of the coated silica is from about 0. 15% to about 4.5% and the pore size of the coated silica is from about 85 A to about 1000 A.

57. An attenuated silica particle material according to any one of claims 25 and / or 50 having an alkaline stability, wherein the alkaline stability' is determined by resistance to degradation of separation performance assessed by column efficiency, peak shape, and peakretention time during continuous challenge with a buffer at pH of at least 10 mixed with an organic solvent, at 50°C, of acidic, basic, and / or neutral analytes, and wherein performance is maintained for at least 2 days of continuous operation.

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